Download a guide-book to biochemistry

A GUIDE-BOOK TO
BIOCHEMISTRY
BY
KENNETH HARRISON
Professor of Biochemistry in the University of Tehran,
formerly Lecturer in BlOchem"try in the
University of Cambridge
CAMBRIDGE
A T THE UNIVERSITY PRESS
1960
PUBLISHED BY
THE SYNDICS OF THE CAMBRIDGE UNIVERSITY PRES,
Bentley House, 200 Euston Road, London, N. W. 1
American Branch: 32 East 57th Street, New York, 22, N.Y.
©
CAMBRIDGE UNIVERSITY PRESS
1959
First Printed 1959
Reprinted 1960
Printed
In
Great Britain at the Ull/versay Press, Cambridge
(Brooke Crutchley, University Printer)
CONTENTS
J
II
INTRODUCTION
ENERGY
page 1
8
III
ENZYMES
22
IV
OXIDATION
46
PHOTOSYNTHESIS
62
CARBOHYDRATE METABOLISM
77
FAT METABOLISM
99
V
VI
VII
VIII
IX
PROTEIN METABOLISM
111
CONTROL OF METABOLISM
134
APPENDIX: COMPLEX FORMULAE
138
INDEX
145
PREFACE
There are several good text-b09ks of biochemistry on the market,
and nobody has much quarrel with them-except that they are far
too long. We cannot blame authors who want to treat every branch
of the subject with justice, and develop a clear picture of the whole;
biochemistry itself is to blame. Still, anyone coming new to it may
be rather daunted by those thick volumes of 500 to 1000 pages. So
there ought to be room for something in the nature of a guide-book
dealing only with the bare bones; a few topics being ignored altogether and others liglltly passed over-in places a little too lightly,
perhaps. But for a more extended treatment, and in particular for
the sort of evidence that shows how living machinery works, the
inquirer will go to bigger and better books: Baldwin, Carter and
Thompson, Fruton and Simmonds, Mitchell, Thorpe, West and
Todd, etc.
In trying to understand biochemistry the reader can take advantage
of two devices that have been forced on biochemists by the very
nature of their subject-matter: a kind of shorthand, and flow-sheets.
As to the first, some very elaborate chemical structures are merely
FP or DPN or ATP in biochemical language, and wherever such
abbreviations are met with it is advisable-at a first glance-to
remember them and the type of process they enter into, even at the
expense of forgetting exactly what they represent. As to the other
device, the pattern of many reactions is more easily grasped by
following carbon atoms, or chemical names, through a series of
changes than by constant attention to the intricacies of chemical
structure. These and similar aids to understanding have been emphasized as much as possible; and here and there) in the interests of
clarity, various short-cuts have been made, so that things appear to
be a little simpler than they really are.
vii
PREFACE
I am grateful to a number of friends-especially H. B. F. Dixon,
G. D. Greville, J. Harley-Mason, D. H. Northcote, and E. C. Webb
-for reading over sections of the manuscript; they are not responsible
for mistakes, clumsy wording, or the wrong slant, wherever such
flaws occur.
K. H.
KING'S COLLEGE, CAMBRIDGE
October 1957
I
INTRODUCTION
Anyone hearing the word biochemistry for the first time may
reasonably ask-' What is it about?', 'What goes on?' These questions cannot be answered in a few, even well-chosen, words. It is true
that definitions of biochemistry have been put forward from time
to time, good definitions as far as they go, very neatly expressed;
yet they all suffer from the drawback of being intelligible enough to
those who already know, but much less helpful to those who do not.
This state of affairs is hardly surprising, because the range and extent
of biochemical interests cannot be packed into a single phrase, and
besides, the frontiers~of .the subject have never been clearly defined
and are continually shifting. Biochemistry must be thought of as
having many' growing points', encroaching on the territory of other
branches of science-for instance, chemistry, physiology and medicine
-and bringing methods of its own to bear' on problems that have
not been solved in orthodox ways. It is, perhaps, by this fresh and
unconventional approach that the biochemist makes his most
characteristic contribution to the increase of knowledge.
The simplest way of getting an insigh.t into biochemistry is to go
through a list of some of the chief topics that are being explored at
the present time. By realizing what sorts of biochemistry there are,
it should not be hard to form a notion of what biochemistry is.
1. The cbemistry of natural products: particularly the structure of
compounds involved in the process of keeping living systems alive.
Here the provinces of biochemistry and of organic chemistry overlap
to a great extent, though not entirely. The organic chemist, discovering a new cell constituent, will not only want to find out its
structure but will also want to synthesize a range of analogues and
derivatives and investigate their reactions, because he is interested
in chemical compounds for their own sake. The attitude of the biochemist is rather different: he is chiefly concerned with the behaviour
INTRODUCTION
of compounds in their natural environment, and how they are geared
into the machinery of life; his job begins where th~ organic chemist
leaves off. There are, of course, biochemists with organic chemical
leanings, spending their time largely on problems of chemical
structure, but they tend to be in a minority.
2. Properties of enzymes. Enzymes are organic catalysts of high
molecular weight (chapter III, p. 22) which occur in cells, or are
secreted by cells. With care, enzymes can be isolated and purified
without damage to their catalytic function, and many have been
obtained in crystalline form. Already the study of their behaviour
has thrown light on what may be called the physical chemistry of
life, and pathways of chemical transformation can be followed in
detail by allowing purified enzymes in the test-tube to catalyse the
reactions they bring about in the cell. It is also becoming clear that
a number of poisons and drugs exert their. action not by virtue of
mysterious or occult powers, but because they interfere with the
normal activities of enzymes. Here, as at other points, biochemistry is beginning to make useful contact with pharmacology
and medicine.
3. Structure of cells. All the chemicals required by a cell must, in
the first place, pass through its cell-wall. There is abundant evidence,
much of which has been obtained by the use of isotopes, that the
cell-wall and the cell-membrane bound up with it do not act merely
as a passive barrier, like a collodion sac; many substances are actively
transported into and out of cells in a very selective fashion. Until
more is known of the nature of the boundary-a complex structllreand of how this 'ferry' system works, our knowledge of vital
mechanisms will be decidedly incomplete. Again, the enzymes within
a cell are by no means evenly distributed throughout the cytoplasm;
some of the most important are found gathered together in small
particles called mitochondria (p. 108), and other kinds of particle
have been recognized, no doubt with specific tasks -to perform.
A great deal of attention has also been directed to the contractile
cells of muscle, in which structure and function must be closely
related. But biochemical methods alone cannot solve all the pro2
INTRODUCTION
blems of cell structure, and increasing use is being made of X-rays
and the electron microscope in this field of research, where biochemistry quickly passes over into biophysics.
4. Chemical microbiology, the study of the chemistry and physiology
of bacteria and of organisms like yeasts and moulds. Quite apart
from their industrial applications-in brewing, for instance; and the
manufacture of antibiotics-and the part they play in causing disease,
all micro-organisms are of importance to biochemistry for the following reason; they grow and multiply extremely fast, given the right
conditions, and hence their turnover of chemical compounds is very
large, or, in other words; their metabolic rate is very high. We should
expect, therefore, that certain kinds "'Of reaction could be more easily
detected in micro-organisms than ip. animals, whose metabolic rate
is lower; and so it has proved. The rapid multiplication of these
organisms likewise affords a convenient approach to the chemical
basis of genetics. Thus in recent years 'chemical microbiology' has
becomi1a portmanteau phrase, covering a wide range of interests.
The mode of action of penicillin and of the sulphonamide drugs has
been largely explained, the fixation of nitrogen by bacteria is well on
the way to being understood, the roles of heredity and environment
in the formation of enzymes are being explored, the kinetics of growth
have received attention from physical chemists; and the tale is not
half told.
5. Tissue metabolism. The cells that make up the tissues and organs
of higher animals are not easily separated from one another without
damage to their structure and chemical functions. In studying the
metabolism of isolated organs like liver, kidney or brain, therefore,
no attempt is made as a rule to detach individual cells; instead, thin
slices of the material are cut off and suspended in a suitable medium.
A few of the cells are damaged in the cutting, but the great majority
are not, and so thin is the sheet of tissue that oxygen and carbon
dioxide can diffuse into and out of it as rapidly as in the intact organ
with its supply of oxygenated blood. By adding sugars or amino
acids or other chemicals to the medium it is possible to find out
whether they are metabolized by the tissue, and to detect the products
3
INTRODUCTION
of metabolism. Naturally the working life of these slices is rather
short-a few hours at most-but with their aid, and by similar
techniques, many reactions of great significance have been discovered.
6. Plant biochemistry. Everyone is aware that green plants, and a
few kinds of bacteria, can use the energy of sunlight to bring about
chemical changes. The ability of plants to fix carbon dioxide and
liberate oxygen is the most important single process in the whole
range of vital activities, for upon it almost all other forms of life
depend. Although some of the details of photosynthesis (chapter v,
p. 62) are still unexplained, the pathway from carbon dioxide to
starch has been made clear, anq' several uncommon sugars that take
,r
part in this process-mere chemical curiosities a few years ago-are
now known to be involved also in the metabolism of animal cells.
Among other activities in plant biochemistry, the isolation and
purification of viruses may be singled out. A number of plant viruses
have been obtaineg. in crystalline form, and the study of their
chemical composition, and of how they multiply within the host, is
likely to resolve some of the fundamental problems of growth and
multiplication.
7. Hormones. These 'chemical messengers', secreted by specific
glands, circulate in the animal body and profoundly influence its
growth and metabolism. The well-being of a great variety of creatures,
from insects to man, depends upon the delicate balance of this
hormonal activity. Although hormo!1es may seem to lie well within
the frontiers of physiology and medicine, the biochemist wants tp
know their structure and how they act, whether by altering the
catalytic behaviour of enzymes, or by regulating the flow of materials
through the cell-walls, or in other ways. Many of the most obvious
questions about hormones cannot yet be answered, in spite of the
fact that several of them are chemically quite simple, because it has
hitherto been difficult enough to demonstrate their action on isolated
tissues, let alone on cell-membranes as such, or on enzymes. For the
most part, then, our insight into the way hormones work is derived
from animal experiments and from the clinical observation of
4
INTRODUCTION
human beings, and we may expect further information from the
study of hormone-like substances that have been shown to occur in
plants.
8. Nutritioe' This is a branch of biochemistry so wide in its applications as almost to have attained the rank of an independent science.
The feeding oflivestock-cattle and sheep, pigs and poultry, and of
man himself-brings the biochemist into contact with economics
and the political scene. For each and every kind of animal a great
deal of information is needed: the composition of food, the constituents re9uired for optimum growth, a knowledge of vitamins and
trace-elements and the disorders that follow from lack of them.
Here, as with hormones, observations on the intact animal must be
made; and the results obtained oy experiment are not always easy
to translate into economic terms, or into language that the farmer
and the politician can make use of. The growth of crops, the nitrogen
cycle, and the metabolism of soil organisms are also matters that fall
within the biochemist's field of study.
Thus the range of biochemistry extends from the slmplest constituents of the cell by way of more cpmplex molecules to the
organization of the cell itself, and finally to the metabolism of plants
and animals considered as intact structures, in health and disease.
In aspiring to understand all these things the biochemist may seem
ambitious, and certainly an individual cannot hope to master more
than a small part of his subject. What makes biochemistry possible,
ho~ever, and saves it from utter confusion, is simply this: that
although Life is complicated, it is not nearly so complicated as it
might be.
. At a first introduction to natural history we learn with astonishment how many sorts and kinds of animals and plants there are:
forty thousand species of fungi, more than half a million species of
insects, and so on. Then, when surprise has worn off, the prodlgality
of Nature sinks into a commonplace to which we seldom give a
thought. Fortunately, however, Nature is not equally prodigal of
chemical machinery. If all the principal orders and divisions of the
animal and plant kingdoms had their own peculiar ways of carrying
5
INTRODUCTION
on life, if chemical variations were evenly matched with variations
of form and structure, the task of biochemistry would be alarming;
but although living things can and do exist in a wide variety of shapes
and sizes, the scope of their chemical behaviour is more limited.
A single example will serve to show how much there can be in
common between creatures far apart in the evolutionary scale.
Yeast is a unicellular organism that breaks down glucose via
pyruvic acid to ethyl alcohol. Human muscle also turns glucose into
pyruvic acid, from which is formed the lactic acid that appears in
the blood-stream during exercise. The transformation of glucose to
pyruvic acid by yeast takes place in eight stages, eight separate
reactions that can be followed in the test-tube with purified enzymes.
Exactly the same eight reactions go on in human muscle, and in the
same order. These facts are not a little remarkable when we reflect
on the ages that have passed since the forerunners of man and the
yeast cell parted company in the course of evolution.
X
A
/
yeast
'"
B
man
(D
In (I) let X represent a bit of primordial slime, or a primitive cell.
Once upon a time, perhaps five hundred million years ago, X divided
into A and B, the respective ancestors of yeast and mankind. Ever
since that division, despite countless mutations, our human stock
has clung to chemical habits picked up in the good old days in the
primeval ooze.
The same point could be illustrated from other creatures and by
other reactions. Thus whereas the outward appearance of plants and
animals is very variable, the inward chemistry to a great extent
follows a set pattern. To a great extent, but by no means entirely;
micro-organisms in particular ha,ve adopted some curious methods
of keeping themselves going, and even among the higher animals
and plants there are significant differences of biochemical behaviour,
so that another branch of the subject, Comparative Biochemistry,
is occupied with their study. Still, when every exception has been
6
INTRODUCTION
allowed for, there remains much ground that is common to all
animals, or all plants, or to both. It is the purpose of this book to
discuss some of these chemical structures and pathWays in common
use, for we may be sure that, after the long testing throughout
geological time, they have been found-if not the best in the best of
all possible worlds-at least serviceable enough for everyday life.
But before looking at the chemical processes that go on, it is
desirable to know why they go on. All animals and plants and
micro-organisms use energy to keep themselves alive; and the kind
of energy they need. and how they get it and make use of it, must
first be explored.
7
II
ENERGY
Since the higher animals maintain themselves at a temperature of
about 37 C., with variations of only a few degrees either way, they
cannot use heat as a source of energy in the manner of a steam engine
because heat can only be made to do work if there is a source of it
at a high temperature, and a condenser, or exhaust, at a much lower
temperature. In the animal body, and in cells generally, appreCIable
temperature gradients do not occur. Nevertheless, animals 'work'
in some useful sense of the word, and so do plants, in a more inconspicuous way; and the reason is that the energy obtained from
chemical reactions can be used to perform work even under conditions of more or less constant temperature and pressure. The
amount of useful chemical work that can be got from a reaction is
tied up with the notion of free energy change; free energy being
denoted by F, the change is symbolized by tlF.
When considering energy changes in a chemical system it is not
immediately obvious that they should be looked at strictly from the
point of view of the substances taking part. For instance, if carbon
is burnt in oxygen we tend to regard the energy produced as a positive
quantity. But, in terms of the reaction itself,
0
C+02 = C02
energy change = -100,000 cal./mole approximately.
The negative sign expresses what is happening to the reactants from
their own point of view: they are losing energy. And chemists and
physicists have come to agree that this way of looking at the system
is the best way, and saves trouble in the long run.
As an approach to the nature of free energy, let us now consider
the decomposition of hydrogen peroxide, 2H20 Z ~ 2H 20+02 •
Pure hydrogen peroxide is a reasonably stable substance which can
be stored for months or even years without signific<l;nt alteration,
but when it is heated to about 150 0 C. a violent explosive reaction
8
6.F
occurs, water and oxygen are formed, and energy is liberated. In (I)
the amount of energy required to activate the molecules and start
the reaction is represented by E a , the activation energy; this quantity
will be discussed later in connexion with enzymes (p. 39). For the
moment we are concerned only with the over-all difference in free
energy content between the products at the end (water and oxygen)
--H,O
-,------
--
--------J~.
--------
{)'F
H,O+
oxygen
x
(1)
and the compound at the start of the reaction (H 2 0 2). This difference
is represented in (I) by 6.F, and amounts to about 25,000 cal./mole.
Since the decomposition is of a violent explosive nature, and since
the reverse reaction-the direct union of water vapour with oxygen
to form H 20 2-is very hard to achieve, 25,000 cal./mole of free
energy are lost when H 2 0 2 breaks up. Consequently l1F is negative.
It is important, however, to realize that a large value of -l1F does
not necessarily mean that the reaction will go on at a rapid rate; it is
only a measure of the initial and final energy contents of the system.
In any reversible system A + B ~ Y + Z, if l1F is negative the
reaction, once started, tends to go spontaneously from left to right.
When equilibrium has been attained clearly l1F = 0, and no further
useful chemical energy can be extracted from the system because it
no longer has a tendency to move in either direction. And if we want
9
ENERGY
to synthesize A + B from Y + Z energy must in some way be supplied,
and AF for this reverse reaction is positive. In brief, under constant
conditions of temperature and pressure:
- AF
Reaction can go spontaneously towards the equilibrium
point
AF = 0 Equilibrium has been attained
+ t!.F
Reaction has to be forced away from equilibrium
A reaction with negative t!.F ~s called exergonic; if - t!.F is large, as
with H 20 2 , and the produets are gaseous, the reaction will be
explosive. Reactions with a positive value of t!.F are called endergonic. In any particular reversible system, at a given concentration
of products and reactants, the t!.F values for the forward and reverse
reaction will be equal and opposite.
Factors which drive reactions
We must now look into the relation between free energy, heat energy,
and entropy. Under conditions of constant temperature and 'Pressure
the free energy of a reversible chemical system is defined by
F = H-(T.S),
where H is the heat content in calories, T the absolute temperature,
and S represents the entropy, whose nature will be discussed in a
moment. In biochemistry we are less interested in the real magnitude
of F, Hand S than in the difference between these quantities before
and after a reaction has taken place, so we can write, at constant
temperature,
AF = t!.H - (T. t!.S).
If a reaction gives out heat, as in the decomposition of hydrogen
peroxide, t!.H will be negative; the system is losing heat to its surroundings. Hence, if the term (T.t!.S) is not too large, t!.F will be
negative also, indicating that the reaction, once started, can proceed
spontaneously to equilibrium. But the term (T.t!.S) must not be
overlooked. The quantity S, the entropy, can be thought of as a
measure of disorder. Solids represent the most orderly state of
matter; liquids, solutions and gases are less orderly, and so are said
to possess more entropy. Changes of state, solid into liquid (or
solution), and liquid into gas, involve increases of entropy, sym10
FACTORS WHICH DRl VB REACTIONS
bolized +~S. Consider, for example, a crystalline salt such as
sodium chloride. In the solid state the atoms are arranged in a
regular orderly fashion in the crystal lattice, but when the salt is
dissolved in water it loses its crystal structure, and hence an increase
of entropy occurs. The equation ~F = ~H - (T. ~S) shows that
reactions tend to proceed when the disorder increases. With NaCl,
the change of entropy when the crystals dissolve is considerable, and
the term within the brackets (T.~S) is larger, in this case, than ~H;
so D.F is negative and the reaction goes forward.
Thus in a very simple process, such as the dissolving of crystals in
water, the entropy change may be the decisive quantity in determining the val_lle of D.F. Considerable changes ~n entropy may also
take place \\!hen a complex compound is hydrolysed, or otherwise
broken d(}wn, to simpler molecules, or when changes of state are
involved; such as the absorption or evolution of gases in a reaction.
But in the majority of biochemical transformations the entropy
change is relatively small compared with D.F or D.H, because the
compounds taking part are of similar shape and size, and are in
solution already.
Changes in D.F with concentration
The more dilute a solution the greater the entropy, and therefore the
entropy change of a reaction in solution varies with the concentrations of the reactants and product~ taking part in it. D.H, however,
is much less sensitive to changes in concentration, so D.F alters with
the concentrations primarily because of the alteration in D.S. The
change in ~F with changing concentrations can be derived from the
equation
D.F = 2.3RT 10
product of molar concentrations of products
glO product of molar concentrations of reactants
-2·3RTlog IO K,
where R is the gas constant and K is the equilibrium constant. In this
equation, the first term on the right-hand side represents the concentrations of products and reactants actually taking part in the reaction;
and K represents, as usual, the equilibrium concentrations.! It will
be observed that at equilibrium D.F = 0, because the two terms cancel
1 The equation properly applies not to concentrations but to • activities'; in
dilute solution, however, these terms are almost equivalent.
11
ENERGY
when equilIbrium concentrations are inserted into the first term.
Suppose now that one product in a reaction is at one-tenth of the
equilibrium concentration, and that all the other products and
reactants are at the equilibrium concentrations. Then the equation
reduces to flF = - 2· 3RT) The value of R is 1·99 cal./molet C. ;
at body temperature, 37° C., T = 310, so
flF = - 2· 3 x 1·99 x 310 cal./mole,
and
-flF
= 1420 cal./mole.
flF is negative since the reaction will proceed towards equilibrium
if one of the products is decreased in concentration. Hence each
tenfold change in the concentration of one product alters flF by
1420 cal./mole, at body temperature.
It also follows that if all the products and reactants are at unit
(molar) concentration, the first term will vanish and
flFO
= -2'3RTlog lO K,
where flFO is the' standard free energy' change. flFo is a constant
characteristic of any particular reaction, because it is the value of
flF when the reactants and products are all present under defined
conditions. This relation is very useful, since the free energy of a
reaction is often difficult to evaluate in any other way.2
When a series of biochemical transformations takes place in the
cell-such as the complete oxidation of a foodstuff-the concentrations of reactants and products are not greatly different, as a rule,
and the molecules are often of similar size and shape. Hence the
entropy term (T. flS) is not very large. and for many practical purposes flF is nearly the same as flH. Take, for instance, the oxidation
of glucose:
1
Because, wnting C for the equilibrium concentrations,
AF=2'3RTloglO i-.C-2·3RTlogIOC
=2·3RT [lOglO ,lo+ log,. C-lOglO C]
=2·3RT(-])= -2-3RT.
2 For bIOchemical purposes, however, AF is sometimes calculated for 0·01 M
solutions (stnctly 'activities', not concentrations) and for gases at the partial
atmosphenc pressure. Inasmuch as these quantities are similar to those occurnng
in the cell, the figure of AF so obtained becomes a closer appro:lUmation to what
may reasonably be expected to happen in llvmg matter.
12
CHANGES IN
!:J.F
WITH CONCENTRATION
If glucose is burnt in a calorimeter, the heat output, !:J.H, is 673,000
cal./mole. A direct measurement of !:J.F is not possible in this case,
but the figure can be calculated and is found to be 691,000 cal./mole.
Thus !:J.H-!:J.F= (-673,000)-(-691,000) = +18,100 cal. (Le.,
T.6.S). The quantity of heat and the quantity of free energy obtainable from the oxidation of glucose are therefore within 3 % of
one another, and the same is true of most other substances that can
be used for food. It is only because !:J.H often approximates to !:J.F
that the calorific value for the oxidation of foodstuffs has any useful
meaning in metabolic studies. Heat changes by themselves have very
little significance for the organism; what really matters is the free
energy that can be extracted from the food and used for chemical
work of various kinds. And the total free energy theoretically made
available by oxidizing food completely to CO2 and H 20 is just the
same, however many stages are involved and however the oxidation
is conducted. If we throw a lump of sugar on the fire it burns up
quickly and the free energy is dissipated; if we give it to the dog,
which burns sugar slowly in a different way, the output offree energy
from each molecule is precisely the same, but is harnessed to perform
chemical work. We must now consider, in general terms, the sort of way
in which free energy becomes available to the organism during the
course of metabolism.
Energy-rich bonds
When a substance is broken down in the cell, most usually by oxidation, all the free energy obtainable from the series of reactions is not
liberated at once. Instead, what happens is that the free energy is
tapped off in stages, bit by bit, as it were, in packets. Also-and this
point is extremely important-these packets of free energy do not
need to be used immediately for chemical work, but can be stored,
for the time being, in various ways. Suppose a compound A is
transformed, through a series of other compounds, B, C, D, E, and
F to G (In. Free energy does not become available to the organism
at every stage during the process; a certain amount of chemical
jugglery and manreuvring takes place en route; and we may suppose,
for the sake of illustration, that free energy is only tapped off at two
stages in the chain, in virtue of reactions B -+ C and E -+ F. This kind
of procedure-the stepwise breakdown of a substance, and the
13
ENERGY
tapping off of its free energy to temporary storage at some of the
steps-is very general in biological systems.
When a chemical compound undergoes a series oftransformations,
as A ... -+ G, the chemical bonds that hold the atoms together are
broken or rearranged to a greater or lesser extent; and in the course
of the process a type of bond can arise which is known as •energyrich'. Such bonds can be considered, for our purposes, as the channel
through which free energy is tapped off; and before discussing the
various types of energy-rich bond that turn up in living systems it is
A-BIC~D-E:F-G
.
I
I
:
I
I
I
I
I
I
I
..,
free energy
tapped off
""
free energy
tapped off
temporary
storage
/
(II)
necessary to be clear about the meaning of the term in biochemical
language. To the physical chemist, bond energy means the amount
of energy needed to separate two atoms of a molecule from one
another; for example, to bre,ak the O-H bond in water requires
about 120,000 cal./mole. The stronger the bond, the greater the heat
energy required to dissociate it. But in biochemistry an energy-rich
bond is so called to distinguish it from an energy-poor bond, and
in each case the energy is the free energy liberated on hydrolysis. Thus
the terms energy-rich and energy-poor have a restricted meaning;
yet the notion of such bonds, introduced by F. Lipmann in 1?41,
plays an indispensable part in our understanding of how the cell
machinery works. The symbol ,..., is often used to denote an energyrich bond, but the convention, though widely employed, must not
be allowed to obscure the fact that free energy changes occur as a
result of reactions between complete molecules. Thus when the
symbol ,..., occurs in a biochemical formula, it only means that the
particular compound can be expected, under the right conditions,
to enter into reactions which may involve free energy changes of as
much as 10,000 cal./mole, or thereabouts.
14
ENERGY-RICH BONDS
Five types of energy-rich bond are known.
1. Pyrophosphates. Many compounds with -OR groups, such as
alcohols or sugars, can react with orthophosphoric acid to give esters
or glycosides:
0
0
II
R-OH+HO-P-OH
~
II
R-O-P-OH + H20
6H
6H
For convenience we write the ester R-,.O-®, where ® stands for
the phosphoric acid residue. On hydrolysis of such compounds the
standard free energy change -D.PO, is about 3000 cal./mole; con!\
versely, to form the compound + D..FO mu~t be the same figure. If
more phosphoric acid is added, by elimination of water we get a
pyrophosphate
0
0
II
II
R-O-P-O-P-OH
I
OH
I
OH
When, however, only the second phosphoric acid residue is hydrolysed, giving R-O-® and inorganic phosphate, -D..FO is found to
be about 8000 cal./mole. Thus the second phosphate residue is
energy-rich by comparison with the first, and is symbolized by ~ ®
instead of -®. So ,,-:e have
R-O-®~®+H20 ~
R-O-®+H20
~
R-O-®+H3P04,
R-OH+H3P04,
-tJ.FO = 8000 ca1./mole
approximately
-t!.Fo = 3000 ca1./mole
approximately.
It is also possible to add more phosphoric acid to the diphosphate
and get R-O-® ~ ® ~ ®, and this type of triphosphate, to
which we shall return later, contains two energy-rich bonds, each
worth about 8000 cal./mole, and one bond that is energy-poor.
2. Acyl phosphates are of the form
o
II
R-C-O~®
These acyl phosphates are energy-rich, more so than pyrophosphates,
-t.Fo being about 12,000 cal./mole on hydrolysis.
3. Enol phosphates arise by the elimination of water
CH20H
I
R-C-O-®
CH2
-H20"
----+ R-C-O~®
h
15
ENERGY
and, as in the previous case, we have an energy-rich bond, - .6.po
being about 12,000 cal./mole. It will be observed that acyl phosphates
and enol phosphates both contain the grouping
"
-C-o~®
4. Guanidine phosphates of the type
NH
II
R-C-NH~®
where the -NH-linking carbon to the phosphate residue replaces
the - 0 - of the preceding types. For this kind of energy-rich bond
-.6.po is about 10,000' c~l./mole.
5. Acyl mercaptide energy-rich bonds are of a different sort, since
phosphate does not enter into them. One way in which they are
formed is by oxidation
OH
0
I
R-C-S-R'
II
-?
R-C-S-R'
h
On hydrolysis of this -C '" S-link, -.6.PO is about 8000 cal./mole.
The acyl mercaptide bond can not only react with water but also,
under appropriate conditions, with orthophosphate (Un. In this
type of reaction, the free energy of the acyl mercaptide bond is not
o
0
"
R-C-S-R'
+
II
R-C-O~®
-+
+
HSR'
0
\I
HO-P-OH
~m
(Ill)
lost, but is retained by the acyl phosphate, R. CO. 0 '" ®, or, in
other words, a transfer of potential free energy has taken place
between one compound and another. Such transfer reactions,
catalysed by enzymes, are of common occurrence in the cell, and
are by no means confined to acyl mercaptides, as we shall see in a
moment.
Formation of ATP
We are now in a position to go more thoroughly int~ the model
sequence of reactions already noticed on p. 13. In the series
16
FORMATION OF ATP
A ~ B, B ~ C ... G, we supposed that a packet or parcel of free
energy, representing a fraction of the total free energy available from
the change A ... ~ G, was tapped off at two points: first by the
reaction B -+ C, and secondly ,by the reaction E -+ F. We are not
here concerned with trying to understand why or how energy-rich
bonds are generated by some reactions and n.ot by others. It is
enough to know that these things do happen.
Let us now suppose that, in the sequence of reactions just mentioned, B is a phosphate ester, and has the formula B-O-®. Then,
without inquiring too closely into the details, the transformation of
B into the next compound C can be written:
B-O-®~C-O"",
®.
Such a reaction might occur in various ways, e.g. the elimination of
water (as for enol phosphates), but, whatever the mechanism, some
of the available free energy from the change A '" -+ G has been
tapped off into the energy-rich bond of C-O ,..., ®. The packet of
free energy is not, strictly speaking, in the energy-rich bond, but is
symbolized as if it were. What has happened amounts to this: that
whereas neither A nor B-O-® can release any of their free energy
in a way that is biologically useful, by contrast C-O ,..., ® is ready
to transfer a portion of its free energy to a suitable acceptor.
We next have to consider how this portion of free energy can be
put into temporary storage, and we can write
c-o '"
® + HO-Z -+ C-OH + Z-O ,..., ®.
Part of the free energy has now been detached altogether from the
sequence of reactions A ... -+ G, and is represented by the free
energy of the energy-rich bond in Z-O ,..., ®. The nature of Z-OH
will be discussed shortly.
As a further illustration, we may imagine that in the reaction
E ~ F of the hypothetical sequence an acyl mercaptide is involved:
E-S-R' -+ F '" S-R'.
Then, by reaction with phosphate, as we have seen earlier,
F '"
S-R'+H3P04~F-O"""
®+HSR',
and again the storage mechanism comes into play:
F-O '" ®+HO-Z~ F-OH+Z-O,.." ®.
]7
ENERGY
In each case, therefore, part of the free energy from the reaction
sequence A ... ~ G has appeared in the substance Z-O ,.., ®,
a phosphorylated derivative of Z-OH.
In the complete range of vital activities, no single compound
fulfils the role of Z-OH, but Nature is close-fisted with chemical
mechanisms, and in a very large number of reactions Z-OH represents adenosine diphosphate, and Z-O ,.., ® adenosine triphosphate,
b~ of which are pyrophosphates of the kind discussed earlier.
/ ~denosine is a compound formed from adenine (a purine base)
and D-ribofuranose (a 5-carbon sugar); such compounds are called
nucleosides. When the -OH group of the sugar at position 5 is
phosphorylated, we get adenosine monophosphate (formula on
p. 138), sometimes known as adenylic acid, which may be written
AMP for convenience. The phosphate residue in AMP is linked to
the rest of the molecule by an energy-poor ester bond. ~hen another
phosphate residue is addeduo AMP by a pyrophosphate linkage, we
get adenosine diphosphate, ADP, which therefore contains one
energy-poor and one energy-rich bond. A further addition of phosphate, again by the pyrophosphate linkage, results in adenosine
triphosphate, ATP, with two energy-rich bonds. The phosphorylated
derivatives of nucleosides are called nucleotides; and the relation
between all these adenosine derivatives can be summarized as follows:
Adenosine
Adenosine monophosphate
Adenosine diphosphate
Adenosine triphosphate
Abbreviation
A
AMP
ADP
Conventional
symbol
A
A-®
A-®~®
A-®~®~®
ATP
Type of
Compound
Nucleoside
Nucleotide
Nucleotide
Nucleotide
Although many other nucleotides occur in nature, composed of
phosphate, bases other than adenine, and sugars other than nribofuranose, the adenine nucleotides just described seem to be far and
away the most important. Indeed, if anyone substance can be called
the Secret of Life, that substance is ATP. It was discovered in 1929 by
K. Lohmann, and synthesized by Sir A. R. Todd twenty years later.
We have observed that when a pyrophosphate linkage is hydrolysed there is a loss of free energy; in the case of ATP,
A-® ,.., ®
('oJ
®+H20~A-®"" ®+H3~04'
-l'!..F0
= about 8000 ca1./mole.
18
FORMA TION OF ATP
Otherwise expressed, the system is very far from equilibrium, and
the reaction tends to go from left to right. Remembering that
-!1FO = 2·3RTlog lO K, at 37° C. we have
and
-8000 = 2·3xl·99x31010glO K
K = 1 X 106 approximately.
This value of K can be arrived at in another· way. In order t9 bring
the system to equilibrium the free energy must be brought to zero.
We recall that each tenfold decrease of reactant concentration or
increase of product concentration adds about 1400 caL/mole to !1F;
since !1Fin this case is negative, we must add about 6 x 1400 cal./mole
to reach zero, and since the relation is logarithmic, K = 1 X 106
approximately. The very high value of K means that ATP is very
ready to part with its terminal phosphate residue, given the right
conditions. It must not be supposed, however, that ATP is an
unstable compound. In the solid state, as the sodium salt, or even
in pure aqueous solution it can be kept for long periods without
much change; but in the presence of such catalysts as acids or alkalis
the energy-rich terminal phosphate residue can react very easily with
the -OH group of water, and in the presence of enzymes with the
-OH groups or -NH2 groups of other sJlbstances. These reactions
fall broadly into two divisions.
(a) Transfer reactions with a relatively small change in free
energy:
A-® ...., ®...., ®+H2N-R
(or HO-R)
~A-®
...., ®+R-NH...., ®
(or R-O ...., ®)
Here, under the influence of appropriate enzymes, another compound is formed with an energy-rich bond. Such reactions are easily
reversible.
(b) Reactions with a fairly large negative !1F. Here the resulting
compound, often a phosphate ester or glycoside, has an energy-poor
bond:
A-®...., ®...., ®+HO-R-+A-®...., ®+R-O-®.
The reaction proceeds readily from left to right, and, although
theoretically reversible, may be regarded for most practical purposes
as irreversible. In such reactions, some of the free energy of ATP is
19
ENERGY
wasted. in the sense that the full amount of useful chemical work
that could be performed is not being performed.!
Thus ATP is a versatile compound, and its central position in the
scheme of free energy changes can be summed up, in very general
terms, as follows.
(1) When a source of free energy, such as a foodstuff, is being
broken down in the cell, small packets of the energy become diverted
into energy-rich bonds in some of the compounds formed during its
breakdown. These compounds-such as phosphorylated derivates
or acyl mercaptides-can react, directly or indirectly, with ADP to
form ATP. There will always be a certain number of ADP molecules
available in the cell, ready to accept '" ® under appropriate conditions. Thus when a cell is actively metabolizing a source of free
energy, ATP is being continually formed from ADP; in short, free
energy is being temporarily stored as ATP. The period of storage
may be extremely short-a fraction of a second-but storage it is.
(2) If this temporary, transient store of ATP is too large for the
immediate requirements of the cell, the free energy can be transferred
to more permanent storage-lasting for periods of minutes or even
hours, perhaps, rather than fractions of a second-as in (a) above.
One storage reaction of this type (p. 132) is of great significance in
the biochemistry of muscular work.
(3) When the cell needs free energy for any purpose, it draws on
its store of ATP, as in (a) or (b) above, and the ADP that is formed
becomes ready to accept '" ® again. Hence the free energy available
from ATP is to be regarded as a driving force behind chemical
reactions in every kind of living cell. 2
In this way, through the mediation of ATP, the free energy from
the breakdown of foodstuffs propels the chemical machinery of life.
These relationships can be put in diagrammatic form (IV).
All these processes, however, would operate to little purpose unless
they were controlled and directed to useful ends. In a complex
organism like the human body much of the over-riding direction of
1 The free energy of ATP can also be converted into mechanical work, as in
muscular contraction (p. 132), and into electrical work, and even into light;
but the reactions are complicated and not yet fully understood.
2 The - ® of ADP can act in a similar manner under certain circumstances,
AMP being formed.
20
FORMA TION OF ATP
metabolism seems to rest with hormones, whose precise mode of
action is still far from clear. But at the cellular level we may regard
enzymes as being the prime directors and controllers of what goes
on, and the how and why of enzyme action must be our next concern.
ATP
Free energy {
from food
breakdown
~ Free energy for
)
ADP
inorganic
phosphate
+
(IV)
21
- chemical work
III
ENZYMES
Every cell contains hundreds of enzymes, and its chemical turnover
depends very largely on their working together in the right way. We
have seen in the previous chapter that chemical energy is derived not
so much from single reactions as from chains or sequences of
reactions, some of which may yield energy-rich compounds and some
of which may not; and in thinking quite generally of the catalytic
function of enzymes within the cell this notion of reaction sequences
must be constantly kept in mind. To take an example, the complete
oxidation of a glucose molecule to carbon dioxide and water may need
the co-operation of over a score of distinct enzymes. Although an
individual enzyme must be studied in isolation if its properties are
to be fully known, in the last resort the biochemist wants to discover
how the whole chain of catalysed reactions is regulated and controlled, that is, how enzymes co-operate in bringing about chemical
changes, and in enabling a supply of free energy to be used to good
purpose. These considerations will become clearer at a later stage,
when the metabolism of particular compounds has been studied in
detail. Meanwhile, something must be said about the chemistry of
enzymes as a class.
All the enzymes that have been obtained in a pure, or nearly pure,
state belong to the group of complex nitrogeneous substances called
proteins. So far as we know, every enzyme is a protein, though this
statement must not be taken to imply that every protein can act as
an enzyme. The molecular weight of an enzyme never seems to be
less than about 10,000, and most enzymes have molecular weights
ranging from 50,000 to 200,000, or even more. The precise structure
of such large molecules must be left to specialists in protein chemistry,
but since the numerous kinds of protein are all variations on a
standard theme, it is not difficult to form a rough idea of the constitution of a typical protein, and how its composition can ~e related
to catalytic activity.
22
ENZYMES
Protein structure
The structural units out of which proteins are built, known as amino
acids. have the general formula (1)1
eOOH
I
H2N -C-H
I
R
(I)
In acid solution these compounds exist as cations,
+H3N . CH(R). COOH,
and in alkaline solution as anions,
H2N. CH(R). COO-;
in neutral solution they are present as dipolar ions, or zwitterions,
+H3N . CH(R).COO-. Rather more than twenty amino acids have
been found in living organisms, but only nineteen of them commonly
enter into the structure of proteins. The simplest amino acid, glycine,
H2N. CH2 . COOH (where R = H) is optically inactive; the others can
exist in D- and L- forms, but nearly always, except in some bacterial proteins, the amino acids of natural occurrence belong configurationally
to the L- se~ies. Although we shall see later that the optical configuration of amino acids, as of other compounds, is of great importance
in cellular metabolism, for the moment it can be disregarded.
When the elements of water are removed from two amino acids
we get a peptide linkage between them, -CO-NH- (II). Such
Rl
/
I
.
CH-CO.OH
/
COOH
HHN-CH
.
L
H2N
Rl
JH
H,N/
"r
NH
"co/
/COOH
R2
(II)
1
The formula does not apply to proline and hydroxyproline (see below).
23
ENZYMES
a compound is a dipeptide; by adding another amino acid we should
get a tripeptide; and by continuing the process we arrive at the class
of compound called polypeptides (III).
Rl
etc. NH
/
~
R3
~
CO
/
~
~
CH
lz
/
ro
~
NH
/
~
~
CO
(Ill)
/
~
~
/
ro~
CH
i4
It is not necessary, in a chain of this kind, that Ri, R2, R3, R4, etc.,
should all be different amino acids; in a long polypeptide chain, with
dozens or even hundreds of amino acids strung together, any particular amino acid may occur several times, or be entirely absent.
The important thing to notice is that the R-groups alternate on either
side of the chain. X-ray studies have revealed the information about
bond angles and the distance, on the average, between the R-groups
shown in (IV). Hydrogens are omitted for convenience. Thus the
R-groups are on alternate sides of the chain, about 3·5 Angstrom
units apart. The picture is complicated by the fact that many polypeptide chains are folded or coiled, so that the R-groups stick out
in all directions from the chain axis; but for the present purposes
these complications can be ignored.
o
+------.3 5 A---~
(IV)
When polypeptide chains are combined together we get proteins.
Taking a purely hypothetical case (V), let A, B, and C be polypeptide
chains of varying lengths, each composed of an assortment of amino
acids, and here represented in a straight uncoiled configuration.
24
PROTEIN STR UCTURE
Between the chains there are inter-chain linkages, represented by
dotted lines in the diagram, that hold the structure together. These
inter-chain linkages between the R-groups will depend on the nature
of those groups, and the possibilities must now be explored.
R
R
R
A
R
R
R
R
R
R
B
R
R
R
R
R
R
C
R
R
(V)
The R-groups of the amino acids of most usual occurrence fall
'Into several categories.
I. A miscellaneous group:
H- glycine
CH 3- alanine
(CH 3)z.CH.CH 2-leucine
CH3 CH 2.CH(CH3)isoleucine
O-CH,-
phenylalanme
(XTCH,-
t"ptophan
H
N=CH
(CH3)z.CH- valine
\ tH
CH=C-CH 2 -
histldme
The amino acid proline is
CH2--CH2
I
CH2
'"
NH/
25
I
CH-COOH
H9
,
ENZYMES
II. Hydroxy amino acids:
The ammo acid hydroxyproline IS
OH
CH3 .CHOH- threonine
Ho-O-
I
CH--CH2
CH,-
"""in,
I
C~
I
/CH-COOH
NH
III. Basic amino acids:
NH2·(CH2)4-iysine
NH2 C(: NH). NH. (CH2h- arginine
IV. Acidic amino acids:
COOH.CH 2- aspartic
COOH.(CH2h- glutamic
These acids are occasionally found as their amides, asparagine and
glutamine, especially in plants.
V. Sulphur-containing amino aCIds:
HS. CH2- cysteine
CH3. S. (CH2)z- methionine
The first category, a somewhat miscellaneous set, where the R-groups
are relatively unreactive, probably plays a rather less important role
in the final properties of the protein than the others do, although
even the feebly basic nitro gens of a histidine residue, for example,
must not be overlooked. As to the second category, the -OH groups
of the hydroxy acids are bound to exercise an influence on the
solubility, and, as we shall see, are of structural significance also.
To the third and fourth categories belong the basic amino acids with
an extra basic group and the acidic amino acids with an extra
carboxyl group. Both can enter into the composition of a protein
in several ways, and its final properties are dictated to a considerable
extent by them. Another amino acid of great structural importance
is cysteine, one of the two sulphur-containing amino acids.
We are now able to consider the kinds of inter-chain linkage that
might exist.
1. Linkages between the basic amino acid residues of one chain,
and the acidic amino acid residues ()f another.
26
PROTEIN STRUCTURE
(a) Wandering of a proton, giving an electrostatic or 'salt'linkage
between chains:
I-NH2
HOOC---j
t
I-NH3+
-OOC--j
(b) Elimination of water, with the formation of a peptide link:
I-NH-co-j
Linkages of type (b), however, are probably rare.
2. The -S-S-linkage between chains is of very common occurrence. If two molecules oJ cysteine are oxidized, by very gentle
means, cystine is formed;
-2H
COOH. CHNH2. CH2. SH + HS. CH2. CHNH2. COOH----+
COOH. CHNH z . CH1. s. s. CH 2. CHNH2. COOH.
Cysteine residues in polypeptide chains behave in the same way:
I-SH
•
HS-j
I-s-s-j
3. Linkages involving -OR groups, thus:
I-COOH
HO-j
t
I-co-O-I
It seems likely that these ester linkages are not at all usual in proteins;
but there is another way in which -OH groups, and feebly basic
-NH groups, come into the picture-through hydrogen bonding.
Hydrogen bonding is essentially electrostatic in nature, and is due
to weak forces that are set up when the electropositive H atoms of
-OR and -NR groups happen to find themselves near an electronegative atom such as the 0 of a -CO group, thus:
I
I-OH ...... OC
or
I
I
I
I
I
NH ...... oc
These forces arise because the hydrogen atom is disposed to share
the electrons of oxygen
.. I
..
.. I
-O:H+ -:O:C ->- -O:H :O:C
.. I
.. I
and the same thing happens with nitrogen. Whereas the heat required
to dissociate covalent bonds is 50,000-100,000 cal./mole, or more,
27
ENZYMES
the heat of dissociation of hydrogen bonds is far smaller, about
5000 cal./mole. But the possibilities of hydrogen bonding in adjacent
polypeptide chains are very numerous; and although the individual
hydrogen bonds are weak in themselves they occur so often as to
contribute very materially to the structure of protein molecules (VI).
It has been shown by L. Pauling and by W. T. Astbury that hydrogen
bonds are of particular importance in holding together the polypeptide chains of proteins like silk, hair and wool, known as fibrous
proteins. But we are here concerned with another class of protein,
commonly called globular, to which the majority of enzymes belong.
These globular proteins are thought to be more or less spherical
or cigar-shaped, rather like a bath-sponge or a loofah.
"
~ ....... oc/
_..OC/
NH....
"'-CH-R
/
R-CH
"
....HrI
"
CO.............HN/
"co....
CH-R
/
/
R-CH
(vD
"
It is not difficult to form an idea of what a small-sized protein
looks like, provided that the picture is not cluttered up with detail.
The diagram (VII) shows a simplified version of the insulin molecule,
the structure of which was ascertained by F. Sanger in 1954; although
insulin is a hormone, and not an enzyme, it will serve to illustrate
a number of points. In the monomeric form insulin contains only
two polypeptide chains, joined together cbiefly by two -S-Slinkages, although other forces, such as hydrogen bonding, will play
a subsidiary part in holding the molecule together and determining
its shape. In the diagram each dot represents an amino acid residue,
everything except tbe terminal portions of the more reactive R-groups
being compressed into the dot, the purpose being not to show all the
details of the molecule but only the most important polar groups,
viz. COO-, -NH3+, and -C(:NH).NH3+ (from arginine), as well
as aliphatic and aromatic -OR groups, and the -"-S-S- linkages.
28
coo-
OH
I
•
NH+
I)
-OOC-o-o-o-o-e-o_o_._._.
L_
I
i
1I J
--------i
NH
--5 ______ 5
HOG-
0
I'
1
NH/
•
•
-OOC-.
•
I
1
I
OH
I
l-OoH
I
•
•
•
-ooc-·
l-OoH
1
I
I
I
.-OH
I
I
•
I
sr--1II iII
L!-s-s-j
I
·-OH
This diagram of the insulin molecule
has been modified to fit the page, and
does not show Its real shape.
HO-·
S
I
I
-ooc-·
•
I
I
•
•
I
•
I
NH)+
I
I
•
•
J
I
•
I
I
•
I
•
NH)+
(VII)
29
Lo-
ENZYMES
It is not necessary to remember this picture of insulin in detail;
what has to be remembered is simply that every protein, whether of
large or small molecular weight, contains numerous chemically
reactive groups scattered throughout the molecule. And it must be
borne in mind that there is nothing arbitrary or random in the
structure of a protein, complex though it may be, and meaningless
though it may appear. The different kinds of mammalia, for instance,
each elaborate their own slightly different versions of the insulin
molecule, but, so far as we can judge, the insulin secreted by any
one kind-the cow or sheep or pig-has always the same composition, and that appears to be true of proteins as a whole.
Before discussing the properties of enzymes in the light of what is
known about protein structure,.a few points of terminology must be
cleared up. It is unfortunate that enzymes have not always been
given names according to an exact and systematic rule. We may
distinguish three sources:
(a) From the type of reaction catalysed, with addition of the
suffix -ase. Thus
is a hydrolysis, and enzymes which catalyse this kind of reaction are
hydrolases.
(b) From the name of the compound acted on-called the substrate
-also with the suffix -ase.
NH2
c6
"
NH2
Here the substrate whose hydrolysis is catalysed by the enzyme is
urea; and hence the enzyme is called urease. It belongs to the class
of hydrolases, but has been given a specific name of its own.
(c) Enzymes christened long ago which have retained the old
names. An example is the digestive enzyme trypsin, one of the class
of proteinases which act on proteins in the gut. Luckily these
unsystematic names are not very common.
It has been mentioned earlier that every cell contains hundreds of
different enzymes, but a cell is not just a bag in which all these
30
PROTEIN STR UCTURE
enzymes move freely about. Broadly speaking the cellular enzymes
fall into two classes. First, those that are more or less firmly attached
t7> the cell w;ll, the nucleus, or small particles such as mitochondria
(p. 108). If we take, for example, a piece of liver or muscle or other
tissue, and grind it up in the presence of a little water or salt solution,
and centrifuge the mixture, the insoluble m.!lterial of the cells carries
down with it a number of enzymes. Many enzymes in this insoluble
fraction are already known to be of great importance in metabolism,
and it is often difficult to coax them into solution without destroying
their catalytic activity. To the se£QlliJ class, however, belong the
enzymes that are readily soluble in water or dilute salt solutions, and
it is these whose properties have been most fully explored. We may
now make a list of the chief characteristic of such enzymes, relating
them as far as possible to their structure as proteins.
Properties of enzymes
1. Solubility. The solubility of an enzyme in water and aqueous
solutions is determined largely by carboxyl and amino groups from
the R-groups of acidic and basic amino acids, and hydroxyl groups
from the hydroxy amino acids. The hydroxyl groups, by virtue of their
ability to form hydrogen bonds with water molecules, will play some
part in taking an enzyme into solution, but a greater importance
attaches to the polar -COO- and -NH3+ groups, and since the
charges on them will depend upon the acidity or alkalinity of the
aqueous solution, it is clear that the solubility of an enzyme will be
affected by the hydrogen-ion concentration.
Pure water at ordinary temperatures dissociates very slightly into
hydrogen and hydroxyl ions, H.OH ~ H++OH-. The product of
the concentrations is constant for a given temperature, and we may
write Kw = [H+] [OH-], where Kw is found to be 1 x 10- 14 approximately. Since the number of hydrogen and hydroxyl ions is equal,
the concentration of each is 1 x 10-7 g.mole/l. In practice we disregard the hydroxyl ions and think only of hydrogen-ion concentration as defining both the acidity and alkalinity of a solution, and it
is customary for this purpose to employ a logarithmic scale of
hydrogen-ion concentration, known as the pH scale. Fure water is
31
ENZYMES
regarded as the neutral point, and since [H+] = 1 x 10-7, we can
write
1
pH = -loglO [H+] = 10glO [H+] = 7.
Thus the pH, or hydrogen-ion exponent, is the negative logarithm
(to the base 10) of the hydrogen-ion concentration. If we add an
acid to pure water, the hydrogen ions are increased in number, and
hence the pH is decreased. Conversely, by adding alkali the hydroxyl
ions are increased and, since the product [H+] [OH-] remains constant, the hydrogen ions are decreased and the pH increases. Table I
shows the relation between [H+] and pH. Throughout the life of most
cells the pH is not allowed to vary by more than a unit or two from
the neutral point, which means that the hydrogen-ion concentration
does not change by more than tenfold or a hundredfold either way,
at most. The pH of some of the fluids in the human body is even more
closely controlled, the blood pH being or~inari1y kept within the
range 7·3-7,5. This process of keeping the pH fairly constant is
known as 'buffering'; and although many buffer systems are known,
and the theory of their action is very important, they can only be
spared a brief mention here.
Table I
[H+]
(moles/litre)
1 x 100
1 x 10- 1
to
1 X 10- 6•9
1 x 10-7 . 0
1 x 10-7 •1
to
1 x 10-14
IOg10 [H+]
0
-1
to
-6,9
-7·0
-7·1
to
-14
pH = -loglo [H+]
~to lJ acid
6'9
7·0 neutral
7·1 }
to alkaline
14
A buffer is any compound which in aqueous solution resists
changes of [H+] that would otherwise occur when an acid or a base
is added to the solution. If we take a small quantity of a strong acid,
such as hydrochloric, and add it to water, it dissociates almost completely into H+ and Cl- ions; a decinorma] solution of HCI has a
pH value of about H, i.e. very acid. Suppose, however, that HCI
is added to the solution of a salt such as sodium flcetate. Whereas
the alkali metal salts of weak acids are highly dissociated, the acids
32
PROPERTIES OF ENZYMES
themselves are only feebly dissociated; consequently, if we add a
small amount of HCI to an excess of sodium acetate
the reaction produces feebly dissociated acetic acid, and the pH
change is smaller than if the HCI had 'been added to water alone.
But now another factor comes into play. When a salt of a weak acid
is mixed in dilute solution with the acid itself, the dissociation of the
acid is decreased. In a mixture of acetic acid and sodium acetate
we have CH3 .COOH ~ CH 3.COO-+H+ from the acid and
CH 3.COONa ~ CH 3 .COO-+Na+ from the salt, the free acid
contributing very few acetate ions to the solution, the salt a great
many. With a high concentration of acetate ions present, the equilibrium of the system CH3 .COOH ~ CH3 .COO-+H+ will be
shifted, in compliance with the Law of Mass Action, from right
to left; hence the dissociation of the free acid is decreased by the salt.
So, returning to the addition ofHCI to an excess of sodium acetate,
we see that until all the sodium acetate has been converted to NaCl
the feebly dissociated acetic acid liberated in the reaction will have
its dissociation still further repressed by the acetate ions provided by
the sodium acetate. When all the sodium acetate has been converted
to NaCl, the buffering power is at an end; but until this point has
been arrived at, addition of strongly dissociated HCI produces
weakly dissociated acetic acid, which is even more weakly dissociated
than if the sodium acetate had not been present; in short, the addition
of a strong acid to a mixture of a weak acid and its salt only produces a small change of pH and the system is .said to be buffered.
Exactly similar considerations apply to the addition of strong bases
to salts of weak bases; and every buffer system has a definite range
of pH throughout which it can act-usually about 2 pH units. In
living cells, which are plentifully supplied with anions like Cl- and
cations like Na+ and K+, the proteins can exert considerable buffering
power, since the weakly acidic -COO- groups can form salts with
cations, and the weakly basic -NH3+ groups with anions. Amino
acids can by themselves act as buffers for the same reason.
We must revert, however, to the factors that influence the solubility
of an enzyme. An inspection of the insulin molecule (p. 29) will
33
ENZYMES
show that it contains a number of ionizable groups, and when the
pH of any protein solution is changed by addition of acid or alkali
the ionization of such groups will be altered and the solubility
affected. Broadly speaking; at low pH values -NH3+ groups will
tend to predominate, and the ionization of -COOH groups will be
suppressed, at high pH values -COO- groups will predominate;
if the molecules carry a net positive or negative charge, they repel one
another, a factor that helps them to go into solution. When the
positive and negative charges on the protein molecule exactly balance,
the iso-electric point is reached where the solubility is at a minimum
because the molecules have the least tendency to repel one another;
for many enzymes, and other proteins, the iso-electric point is in the
neighbourhood of pH 5. The solubility of an enzyme in aqueous
solution is also dependent on salt concentration, for rather complicated reasons. Whereas some enzymes are readily soluble in distilled water, and are thrown out of solution-' salted out'-by the
addition of salts such as KCl or (NH4)2S04, others behave ip. the
opposite fashion. A class of proteins called globulins, to which a
few enzymes belong, is distinguished by being relatively insoluble in
pure water but much more soluble in dilute salt solutions; at high
salt concentrations, however, globulins are also salted out. These
relationships are of great value in purifying enzymes, many of which
have been obtained in crystalline form.
2. Active centres. Although a typical enzyme contains a great many
chemically reactive groups, its catalytic activity is confined to a
relatively small number of spots on the surface, called the active
centres. Thus the digestive enzyme trypsin, in spite of its molecular
weight of 34,000, has been shown to possess but one active centre;
and in general it may be said that although the molecular weight
may run into hundreds of thousands, catalysis will only occur at a
few places on the enzyme surface. Much of the evidence for this
conclusion has been derived from substances that stop, or reduce,
enzyme activity (p. 41). But in spite of there being only a few active
spots on its surface, the catalytic power of an enzyme can be very
considerable. What is known as the 'turnover number' is defined as
the number of molecules of the substrate acted upon by one molecule
34
PROPERTIES OF ENZMYES
of the enzyme in 1 minute. Turnover numbers range from about 100
or less (a very lethargic enzyme) to over a million (a very active one).
Like all other catalysts, therefore, enzymes are effective in small
amounts, and, ideally, they are unchanged in the reaction, though
due allowance must be made for the fact that proteins are chemically
somewhat delicate. Enzymes also resemble other catalysts in that
they do not influence the equilibrium of an isolated reversible
reaction, but only hasten its attainment. We have already seen that
free energy considerations determine whether a reaction can go
forward or not. Reactions involving a loss of free energy, - tlF, can
proceed of themselves, but perhaps only at a rate too slow to be
measured; in the presence of an appropriate enzyme, such reactions
are speeded up, though the position of final equilibrium is unaffected.
In order that a reaction may proceed, the enzyme and its substrate
must be brought into close conjunction with one another. A kind
of compound or complex is believed to be formed at first, which then
breaks up:
Enzyme+Substrate "" Enzyme-Substrate Complex.
Enzyme-Substrate Complex"" Enzyme+Reaction Products.
In sum: Substrate "" Reaction Products.
Thus the enzyme takes hold, as it were, of a substrate molecule, and
causes it to undergo reaction, and at the end the enzyme emerges
unchanged, ready to repeat the whole process.
Unfortunately we do not know as much as we should like about
the ways in which enzyme-substrate complexes or compounds are
formed at the active centres. It is possible, however, to develop a
rough picture of the kind of thing that can happen in particular cases.
(a) From the diagram of the insulin molecule (p. 29) it will be
seen that the structure is to a great extent held together by -S-Slinkages, and if insulin is reduced, so that -SH groups are formed,
the physiological activity of the hormone is destroyed. A number
of enzymes, however, in complete contrast with insulin, are active
only if one or two -SH groups on the surface are in the reduced
state. Such groups can sometimes react with aldehydes:
H
I
R-CHO+HS-@ "" R-C-S-@
I
OH
35
ENZYMES
where HS-® represents the enzyme molecule. A reaction that is
thought to take place in this way will be found on p. 87. Here,
therefore, the substrate becomes a compound with the enzyme, very
temporarily of course, by means of the -C-S linkage; we saw
earlier (p. 16) that such linkages are involved in the formation of
acyl mercaptides. Not all -SH groups, however, behave in this
fashion, and the part they play in the activity of some enzymes is
still unknown.
(b) Polar groups are likely to enter into the active centres of many
enzymes. It is easy to see that substrates provided with polar groups
could be anchored to enzyme surfaces with oppositely-charged
groups:
Substrate
R-COO-
or
-OOC-j
When electrostatic forces are involved, the pH of the solution will
clearly be important, since the charges on both enzyme and substrate
will be affected by [H+J; and indeed it appears that for all enzymes
there is a more or less well-defined region of pH at which they are
most active-the optimum pH. Pepsin, for example, which hydrolyses proteins in the stomach, works best at about pH 2, trypsin,
working in the less acid surroundings of the duodenum, has a pH
optimum of about 8, and most other enzymes lie between these
extremes. It is important to realize that a shift of one or two pH
units from the optimum will often render an enzyme inactive.
(c) Weaker forces, such as hydrogen bonding, will also take part
in the union of an enzyme with its substrate. In certain cases, too,
metal ions are thought to be involved (p. 44). So if the subs~rate
molecule is large and complicated, which often happens in biological
systems, we must imagine a variety of linkages, some strong and
some weak, between it and the enzyme; linkages of which the foregoing examples can only give an inadequate notion.
But although we cannot yet say exactly how any particular enzyme
combines with its substrate, or define precisely the forces concerned,
it is possible to make comparisons between one enzyme-substrate
complex and another by means of what is comm,only called the
Michaelis constant, K m , which is defined as the concentration in
36
PROPERTIES OF ENZYMES
moles/litre of the substrate at which the rate of reaction is half the
maximum rate. For the derivation of this constant, first introduced
by L. Michaelis and L. M. Menten in 1913, the advanced text-books
must be consulted; what interests us now is only the light that it
throws on enzyme-substrate complexes. When the concentration of
the substrate is much greater than that of the enzyme, which is the
usual condition, we can write
k.
E+S
~
k,
ES -+ E+P,
k,
where kl and k2 are rate constants for the reversible formation of the
complex ES from enzyme and substrate, and k3 is the rate constant
for the decomposition of the complex into enzyme and products of
reaction, here assum~d to be for all practical purposes irreversible.
The Michaelis constant, which is characteristic for any given enzyme,
is in the form of a ratio:
k2 + k3
Km = ----rt.
For some enzymes k3 is small compared with k2 and we can put
Km
k2
= k 1'
the value of the constant being occasionally as low as 1 x 10-1. This
very low figure means that the rate of formation of the complex ES
is far faster than its reverse breakdown into E and S, and implies
that the enzyme binds the substrate very firmly indeed. Conversely,
a high figure for K m , in the neighbourhood of 1, would imply a low
affinity between enzyme and substrate. The fact that Michaelis constants for different enzymes can vary by as much as ten thousandfold
will seem less surprising, however, if we remember that hitherto we
have considered only the nature of the forces that can help to unite
an enzyme with its substrate, but not their distribution in space. In
other words, the arrangement of atoms in both enzyme and substrate
molecules will be of importance in determming whether a complex
can be formed: and so we are led to another, and very significant,
feature of enzyme action.
3. Specificity. Whereas inorganic catalysts-such as the platinum
metals-are often useful in speeding up a variety of chemical changes,
37
ENZYMES
an enzyme can usually catalyse only one type of reaction, and sometimes can act only on one particular substance. An example of the
latter kind of enzyme is urease (p. 30). If any of the hydrogens in
urea is substituted by, for instance, a -CH3 group, or any other
group, the enzyme will not work. Thus urease is said to be absolutely
specific towards its substrate, and there must be a very subtle alignment of molecule and active centre, so that the smallest change in
the size and shape of the former is enough to stop the enzyme acting.
A number of enzymes work so slowly on any but the naturally
occurring substrate that they may be regarded as absolutely specific
for practical purposes.
Going a little further down the scale of specificity, we may next
consider a class of enzymes well represented by the p-glycosidases of
plants, which hydrolyse p-glycosides to glucose and an alcohol.
Disregarding most of the glucose molecule, we can write
H
O-R
y.
+H20
~
H
Y
OH
+R-OH
/"'-
/"'-
p-glycoside
glucose
But if these plant enzymes are given the isomeric a-glycosides to
work on
R-O
H
"'-/
c
/"'-
O\-glycoside
the reaction does not occur at an appreciable rate. Provided, however, that a glycoside has the p-configuration, the nature of the
R-group is not very material; thus, in the presence of a large excess
of water, p-phenyl-o-glycoside (R = C6HS-) is hydrolysed ten times
faster than p-methyl-o-glycoside (R = CH 3- ) , but both are readily
attacked. This behaviour of p-glycosidases is typical of a large number
of enzymes, perhaps the majority; they are highly specific towards
one part of the substrate molecule, sometimes the bulk of it, but will
tolerate minor changes of structure in the other part. When such
changes are made, as in the substitution of a methyl for a phenyl
group in p-glycosides, the reaction will still go forward, although its
rate is altered; a fact which should help to emphasize the complex
nature of the forces that bind the substrate to the activ'e centre.
38
PROPERTIES OF ENZYMES
Another, and not very common, type of specificity is shown by
esterases, which belong to the class of hydro lases, catalysing
R.COOH+HO.Rl ? R.CO.O.Rl+H20.
Here the nature of Rand Rl affects the rate of reaction to some
extent, but the determining factor-whether the enzyme will act or
not-is the presence of groups necessary to form or hydrolyse an
ester linkage; an esterase can make or break esters, but not ethers,
R-O-Rl. Such enzymes exhibit the lowest degree of specificity.
It will have been observed that the specificity of fi-glycosidases is
determined by the position of the groups round a carbon (carbon 1)
of the glucose residue. Whenever a substrate contains an asymmetric
centre, represented by anomers or by enantiomorphism of the whole
molecule, some degree of specificity will be exhibited by the enzymes
attacking it. This fact is not surprising, for the amino acids out of
which proteins are built, except the optically inactive glycine, nearly
always belong to the L-series; hence the R-groups in the neighbourhood of the active centre will be distributed in space in a definite
way. It follows that the 'goodness of fit' of an enantiomorph on
the active centre will always depend to some extent, and perhaps
decisively, on its configuration. Optical specificity is occasionally
absolute, the reactions of one enantiomorph being vigorously
catalysed, and of the other not at all; more often it is displayed in
the different rates of reaction of the enantiomorphs.
These and other aspects of specificity will become clearer as particular examples are studied; meanwhile, one other point deserves a
brief mention. An enzyme called the Schardinger enzyme, after the
man who discovered it in 1902, exhibits double specificity: it not
only oxidizes xanthine (a purine) to uric acid, but also aldehydes to
the corresponding carboxylic acids, R. CHO -+ R. COOH. No explanation can be offen~d for this curious behaviour.
4. Activation energy. We saw in the previous chapter (p. 9) that
before hydrogen peroxide can decompose its molecules must be
'activated'. The function of any catalyst is to lower the activation
energy, and so enable a reaction to proceed at a faster rate than it
would otherwise have done. How a catalyst performs this function
is not easily explained; in the last resort explanation must be in terms
39
ENZYME~
of wave mechanics and other matters that the ordinary biochemist
is happy to leave to other people. But by comparing the activation
energies, E a , it is possible to compare the efficiency of enzymes with
inorganic catalysts. An enzyme called catalase accelerates the decomposition of H 20 2 , and its efficlency can be judged by the
following figures:
Ea (cal./mole approx.)
No catalyst
Platinum catalyst
Catalase
18,000
12,000
2,000
The relation between the velocity constant of a reaction and the
activation energy is given by an equation due to S. Arrhenius, which
can be written
B represents a constant which we can assume to be of the same order
of magnitude for both catalase and platinum; and taking a temperature of 37° C. (= 310° K.), and calling ko the velocity constant
in the presence of catalase, and k1) that for platinum, we have
ko
10glO
and
k
1)
kO
k
12,000 - 2000
= 2.3 x 1·99 x 310 = 7·1,
= 1 X 107 roughly.
1)
Thus at 37° C. catalase decomposes H 20 2 about ten million times
faster than platinum does. The action of catalase on a dilute solution
of H 20 2 at this temperature is dramatic: a trace of enzyme makes it
fizz like champagne. Although this is an extreme case, enzymes are
in general incomparably more effective than any other kind of
catalyst within the range 0°-50° C., outside which limits of temperature living matter-except viruses and micro-organisms-cannot
survive for any length of time.
5. Denaturation and inhibition. One of the reasons why cells cannot
endure high temperatures is that proteins are very sensitive to heat.
When a solution of a protein in water is heated, the thermal agitation
of the polypeptide chains tends to make them fly apart, and at the
same time some of the inter-chain linkages are hydrolysed. Thus the
structure becomes thoroughly disorganized, and the protein is said
40
PROPERTIES OF ENZYMES
to be denatured. The commonest sign of denaturation is that the
protein becomes insoluble as the polypeptide chains tangle together
in disorder, and are no longer kept in position by the inter-chain
linkages. As a rule this process IS irreversible-an egg once boIled
stays boiled, and, with very few exceptions, enzymes that have been
heated to boilIng for a minute or two lose all their activIty. But
by gentle lIeating, under carefully controlled conditIOns, a reversible
denaturation can sometimes be demonstrated. For such reversible
reactions it is found that the free energy change is very small, but the
change in entropy (T . tl.S) is very large-a good indication that the
molecular structure has become disorganized.
Enzyme-catalysed reactions are no exception to the general rule
that for every 10° C. rise in temperature the rate of reaction is
roughly doubled; a rule conventionally expressed by saying that
QIO = 2 approximately. But with increasing temperature the rate
of denaturation also increases. Hence at 80° C. an enzyme may be
extremely active, but only for a very short space of time-pOSSIbly
a few seconds, whereas at 20° C. its activity will have dropped to
about one-sixtieth of the former value, yet that degree of activity
may be retained for several hours, or even days. Thus, for any given
period of time, there will be an optimum temperature at which the
enzyme can work at full capacity before denaturation sets in.
Enzymes in solution are not only inactivated by heat, but sometimes
also by mechanical means such as repeated freezing and thawing, or
violent stirring in solution. Many chemical substances can likewise
put enzymes out of action; we must confine ourselves to those that
throw light on the constitution of active centres. Two classes of
inhibitor are of particular interest.
(a) Metals. The poisonous nature of metals such as arsenic,.
mercury, and silver can be partly explained in terms of enzyme
inhibition. There is good reason to suppose that the activity of a
number of enzymes depends on a pair of -SH groups at the active
centre or in its immediate neighbourhood. When a very small
quantity of, for instance, a mercury salt is added to such an enzyme,
we may picture this kind of reaction:
t:: + HgCl
2
-+
41
t:>Hg + 2HCl
BNZYME'S
A similar reaction takes place with salts and organic derivatives of
arsenic, and it has been shown by Sir R. A. Peters, L. H. Stocken,
and R. H. S. Thompson that the inactivated enzyme can be revived
by the addition of dimercaptopropyl alcohol:
s
As-R
t S)
+
HS-CH2
I
tSH
-+
HS-CH
SH
~H20H
+
R-As
<S-CH2
I
S-CH
~H20H
Poisoners who use arsenic are nowadays at a double disadvantage:
they are almost sure to be found out, and their victims can often be
restored to health.
Metals can also combine with enzymes in other ways, and by
determining the least number of ions that can cause complete inhibition, an estimate of the number of active centres can be arrived at,
provided the molecular weight of the enzyme is known. Urease is
completely and irreversibly inactivated by four silver ions per molecule, from which we may conclude that not more than four active
centres exist. Clearly the degree of inhibition in such cases depends
entirely on the amount of inhibitor, and not on the concentration of
the substrate, so the type of inhibition produced by metals is said to be
non-competitive, in order to distinguish it from that brought about by
(b) Competitive inhibitors. The rate at which enzyme catalysis can
occur is determined, at fixed temperature and pH, by the concentration of both enzyme and substrate in the reaction medium. For a
given substrate concentration, the more enzyme is present the faster
the reaction goes, and for a given amount of enzyme, increases in
the substrate concentration increase the rate of reaction until a point
is reached when all the active centres of all the enzyme molecules
are working as fast as they can. Competitive inhibition arises in the
following way. Suppose we have a chemical compound X whose
structure is very similar to that of the right and proper substrate S,
and which can combine reversibly with an enzyme E. Then if Sand
X are both present the enzyme will be deceived, as it were, by the
similari ty of structure; at some of the active centres the right enzymesubstrate complex will be formed, ES, and at others the wrong one,
EX. Thus X competes with S for the available active centres; the
rate of attack on S is slowed down; and the enzyme is partially
42
PROPER TIES OF ENZYMES
inhibited. But if the concentration of S is now raised, S will displace
X from the enzyme, since both compounds can combine with it
reversibly in more or less the same way, so an increase in the concentration of S will lower the inhibition produced by a fixed concentration of X. Thus the extent of competitive inhibition depends on
the concentration of the substrate S as well as on the concentration
of the inhibitor X.
One of the simplest examples of competitive inhibition has been
furnished by studies on the bacterium Staphylococcus aureus, some
strains of which will not grow and flourish unless they are supplied
with several amino acids. If to a culture of S. aureus the structurally
similar sulphonic acids are added, the organism stops growing, or at
least grows more slowly, the sulphonic acids being of no value to it.
We may infer that these acids anchor themselves to enzyme surfaces,
in the way already indicated (p. 36), by means of one polar group
or both polar groups (VIII); and however more elaborate the whole
R
I
CH
/
"'-NH3+
S03(VIII)
story may be, it is evident that the sulphonic acids, by their similarity
of structure to amino acids, can neatly block the active centres concerned. These observations on S. aureus, and many others of the
same kind, help to confirm a theory advanced in 1940, by D. D.
Woods and Sir Paul Fildes, that the sulphonamide drugs exert their
bacteriostatic effect by the competitive inhibition of enzymes. Many
bacteria need p-amino benzoic acid for growth, and can only flourish
in the animal body because traces of it occur in the blood and tissues;
and the sulphonamide drugs act by liberating sulphanilamide, or
closely related compounds, which can form an enzyme-substrate
complex with the enzyme concerned in p-amino benzoic acid metabolism (IX). It is obvious that drugs which behave in this way must
be present in high concentration if they are to be effective, since the
inhibitor ought to occupy as many active centres as possible, and is
in competition with the natural substrate, for which the enzyme may
well have a rather higher affinity (lower Km).
43
ENZYMh~
6. Co-enzymes. Although metals such as arsenic and mercury can
inhibit enzyme action, there are other metals without which certain
enzymes cannot work. For example, the amide of L-leucine is
hydrolysed to the amino acid and ammonia by an enzyme from
mammalian tissues, but only in the presence of Mn++ or Mg++ ions;
if these ions are removed by dialysis, the activity of the enzyme is
COOH
NHz
sulphanilamide
NH2
p-amino benzoic acid
(IX)
lost. E. L. Smith has proposed that the metal acts as a link between
the enzyme and its substrate (X). The enzyme surface in (X) must
be imagined as lying below the plane of the paper; the dotted lines
represent forces whose nature need not be precisely defined. This
enzyme exhibits stereochemical specificity, since D-Ieucinamide is not
acted on; so the substrate is presumably attached to the enzyme not
only through the metal but also by the R-group; and from reasoning
of this kind we infer that many substrates are bound to active centres
at several points and by several kinds of linkage .
. R--CH--NH 2
I
CO--NH2"
'
Mn++ '
(X)
By no means all enzymes, however, need the co-operation of metal
ions, or anything of a non-protein nature in their work. But when
a metal is necessary it is called a co-enzyme; and that name has also
been applied to other substances whose function will be described
in the next chapter. We shall find, indeed, that the chemical behaviour
of co-enzymes throws light on many reactions of fundamental
importance to the cell.
44
PROPER TIES OF ENZYMES
Meanwhile it is desirable to emphasize two aspects of enzyme
action. The first arises from the great efficiency of enzymes as
catalysts: they are able to bring about reactions at low temperatures
which would not ordinarily occur. Were it not for this property, the
complicated series of chemical changes which we call Life could not
go on. The second aspect is their specificity: they transform definite
compounds, or types of compounds, in 100' % yield to other definite
compounds. Hence an enzyme exerts a directive force on its substrate, turning substance X into substance Y but not into substance Z;
and the sum of these directive forces, exerted by all the enzymes,
enables a cell to make use of free energy in maintaining itself. If this
statement sounds a little obscure at the moment, it will be clearer at
the end of the following chapter, when we have discussed in detail
the means whereby _a chain of enzyme-catalysed reactions can
generate energy-rich bonds.
45
IV
OXIDATION
Apart from some of the simplest organisms, most forms of life get
a supply offree energy by oxidizing their food with molecular oxygen.
Plants are no exception to this rule; they differ from animals in that
they first make food and then oxidize it-the animals feeding on
what is left over. It would appear, then, that the most fundamental
process of all must be reduction and not oxidation; something must
first be reduced before oxidation can take place. We shall consider
in due course how the energy of sunlight is used by plants, through
a reductive process, to manufacture sugars and other foodstuffs; but
the understanding of photosynthesis is greatly helped by a knowledge of how oxidation goes on in living matter, because the first step
in the oxidation of a metabolite usually involves the simultaneous
reduction of something else.
Take, for instance, the oxidation of lactic acid to pyruvic acid.
Lactic acid appears in the blood-stream during vigorous exercise,
but disappears when the animal is at rest; and its removal to a great
extent depends on' an oxidative process that goes on in the tissues.
There are two ways in which this oxidation might occur:
o
(i)
CH 3.CHOH.COOH - - + CH3.CO COOH +H20;
lactic acid
pyruvic acid
(ii)
CH3.CHOH.COOH - - +
-2H
CH3 CO COOH.
The subsequent fate of the pyruvic acid will not concern us until
later (p. 91); we deal now only with the mechanism of lactic acid
oxidation.
The first reaction is simple and obvious: given a source of' active'
oxygen, such as permanganate or persulphate, lactic acid can readily
be oxidized to pyruvic in the test-tube, and it might be supposed that
if an enzyme could cause its substrate to react with molecular oxygen
the same process would take place in the cell. Enzymes that perform
this function are in fact known; they are called oxidases, and one of
46
OXIDATION
them, we shall see, is extremely important in another connexion. But
nobody has yet discovered an oxidase specific to lactic acid, and so
we must tum to (ii), a process of dehydrogenation. An enzyme has
been isolated from animal tissues, lactic dehydrogenase, whose function is to detach the hydrogens and transfer them to its own
co-enzyme. So there is a reversible reaction
lactic acid + co-enzyme "" pyruvic acid + reduced co-enzyme,
which is only one example out of many that are known, and for the
majority of biological oxidations the first step can be written
metabolite+co-enzyme "" oxidized metabolite+reduced co-enzyme.
It is important to notice that, with few exceptions, every ordinary
metabolite is oxidized by an enzyme (protein plus co-enzyme) specific
to it; in other words, each oxidizing enzyme present in the cell
oxidizes a definite substrate to a definite product-a good illustration
of the directive influence that has been alluded to. Lactic dehydrogenase produces pyruvic acid, and nothing else; it even exhibits
optical specificity, the D-enantiomorph being oxidized far more
slowly than the L-enantiomorph.
Biological oxidations as a rule, then, involve the transfer of
hydrogen from a hydrogen donor to a hydrogen acceptor. It does
not always happen that each atom of hydrogen (proton plus electron)
is transferred as a whole; sometimes the process is one of electron
transfer only, but the principle remains the same. Also, the oxidation
of any particular substance is seldom accomplished in a single stage,
but more often by a chain of reactions at the end of which is the final
hydrogen acceptor-oxygen. This reaction chain brings to our notice
a number of complex substances-pyridine nucleotides, flavoproteins,
and cytochromes-whose properties must be considered one by one.
Pyridine nucleotides
We can now look a little more closely at the phrase 'reduced
co-enzyme' of an earlier paragraph. Lactic dehydrogenase is made
up of two distinct components, a protein and a co-enzyme. When
the co-enzyme is removed from the protein by dialysis, the dehydrogenase activity vanishes. Many other dehydrogenases behave in the
47
OXIDATION
same fashion, the protein alone being unable to act as a catalyst, and
the activity of such enzymes is not restored by metal ions (p. 44).
Instead, the co-enzyme turns out to be far more complicated. We
will first consider the co-enzyme of the lactic dehydrogenase that
occurs in animals, the structure of which is given in the Appendix
(p. 142). Its biochemical name is diphosphopyridine nucleotide,
abbreviated to DPN; and it contains the following residues:
adenine-o-ribose-two phosphates-D-ribose-nicotinamide.
Hence it consists of adenosine (p. 18) to which are attached two
phosphates, another D-ribose residue, and nicotinamide, the latter
substance (I) being the amide of pyridine-3-carboxylic acid (nicotinic
acid). The existence of DPN was first demonstrated by Sir A. Harden
~'NB'
(I)
and W. J. Young as long ago as 1904, but its importance did not
become fully apparent until the work of O. Meyerhof and of H. von
Euler and K. Myrback in the nineteen-twenties, who showed that it
can act as a 'universal aunt' to a family of dehydrogenases, of which
lactic dehydrogenase is one. DPN has therefore received the name
co-enzyme I, to distinguish it from another complicated co-enzyme
discovered by o. Warburg and W. Christian in 1932. This latter
substance, whose structure will be found in the Appendix (p. 143),
is called triphosphopyridine nucleotide, TPN, otherwise co-enzyme II;
it consists of
adenine-o-ribose-two phosphates-D-ribose-nicotinamide,
I
phosphate
and can be regarded as adenosine plus three other phosphates,
D-ribose, and nicotinamide. TPN behaves as a 'universal aunt' to
another family of dehydrogenases; in general, the DPN-linked
enzymes will not act with TPN, and vice versa, although there are
some exceptions to this rule. Lactic dehydrogenas~ prefers DPN as
its co-enzyme, but will work slowly with TPN; F. B. Straub showed
48
PYRIDINE NUCLEOTIDES
in 1940 that the oxidation of lactic acid is over 100 times faster with
DPN than with TPN.l
Both DPN and TPN enter into dehydrogenation reactions in the
same way, and we can luckily ignore most of the molecule except the
nicotinamide residue, which behaves as a weak quaternary base and
is readily oxidized and reduced (II). When lactic dehydrogenase
attacks its substrate, the protein part of the enzyme is chemically
/C~
~H
iCO.NH2
CH +....:::::CH
,~
/CI:!z
fiH
II-CO.NH2
+2H
~
CH
~/
-lli
1
I.
I
D-ribose
)=1-I
0
CH
D-nbose
-
I-OH
0
I
I
(II)
etc.
etc.
unaffected, but the co-enzyme (DPN) is reduced, one of the hydrogens
entering the pyridine ring in position 4 (para to the nitrogen). The
other hydrogen can be regarded as splitting into a proton and an
electron; the latter pairs off with the charged nitrogen in the ring,
and the former becomes attached to the -0- of one of the phosphoric acid residues. It is, however, far simpler to write
DPN +H2 "'" DPNH2 ,
though in some books the oxidized and reduced forms of the
co-enzyme are symbolized DPN+ and DPNH respectively. We can
now build up a diagram of what happens in the oxidation of lactic
acid by the dehydrogenase (III). A molecule oflactic acid approaches
the enzyme, and the enzyme-substrate complex is formed at the active
CH1' CHOH. COOH --------OfN
!
,i
enzyme protein
DPNtIg
1
i'
(III)
enzyme protem
1 Besides co-enzymes I and II, a similar co-enzyme
importance has not yet been defined_
49
III
is also known, but its
OXIDATION
centre; in some way, which we need not examine here, the enzyme
activates the hydrogens of the substrate, and they pass to the
co-enzyme; the pyruvic acid and reduced co-enzyme are liberated
from the surface and the reaction is over.
Thus lactic acid has been oxidized to pyruvic and DPN reduced
to DPNH 2 • But the reduced co-enzyme cannot react directly with
oxygen, and since it is observed that the oxygen consumption of
tissues is increased by adding lactic acid to them, something else
must come into the picture. In fact, several more reactions are known
to occur before the hydrogen of the lactic acid finally unites with
oxygen to form water-reactions involving the fiavoproteins and
cytochromes.
Flavoproteins
These substances, as their name implies, are yellow in colour, differing
in that respect from most other proteins; they are sometimes known
as 'yellow enzymes'. They are readily soluble in water and seem to
occur in every kind of living cell. Much of the pioneer work on
fiavoproteins was carried out by O. Warburg and H. Theorell, and
our detailed knowledge of their chemical behaviour largely dates
from 1933 to 1935, when the nature of the colouring matter was
investigated by R. Kuhn and by P. Karrer. Flavoproteins are
dehydrogenases, each with its specific substrate, and they fall into
two classes: (1) those which remove hydrogen from DPNH2 or
TPNH2; (2) those which remove hydrogen from other substances.
(IV)
The yellow colour is due to a heterocyclic compound, 6 :7-dimethylisoalloxazine, which can be reversibly oxidized and reduced (IV).
This compound is the active part of two complicated molecules whose
formulae will be found in the Appendix (p. 142): flavine mononucleotide, written FMN, and flavine adenine dinucleotide, FAD.
All flavoproteins so far discovered appear to contain FMN or FAD.
The former consists of
phosphate-o-ribitol-6:7-dimethyl-isoalloxazine:
50
FLA VOPROTEINS
the D-ribitol residue being firmly linked to the isoalloxazine via the
nitrogen in position 9. FAD is a little more complicated:
adenine-o-ribose-two phospha tes-o-ribitol-6 :7-dlmethy I-isoalloxazine.
The resemblance of FAD to DPN and TPN will be noticed, in so
far as it contains the elements of adenosine. But whereas DPN
and TPN have received the name 'co-enzyme', both FMN and
FAD are usually called 'prosthetic groups', because they are more
securely attached to the protein part of the enzyme, and cannot as
a rule be removed by dialysis alone. The distinction between coenzymes and prosthetic groups need not worry us, nor need we, when
thinking of the role of flavoproteins in oxidation, worry whether a
particular yellow enzyme contains FMN or FAD. Representing any
flavoprotein by FP, we can simply write
FP+2H.= FPH 2 •
Reverting now to the oxidation of lactic acid, we have seen in the
last paragraph that some flavoproteins can activate the hydrogens
of DPNH 2 and TPNH 2 , and remove them:
(i) Lactic+DPN.= pyruvic + DPNH2 ;
(ii) DPNH 2 +FP.= DPN+FPH2 •
Hence in a system containing lactic acid, lactic dehydrogenase (with
DPN as co-enzyme), and the specific flavoprotein, there is a coupled
reaction resulting in the reduction of the flavoprotein. What happens
next, in the chain of reactions linking metabolites with oxygen, we
shall see in a moment.
The curious enzyme discovered by F. Schardinger has already been
referred to (p. 39) on account of its double specificity. It is a flavoprotein of particular interest for two other reasons. In the first place,
it represents a class of flavoproteins which in the reduced state can
react directly with oxygen; when xanthine is the substrate
Xanthine + FP .= uric acid + FPH 2 ;
FPH2 + O2 .= FP + H 2 0 2 •
The enzyme has been called xanthine 'oxidase', but the term 'aerobic
dehydrogenase' is often used for such enzymes, as helping to distinguish them from 'anaerobic dehydrogenases' which in their
reduced state do not react directly with oxygen at an appreciable
rate. The anaerobic dehydrogenases include not only the DPN- and
51
OXIDATION
TPN-linked enzymes, but also the tlavoproteins that remove hydrogen
from reduced pyridine nucleotides.
The second point about the Schardinger enzyme is that it contains
iron and molybdenum in addition to the FAD prosthetic group, and
iI these meu;.ls are removed the catalytic activity is impaired.
A number of similar enzymes---<::alled metallotlavoproteins-have
been recognized; one of them, succinic dehydrogenase, is of considerable importance. The succinic enzyme belongs to the anaerobic
class, and will not react with oxygen; it was isolated in 1956, by
T. P. Singer and E. B. Kearney, and shown to be a metalloflavoprotein containing iUln. The metal (four atoms per molecule) is
rather firmly bound to the enzyme, and seems to be associated with
the prosthetic group, since removal of it lowers the activity. The
reaction catalysed is represented in (V) and its significance will be
discussed later (p. 95). This enzyme is specific to succinic acid and
will not attack DPNH2 or TPNH2 , either alone or united to enzymes.
COOH
COOH
bH2
I
CH2
+FP
~ ~
II
CH
tOOH
bOOH
succinic acid
fumaric acid
(V)
Thus there are at least three kinds of anaerobic dehydrogenase.
(a) Those, like lactic dehydrogenase, which are not tlavoproteins,
and require DPN or TPN as co-enzymes to effect dehydrogenations.
(b) Metallotlavoproteins such as succinic dehydrogenase.
(c) Flavoproteins whose substrate is DPNH2 or TPNH2 • It happens that several enzymes of this kind are also metallotlavopn;>teins,
and contain iron, as was shown by H. R. Mahler in 1954. They are
known as cytochrome-c reductases. Some of them are specific to
DPNH2 and some to TPNH 2 , and their function is to link the
reduced pyridine nucleotides with a substance called cytochrome-c.
Cytochromes
Ever since 1925, when the importance of these compounds was made
clear by D. Keilin, there has been something of a ~ap between what
we should like to know about them and what we do know. The
CYTOCHROMES
reason for this state of affairs is that cytochromes tend to be associated with the 'insoluble' material of a cell (p. 31), and as a rule
can only be separated with difficulty, if at all, from that material.
But, as their name implies, they are coloured substances, possessing
well-defined absorption spectra; and by the use of optical as well as
chemical methods a good deal of information has been obtained.
The different cytochromes are conveniently distinguished, on the
basis of differences in their absorption spectra, by the letters a, b, c,
etc. Two of them playa very important part in biological oxidations.
Cl'tocbrome-q is one of the few cytocbromes to have been isolated
in a pure condition. It is a pinkish compound of molecular weight
13,000, implying about 50-100 amino-acid residues, and so rather a
small protein. The colour is derived from a prosthetic group whose
detailed structure we can ignore, except to say that it consists of a
porphyrin united with iron (cf. p. 144). Each molecule of cytochrome-c is readily oxidized and reduced by chemical reagents; the
change can be represented
cyt.-c Fe+++
cyt.-c Fe++.
oxidized
reduced
In the oxidized state, the absorption spectrum shows a rather diffuse
band at 500-600 m,u; on reduction, two sharp bands appear at 520
and 550 m,u, by means of which the pigment has been identified in
practically all living cells except the strictly anaerobic bacteria. Its
absence from these orgamsms, which will not grow in the presence
of even a trace of oxygen, strongly suggests that it is concerned in
oxidation reactions. But it acts only as an electron carrier, and not
as a hydrogen carrier like-DPNor TPN, or the flavoproteins;
furthermore, it is not an enzyme.1
The other compound of importance belonging to this class is
cytochrome-a3, commonly known as cytochrome oxidase. This name
accurately expresses its enzymic function, for it is a true oxidase,
employing molecular oxygen as its hydrogen acceptor. In the reduced
state, cytochrome-c is quite unaffected by oxygen; when cytochrome
oxidase is added, however, the reduced cytochrome-c is rapidly
oxidized. Unfortunately, cytochrome oxidase is very firmly bound
lOr, at least, its catalytic functions are not easy to define.
53
OXIDATION
to the 'insoluble' fraction of cells and attempts to detach and purify
it have not been very successful, but the prosthetic group of the
enzyme is known to be somewhat similar to that of cytochrome-c,
and to contain iron.
Between the reduced flavoproteins and oxygen, therefore, an
electron-carrier system operates (VI). Reduction of cytochrome-c is
catalysed by the flavoprotein (cytochrome-c reductase), oxidation by
cytochrome oxidase. The importance of this system may be judged
from the fact that when a cell is poisoned with hydrogen cyanide its
oxygen uptake falls by about 90 % and the bands of reduced cytochrome-c become visible. Cyanide is therefore poisonous because it
inhibits cytochrome oxidase and blocks the main pathway through
which metabolites are oxidized.!
FPH 2
(Cyt.-c Fe
)
H
)
(H020
cyt -c Fe+++
FP
(VI)
H 20
t
oxygen
2H+ (
tI
2e -
r
M
cytochrome-c and
cytochrome oXidase
Via
:::PN " TPN
metabolIte
(VII)
Considering the whole series of reactions which began with the
dehydrogenation of lactic acid, we observe at the flavoprotein level
a switch from hydrogen transfer to electron transfer (VU)_ In the
reduction of DPN or TPN by lactic acid (and many other metabolites) one of the hydrogens enters the pyridine ring, and the other
is taken up by the remainder of the co-enzyme molecule in the
manner already described (p. 49). When DPNH 2 or TPNH2 reacts
with a cytochrome-c reductase, the prosthetic group of the flavoprotein is likewise reduced by a pair of hydrogens which enter the
1
The retina of the eye is peculiar in bemg relatively insensitive to cyanide.
54
CYTOCHROMES
isoalloxazine ring (p. 50). At the next step, both hydrogens must
be thought of as splitting into protons and electrons; the latter reduce
cytochrome-c from the ferric to the ferrous state. Possibly the iron
attached to the prosthetic group of cytochrome-c reductases is
involved in this process of electron transfer, and since cytochrome-c
can only accept one electron at a time, it is reduced and oxidized
twice as each pair of hydrogens passes f:t:om the flavoprotein to
oxygen. Finally, cytochrome oxidase, taking the electrons one at a
time from cytochrome-c, causes them to unite with the pair of protons
and with oxygen to form water. Until cytochrome oxidase has been
obtained in a pure state the details of this complex reaction are likely
to remain obscure.
oxygen
~cytowl~~
fiavoprotems
(e.g. succiniC
dehydrogenase)
t
a few metabolites
(, g 'uoc;,k ,dd)
fiavoprotems
(cytochrome-c
reductases)
t
"Tm",,=,
DPH- and TPNh,kox!
flavoprotems
(e g. ~anthine
oXidase)
t
a few metabolItes
("g, ,,",h;",l
the maJonty of metabolites
(VIII)
We can now make a summary of the chain of reactions with
which metabolites of various kinds can enter when they are oxidized,
the arrows showing the direction of hydrogen (and electron) flow
(VIII). In contemplating this diagram the distinction between aerobic •
and anaerobic dehydrogenases must be kept in mind. The former, of
which the Schardinger enzyme (xanthine oxidase) is an example,
require no cytochrome to mediate between them and oxygen; the
product of reaction is H 20 2 , but since all cells contain catalase
(p. 40), the eventual outcome is that the substance is oxidized to
product plus water. Such enzymes are not common, and the chief
55
OXIDATION
part in oxidative metabolism must be assigned to the anaerobic
dehydrogenases.
The first step in the oxidation of most metabolites is dehydrogenation by DPN- or TPN-linked dehydrogenases, which then react
with oxygen through the cytochrome-c reductases (some specific to
DPNH2 and some to TPNH2) and the cytochromes. Hence the
chain of reactions (IX) may be taken as typical of biological oxidations in general.
:~:')
metabohte
(NH') (FPH') ( F," ) (0
'y< -c
DPN
FP
(IX)
cyt.-c Fe+++
0
But two deviations from the standard path must not be overlooked.
The first arises with certain fiavoproteins which do not themselves
react with oxygen, and therefore fall into the class of anaerobic
dehydrogenases. Succinic dehydrogenase, for instance, can remove
hydrogen from its substrate without the intervention of DPN or
TPN; and, as D. Kellin has recently shown, this enzyme under
appropriate conditions can reduce cytochrome-c.
The second deviation is of a different kind. Whereas lactic acid is
oxidized in animal tissues by a DPN-linked dehydrogenase, in yeast
another enzyme comes into play. This enzyme was crystallized by
R. K. Morton in 1954, and shown to be both a flavoprotein and a
cytochrome. To a single protein molecule are anchored a prosthetic
group characteristic of flavoproteins (in this case FMN) and a
prosthetic group characteristic of cytochromes (an iron-porphyrin
complex). The yeast enzyme is known as cytochrome-b2 , a!ld is
capable of reducing cytochrome-c. Representing the flow of protons
(and electrons) by arrows, the sequence of reactions is
Lactic acid
->-
FMN
->-
Fe-porphyrin ...... cyt.-c
protein
(cytochrome-b2)
Here, then, we have a portmanteau enzyme, two separate prosthetic
groups forming part of the same molecule; such compounds have
been christened <fiavocytochromes'.
56
OXIDA TION
The rH scale
Some of the chief aspects of biological oxidation have now come
under review, and it is time to inqulfe what useful purpose is served
by such processes; in particular, how oxidation is related to the
supply of free energy needed by the cell. When gaseous hydrogen
and oxygen react together to form water, with explosive vi<?lence,
there is a large loss of free energy, -t:J.F = about 58,000 cal./mole.
Yet in living tissues the hydrogen from metabolites is made to react
with oxygen smoothly and quietly, through the systems of hydrogen
(and electron) transport outlined above. The free energy, instead of
being wasted in the heat of an explosion, must somehow be diverted
-in part, at least-to other ends. The manner in which this diversion
comes about is not yet fully understood, but the approach is made
easier by considering the rH scale advocated by M. Dixon in 1949.
In its simplest form, without reference to enzymes, the oxidation
and reduction of a pyridine nucleotide, such as DPN, can be written
DPNH2 ~ DPN+2H.
On paper, therefore, we can imagine the reduced nucleotide dissociating into oxidized nucleotide and hydrogen. In practice, of
course, DPNH 2 does not evolve hydrogen spontaneously; nevertheless, as a reducing agent it has a tendency to part with hydrogen,
and this tendency can be measured. The greater the readiness to part
with hydrogen, the more powerful will a reducing agent be. Since
DPNH 2 can reduce a cytochrome-c reductase, it must be the stronger
reducing agent of the two. I In reversible reactions of this kind it is
convenient to think of each reactant as exerting a definite hydrogen
pressure; DPNH2 reduces FP easily, but FPH2 reduces DPN very
slightly-hence DPNH2 exerts agrea ter hydrogen pressure than FPH2 .
It is a familiar fact that a few substances will part with their
hydrogen to metallic platinum. One such is hydro quinone (X). If
we dip a platinum foil into a dilute solution of hydroquinone, a
reaction takes place, to a very small but definite extent, with the
formation of quinone. The hydrogen atoms from the hydroquinone
dissociate at the platinum surface into equal quantities of hydrogen
ions and electrons, and if the foil is connected with a standard cell,
t
3
Provided that the concentrations of reactants are not widely different.
57
HB
·
OXID A TION
the potential set up-the electron pressure-is a measure of the
degree to which the reaction can occur, or in other words, of the
hydrogen pressure generated by the hydroquinone. Many compounds of biological interest-metabolites, pyridine nucleotides, etc.
-do not react with platinum, in the manner of hydroquinone
(although cytochrome-c does), but indirect measurements can be
made. Directly or indirectly, therefore, the strength of a reducing
agent can be ascertained in terms of the hydrogen, or electron,
pressure exerted by it.
OH
+Pt
+Pt H2
OH
hydroqumooe
quinone
(X)
If hydrogen itself is bubbled through pure water at atmospheric
pressure some of the atoms adsorbed on the platinum dissociate into
hydrogen ions and electrons, and the potential thereby set up furnishes
a standard of reference. Nearly all reducing agents are far weaker than
hydro gen; and in forming a com para ti ve scale ofhydro gen pressuresthe rH scale-it is customary to use the negative logarithm
1
rH = -logio [H 2] = 10glO [H
]
2
For hydrogen itself at 1 atmosphere the rH = O. For a mixture of
hydro quinone and quinone in equal quantities at pH 7 the hydrogen
pressure is 1 x 10-23 atmospheres, hence rH = 23.
Returning noW to the oxidation and reduction of a pyridine
nucleotide, DPNH2 ~ DPN + H 2 , the free energy of the reaction is
the difference between the free energies of DPNH2 and DPN, which
is proportional to the difference between the hydrogen pressure
exerted by the system containing equal amounts of the two components and hydrogen at 1 atmosphere, the proportionality being
expressed by
-t::..F = 2'3RTxrH = 1420xrHcal. (at 37°).
58
THE rH SCALE
As an illustration we may consider the oxidation of hydrogen by
molecular oxygen to form water. Because of the reaction
O+H 2 0+2e- ~ 20H-,
oxygen in aqueous solution at 1 atmosphere exerts an extremely
small electron pressure, and hence occupies a definite position on
the rH scale; the rH value is 41. So
40when hydrogen and oxygen react-for
-so,OOO
example, at a platinum surface-to form
35water,
-!l.F = 1420x41 = 58,000 cal./mole.
30-40,000
This figure is a measure of the maximum
free energy that can become available
when a pair of hydrogens is oxidized by
25molecular oxygen; what is actually r'H
.c
f-30,000
available in the cell will depend on the
20rH value of the dehydrogenase system
that initiates the oxidation of a metabolite.
15-20,000
In biological oxidations the compounds involved nearly always react as
10ions, and hence the pH must be taken
1-10,000
• into account. The rH of a half-reduced
ionizing system is represented by r'H;
5and in (XI) some typical figures are given
for r'H at pH 7. Thus the lactic dehydro0-'---'--1---'-genase of animal tissues has an r/R
70
pH
value of about 8 at 37° C., and other
(Xn
DPN- and TPN-linked dehydrogenases
have rather similar values, represented by D in (XI). Flavoproteins
(F in XI) lie higher up the scale, in the region of r'H 14, and
cytochrome-c higher still (C in XI). The linear relation between r'H
and 6.F enables the free energy of each step in hydrogen (or electron)
transport to be read off the scale:
-llF, cal./mole approximately
Lactic dehydrogenase to flavoprotein
(cytochrome-c reductase)
Flavoprotein to cytochrome-c
Cytochrome-c to oxygen
59
9,000
13,000
25,000
,
OXIDA TION
Oxidative phosphorylation
When electron (or hydrogen) transport occurs in living cells there is
a simultaneous phosphorylation of ADP to ATP. This process, called
'oxidative phosphorylation', can only be demonstrated by refined
experiments, under carefully controlled conditions; that it is known
to occur at all is due to the work of H. M. Ka1ckar, S. Ochoa,
V. A. Belitser, F. Lipmann, H. A. Lardy, and A. L. Lehninger,
among others. What seems to emerge from their experiments is this:
when a metabolite like lactic acid is oxidized, three energy-rich
phosphate bonds, ,.., ®, i.e. three molecules of ATP from ADP and
inorganic phospate, are formed from each pair of hydrogens passed
up to oxygen by the chain of reactions just described. This finding
is commonly expressed by saying that the phosphorus-oxygen ratio
is three, P:O = 3. Not all metabolites behave in the same way;
with succinate, for instance, p:o = 2 only. We have seen that the
free energy change in an oxidative process depends on the rH of the
enzyme system which initiates it. Evidently the free energy appears
in energy-rich bonds at three steps in the reaction chain:
(a)
(b)
\ (c)
DPNH2+FP
FPH2+Cyt.-c Fe+++
cyt.-c Fe+++O
-->-->-->-
DPN+FPH2
FP+cyt.-c Fe++
cyt.-c Fe++++H20
~®;
~®;
~®.
A few points are worth noting about these equations. First, that the
free energy change at every step is more than enough to generate a
pyrophosphate bond of about 8000 cal./mole. Secondly, that the
reduction of DPN to DPNH2 as such (or TPN to TPNH 2) does not
yield ,...., ®. Thirdly, that the whole process is sometimes called
'electron transport' phosphorylation, in order to distinguish it from
another type of oxidative phosphorylation (p. 88); the term
'respiratory chain' phosphorylation is also used
Unfortunately the mechanism of this process is still obscure.
It is far from simple, and we cannot even be sure that precisely the
same type of reaction occurs at each stage, but in a rough way, and
without resorting to detail, it is not hard to symbolize what goes on:
X-OH+inorganic phosphate
X-O-® -2e-
~
(b)
(c)
X++-O~®+ADP
~
(a)
(d) X++-OH+2e-
~
60
X-0-®+H20;
~ X++-O~®;
X++-OHtATP;
X-OH.
OXIDATIVE PHOSPHOR YLATION
The starting-point is an intermediate substance X-OR, capable of
reacting with inorganic phosphate to give an energy-poor compound
X-O-®. We do not know the nature of X-OR, but it is likely to
be rather complicated. When X-O-® is oxidized, by cyt.-c Fe+++,
let us say, it loses electrons, and becomes X++-0"" ®, the energyrich bond of which is then transferred to ADP to yield ATP; such
transfer reactions are known to occur (p. 19). Finally, X++-OH
is reduced back to X-OR, which is then ready to enter the cycle
again.
At all events, when electrons 'flow over' the prosthetic groups of
the metalloflavoproteins and cytochromes, part of the free energy of
the process is tapped off into the terminal energy-rich bond of ATP.
Given a supply of reduced pyridine nucleotide, and the means of
oxidizing it, through flavoproteins and cytochromes, three energyrich bonds are produced for every pair of hydrogens oxidized to
water, that is, three molecules of ATP are formed from ADP and
inorganic phosphate. The amount of pyridine nucleotide in living
cells is small, only a few milligrammes per kilogramme of tissue
(wet weight), yet the process of reduction and oxidation is so rapid
as to furnish an adequate supply of A TP for synthetic reactions. In
the next chapter we shall see how ATP goes to work.
61
v
PHOTOS YNTHESIS
Oxygen is constantly being removed from the air through the
weathering of rocks and minerals, and by escape from the earth's
atmosphere into outer space. But setting aside these losses the
amount of oxygen primarily depends upon a balance between
photosynthesis
organic matter+ 02.
oxidation
During photosynthesis the energy of sunlight is stored in the form
of carbon compounds, largely sugars and polysaccharides; the overall process is often represented as
energy
6C02+6H20 - - C6H 1206+ 602.
This equation, however, gives an imperfect and rather misleading
picture of what goes on, because it does not distinguish between
different kinds of reaction: those for which light is needed, and those
for which it is not.
Photolysis of water
When light falls on green plants the greater part of the energy needed
for photosynthesis, if not all, is absorbed by small particles called
chloroplasts. The number of chloroplasts in each cell is variable: in
some of the green algae, such as ChIarella, a cell may be furnished
with only one or two chloroplasts; in a leaf cell, on the other hand,
there may be several hundred. Chloroplasts are usually more or less
ellipsoidal in shape, and measure about 5 p across; they cohtain an
assortment of pigments, chiefly chIorophylls and carotenoids, whose
structure is too complicated to discuss here. The colouring matter of
widest distribution is chlorophyll a, which has strong absorption
bands at the blue and red ends of the spectrum, and is chiefly
responsible for the green colour of plant material. These pigments
turn the energy of visible light into chemical energy, although the
mechanism of the process is not known. We have seen (p. 31) that
when glucose is oxidized the change of free energy, -!:1F, is about
62
PHOTOLYSIS OF WATER
691,000 cal{mole, and in photosynthesis the same quantity of free
energy must be stored up, or about 115,000 cal./mole of oxygen
produced. It can be calculated that 3 quanta of red light are enough
to supply the energy required for each molecule of oxygen, but in
practice the plant seems to need about 8-10 quanta; thus the
'thermodynamic efficiency', in terms of oxygen production, is about
30 %. However that may be, the radiant energy absorbed by chloroplasts is employed to break up water molecules.
In 1937, R. Hill showed that isolated chloroplasts, in the presence
of light, can act as reducing agents while simultaneously oxygen is
evolved. Soon afterwards S. Ruben and M. D. Kamen, using the
isotope lBO, proved that the oxygen is derived from water and from
no other source; and in 1951, W. Vishniac and S. Ochoa found that
pyridine nuc1eotides are reduced by illuminated chloroplasts. Hence
the photolysis of water can be represented by
light
H20+DPN
----+
DPNHz+t02'
It appears, however, from the work of A. A. Benson and M. Calvin
that the reduction of DPN may involve another compound, a-lipoic
acid (6: 8-thioctic acid), which is readily oxidized and reduced (I).
/C~
yHz
/C~
YH.(CHzkCOOH ~ 1Hz
s--s
-2H SH
1H.(CH2kCOOH
SH
(I)
-chl.+
(II)
DPN
-
(III)
The rH of this system is probably a little lower than that of the
pyridine nucleotides; in other words, the reduced form of lipoic acid
can reduce DPN. Representing the light-excited state of chlorophyll
as chl. * we can represent the process as in (II) and (III). No doubt
63
PHOTOS'YNTHESIS
a great deal more goes on than this simplified scheme would suggest;
at all events, water splits open the ring of lipoic acid, producing a
dithiol and oxygen; then the dithiol reduces DPN.
The significance of these reactions will now be apparent: the plant
has obtained a store of reduced pyridine nucleotide, some of which,
when oxidized through the flavoprotein-cytochrome pathway, furnishes ATP.1 A molecule of pyridine nucleotide is reduced for each
atom of oxygen evolved. But the oxidation of DPNH 2 yields three
molecules of ATP (p. 59), therefore some of the radiant energy of
sunlight has appeared in the energy-rich bonds of ATP:
light energy
(via chlorophyll, etc.)
DPNH 2+3ADP+3®OH+!Oz -+ DPN+3ATP+4H 20.
Armed with this store of ATP the plant proceeds to fix carbon
dioxide. 2
Fixation of CO2
Before discussing how CO 2 is drawn into the net of synthetic
reactions, it is desirable to have a rough idea of what is going to
happen. The building up of starch from CO 2 involves, in its early
stages, a cyclic process which can be illustrated by a carbon flowsheet (IV). Three molecules of the C 1 compound (C0 2) react with
'C,
+
'~/6C'~
5C3
lC 3
condensation
sugar and
starch
(IV)
three molecules of a Cs compound to give, after some curious
chemistry, six molecules of a C 3 compound (or rather, a mixture of
C 3 compounds). From this C3 pool, one-sixth of the molecules
follow a condensation pathway, ending finally in starch; five-sixths,
by devious routes, return to the Cs compound. The net result is that
the C 1 compound (C0 2) flows into the cycle at one point, and sugar
and starch emerge at another.
1 The remainder of the DPNH2 is used, as such, at a later stage in the process
(p.66).
z It should be added that ATP can also be produced in plants by a slightly
different mechanism, involving reduction of TPN to TPNH2, followed by oxidation of TPNH2; but the details are not yet clear.
'
64
FIXATION OF C02
It will be convenient to represent the sugars and their derivatives
that participate in this cycle by straight-chain formulae,! and to
begin with the driving force behind the whole process-the action
of A TP on a C s compound, ribulose-5-phosphate (V). The phosphate
resid ue from ATP becomes attached to carbon 1 in the ribulose
phosphate by an energy-poor bond, hence about 5000 cal./mole of
free energy are 'wasted', so the equilibnum point of the system is
1
CH 20H
bo
I
2
3 RCOR
I
CH20®
bo
I
ATP
-
RCOR
4 RCOH
HCOR
bH20®
5
+ADP
I
bH20®
ribillose-5-phosphate
ribulose-I: 5-diphosphate
(V)
+
COOH
I
I
HCOH
CH 2 0®
3-phosphoglyceric acid
(VI)
pushed far to the right, and the reaction is not easily reversible. The
enzyme bringing about the phosphorylation is called a phosphokinase, that name being given to those enzymes which transfer
phosphate groups from ATP to another compound without the
liberation of free phosphate; and since the substrate in this instance
is a pentose phosphate, the enzyme is called phosphopentokinase.
Ribulose-l: 5-diphosphate now reacts with water and CO 2 (VI).
1
They belong configurationally to the D-series.
65
PHOTOSYNTHESIS
Besides the addition of CO 2 , this complex reaction involves an
oxido-reduction, or dismutation, one part of the ribulose diphosphate
(below the dotted line) being oxidized and the other part reduced;
the enzyme responsible is therefore called carboxydismutase. The
product is two molecules of 3-phosphoglyceric acid, a derivative of
glyceric acid which can be synthesized in the laboratory by the
oxidation of glycerol.
CH20®
I
I
HCOH
+2H
COOH
3-phosphoglyceric acid
3-phosphoglyceraldehyde
(Vm
CH20®
HtOH
I
+ATP
+ADP
CO-O~®
1 : 3-diphosphoglyceric aCId
(VIm
CH20®
I
HCOH
CH20®
I
+ DPNH2
to-o~®
HCOH
+DPN+®OH
tHO
(IX)
The next step is a reduction (VII), the product being 3-phosphoglyceraldehyde. On following this reaction in detail it is found that
ATP is concerned; another phosphokinase-the phosphoglyceric
phosphokinase-catalyses (VIII). This reaction is reversible. In its
turn, 1: 3-diphosphoglyceric acid is reduced by DPNH2 in the presence of an enzyme (IX). We have already seen that'a store of
reduced phosphopyridine nucleotide is made available by the photolysis of water: part of this store is oxidized to form ATP; the
remainder is then used in the reduction of 1: 3-diphosphoglyceric
acid.
For simplicity we shall not now inquire into this process of
reduction, which presents some curious features. The substance
3-phosphoglyceraldehyde belongs to a class of compounds known as
triosephosphates, and the enzyme responsible for its synthesis is a
66
FIXATION OF C02
DPN-linked triosephosphate dehydrogenase working in reverse. 1 At
a later stage the importance of this enzyme, and its mode of action,
will be discussed in detail (p. 87).
When 3-phosphoglyceraldehyde is formed, it is immediately
attacked by a triosephosphate isomerase, producing the isomer
dihydroxyacetone phosphate (X). The standard free energy change
CH 20®
CH 2 0®
I
HCOH
I
CHO
3-phosphoglyceraldehyde
toI .
CH 20H
dihydroxyacetone
phosphate
(X)
in this reaction is relatively small, - D..PO = about 2000 ca1./mole; at
equilibrium the mixture contains about 95 % of dihydroxyacetone
phosphate and 5 % of 3-phosphoglyceraldehyde. This equilibrated
system of triosephosphates represents the C3 pool mentioned earlier,
and we may thus sum up the story as far as it has gone:
3ATP
3 ribulose-5-phosphate
6ATP
3 ribulose-I: 5-diphosphate
t 3C0 2
6 3-phosphoglyceric acid
6 triosephosphate
6DPNH 2
Reactions of triosephosphate
One-sixth of the molecules in the triosephosphate pool are destined,
we have seen, to form starch; the remaining five-sixths return to
ribulose-5-phosphate, through a maze of reactions which can be set
out in terms of carbon (XI). Clearly this process divides into four
main steps:
(a) C 3+Cr >-C 6;
(b) C3+C6 ->- C4+ C s ;
(c) C3+C4->-C7;
(d) C3 + C7 ->- Cs + Cs.
Reaction (a) is simply a condensation of one molecule of each of
the triosephosphates, under the influence of the enzyme aldolase
(XII), the process being a straightforward aldol condensation. It
must be observed that when aldolase removes 3-phosphoglycer1 Plants contain both DPN- and TPN-linked triosephosphate dehydrogenases;
we here consider only the former.
67
,
PHOTQSY NTli ESIS
aldehyde from the triosephosphate mixture, the isomerase immediately restores the equilibrium; we saw (p. 12) that each tenfold
diminution of product concentration alters 6.F by about 1420 cal./
mole, and since the standard free energy change is small in this
reaction, equilibrium is readily achieved. The fructose-I: 6-diphosphate is then attacked by a hydrolytic enzyme-a phosphatasewhich selectively removes the phosphate from carbon 1, yielding
fructose-6-phosphate.
C3 :
C3~-------------------------
C,
C.~
r
C
'
'
C3~.---------~~~---------------------------- ~C5
CH20 ®
to
__
(XI)
HOtH
HtOH
HtOH
tH20 ®
fructose-I: 6-diphosphate
(XII)
Reaction (b) involves fructose-6-phosphate and another molecule
of 3-phosphoglyceraldehyde (XIII). Here the CH20H-CO----; group
(and a hydrogen) are transferred from fructose-6-phosphate to
3-phosphoglyceraldehyde; the group is both a ketone and an alcohol,
hence the enzyme responsible for the transfer is called a transketo lase. 1 As a result of this reaction, which appears to be readily
reversible, the 4-carbon compound erythrose-4-phosphate is produced, and also xylulose-S-phosphate. The latter, however, does not
possess the configuration required for reaction with phosphopento1 This enzyme requires thiamine (formula in the APPendix, p. 142) as coenzyme, in the fonn of its pyrophosphate.
68
REACTIONS OF TRIOSEPHOSPHATE
kinase, hence an enzyme is needed to switch round (or epimerise) the
hydrogen and hydroxyl attached to carbon 3 (XIV). By the action
of this phosphopentose epimerase the first of the three ribulose5-phosphate molecules is arrived at.
CHzOH
CHO
CHzOH
I
I
I
+ HCOH
co
HOtH
CO
CHO
tH20®
I
HatH
I
HCOH
HCOH
HtOH
HtOH
I
+
I
CHzO®
tHzO®
erythrose-4phosphate
CHzO®
fructose-6phosphate
HtOH
xylulose-5phosphate
(XIII)
CH2 0H
CHzOH
2
3
4
I
co
to
epimerase
HtOH
HOtH
I
HCOH
I
HtOH
5
CH 2O®
xylulose-5-phosphate
tH 2 O®
ribulose-5-phosphate
(XIV)
Reaction (c), like reaction (a), is an aldol condensation, catalysed
by aldolase. It takes place between erythrose-4-phosphate and
dihydroxyacetone pbosphate (XV). The product is a 7-carbon sugar,
sedoheptulose, phosphorylated at positions I and 7. This compound
undergoes hydrolysis by a phosphatase at position 1, yielding
sedoheptulose-7-phospbate.
CHzO®
CH20®
I
co
to
I
I
CH20H
HOCH
+
CHO
HtOH
HCOH
HCOH
I
I
I
HCOH
tHzO®
HtOH
tHzO®
sedoheptulose-! :7-dlphosphate
(xV)
69
PHOTOSYNTHESIS
Reaction (d) then takes place between this compound and another
molecule of 3-phosphoglyceraldehyde (XVI). Once again the transketolase is responsible. The xylulose-5-phosphate is promptly converted by the epimerase into ribulose-5-phosphate, as before. And
an isomerase converts the ribose-5-phosphate into ribulose-5-phosphate also (XVII). Thus are produced two other molecules of the
C s compound that reacts with ATP.
CH 20H
bo
HOtH
CH2 0H
HtOH
CHO
bo
HtOH
HCOH
+ HOtH
HtOH
HCOH
I
I
I
HCOH
I
bH 0®
HCOH
2
bH 0®
2
bH 0®
2
ribose-Sphosphate
xylulose-Sphosphate
(XVI)
CHO
CH20H
HtOH
HboH
isomerase
toI
HCOH
I
I
HtOH
HCOH
tH 2 0®
CH 20®
ribulose-5-phosphate
ribose-5-phosphate
(XVII)
The carbon flow-sheet for these reactions can now be written in
more detail (XVIII). This complex series of reactions has been largely
worked out by B. Horecker, E. Racker, and J. A. Bassham, as well
as by others whose names have been mentioned earlier in this chapter.
Stripped of all its complexities, the 'steady state' of the photosynthetic cycle may be represented by (XIX): where PG = 3-phosphoglyceric acid and C 3 ® = triosephosphate, as before. In the
form of an equation:
3C02 + 6DPNH2 + 9ATP + 5H20 ......
6DPN+9ADP+8®OH+l triosephosphate.
70
C5~
(xylulose)
E
Cs ®
Cs ®
(nbose)
(xylulose)
~l
Cs~
~E
C5~
all nbulme
(XVIII)
Explanation of symbols
aC 3® = aldehyde isomer of tnosephosphate, viz. 3-phosphoglyceraldehyde
kC 3 ® = ketone isomer of tnosephosphate, viz. dihydroxyacetone phosphate
A=aldolase
E=eplmerase
P=phosphatases
TK= transketolase
I=isomerase
71
·
PHOTOSYNTHESIS
Reverting now to the equations for the photolysis of water and
formation of ATP and DPNH 2 , we have:
(1) 9H 20 + 9DPN __,. 9D PNH2 + 4-!-02,
3DPNH2+9ADP+9®OH+ HOz __". 3DPN + 12HzO+9ATP
(2) 6DPN +9ADP+9®OH --+ 6DPNHz+ 9ATP+3H zO+30;-:-
Then for the fixation reaction,
(3) 3C02+6DPNH2 +9ATP+ 5H zO __,.
6DPN + 9ADP+ 8®OH + 1 triosephosphate.
And summing (2) and (3),
3C0 2 + 2H 20+ ®OH --'>-1 triosephosphate+ 302.
It only remains to follow the comparatively simple series of chemical
changes by which starch is obtained from the triosephosphate pool.
3C s®
( +2H]O,
-2®OH)
1
3ATP
(+3H zO)
5+1C3 ®
I
3Cs dl-CPJ
6ATP
3DPNHz
(-6®OH)
t-
3CO
,
6PG
starch
(XIX)
Formation of starch
The first step covers familiar ground-Reaction (a), forming fructosel:6-diphosphate from a molecule of each triosephosphate undefthe
influence of aldolase (p. 67). From tbis point onwards it is preferable
to use the correct formulae for the sugars and their phosphates.
And, as before, a phosphatase removes the phosphate residue from
carbon I (XX), yielding fructose-6-phosphate. An isomerase (the
phosphohexo-isomerase) now converts this compound into glucose6-phosphate, whereupon another enzyme (the phosphoglucomutase)
transfers the phosphate group to positlOn 1,1 resulting in glucoseThis reactIon is a lIttle more complicated than it is made to appear here.
72
FORMATION OF STARCH
I-phosphate (XXI). The standard free energy change is only about
-1740 cal./mole, and the reaction is readily reversible; at equilibrium
roughly 95 %of glucose-6-phosphate and 5 %of glucose-I-phosphate
are present. It will be observed that whereas glucose-6-phosphate IS
an ester, glucose-I-phosphate is a glycoside.
+®OH
fructose-J .6-diphosphate
fructose-6-phosphate
(XX)
CH20H
-
~
O
H
HO
glucose-6-pbospba te
H
O-®
OH
glucose-I-phosphate
(xxD
ro"": . . . . . . .
~~o-®
:rO\i:. . . . . . . .v+--
Y{----°"J: ....... .
W~1o-®
L............ l
:............... !
I~
H,O~lO-®
H~
:........... :
phosphorylase
(XXII)
Glucose-I-phosphate possesses the a-configuration, and under the
influence of a type of enzyme called phosphorylase can condense,
with loss of phosphate, to form the particular kind of starch that
goes under the name of amylose (XXII). The synthesis of amylose
was achieved in 1940 by C. S. Hanes, and represents it process called
73
etc.
PHOTOSYNTHESIS
transglycosidation: the glycoside bond simultaneously loses phosphate and forms a new glycosidic link with the -OR group in
position 4 of another glucose residue; at no time is the free sugar
liberated. For the reaction -/)•.£0 = approx. 400 cal./molc (of
glucose residue), and hence the synthesis of the polysaccharide is
excrgonic.
The phosphorylases from plant sources that make amylose are
often referred to as P-enzymes. They do not act unless there is
present, in addition to glucose-I-phosphate, a 'starter' in the form
of 4 or 5 glucose residues already linked together. Once started,
however, these phosphorylases will add on glucose residues almost
indefinitely; chains of 1000 glucose residues or more have been made.
The synthetic amyloses differ from the natural ones in that they
contain only 1:4-lX-links in the chain, whereas amyloses in the plant
contain occasional jJ-links as well; hence another enzyme must come
into play. Amyloses form 'colloidal' solutions in hot water, from
which on cooling they sometimes separate in more or less crystalline
form.
HOOH2C ......... .
HO
;o-® :
·
.
··
..
What we call 'starch', however, contains besides amylose another
polymer, amylopectin. Rice and potato starch, for instance, contain
about 20 % of amylose and 80 % of amylopectin; starch from other
plants may contain as little as 5 % of amylopectin. The synthesis of
amylopectin is determined by phosphorylases known as Q-enzymes,
which form 1:6-a-linkages between glucose-I-phosphate molecules
(XXIII). Hence Q-enzymes can introduce branches into the amylose
type of straight chain (XXIV). Amylopectins with the~r branching,
tree-like, structure often incorporate over 1000 glucose residues, each
74
FORMA TION OF STARCH
branch usually constituted of about 20-30 residues. They are less
soluble in water than amyloses.
Besides starch, many plants store carbohydrate in the form of
fructose polysaccharides (inulins and levans). Disaccharides also
. occur, of which the most familIar is sucrose, and L. F. Leloir has
shown that it arises from fructose-6-phosphate, or free fructose, and
etc;.-~4
CH,OH
1Hz
CH,OH
etc-~~~'"
(XXIV)
a complex substance called uridine diphosphate glucose (UDPG)
whose structure will be found in the Appendix (p. 143). Sucrose is
found in all photosynthetic plants, but appears not to occur in yeast
and fungi.
Cellulose
" As opposed to starch, which acts as a food reserve for plants and
their seeds, cellulose is a polysaccharide of purely structural importance, entering into the composition of cell walls, and forming
~CH20H
0
~CH20H
0
CH,OH
o
0
00
o~
£
(XXV)
the fibrous or woody part of vegetable matter. Cellulose is the most
abundant organic compound of all, since about half the carbon of
plants is locked up in it. The glucose residues are here /l-linked in
the 1: 4-position (XXV). For stereochemical reasons such a chain
can exist as a long, rod-like structure, and, as in proteins, hydrogen
bonding can occur between the -OR groups of adjacent chains,
75
PHOTOSYNTHESIS
which helps to give the molecule a considerable degree of tensile
strength. The length of an individual chain may be as much as 2500
glucose residues. Cellulose does not dissolve in water, is chemically
very inert, and, as we shall see, only a limited range of organisms
possess enzymes capable of attacking it.
lt will not be supposed that the synthetic ability of plants is confined to carbohydrates. They manufacture proteins and fats also, as
will appear in due course, and many other kinds of complex molecule.
One of these activities must be briefly mentioned here-the synthesis
of vitamins. From the pioneer work of H. Eijkman and of Sir F. G.
Hopkins it has become clear that many organisms-and by no means
only the higher animals-need in addition to the major foodstuffs a
small supply of certain substances which they cannot manufacture
for themselves. What distinguishes these substances from ordinary
articles of food is the smallness of the amount required-perhaps
only a few milligrammes a day for a large animal. The terms
'accessory food factor' or 'growth factor' or 'vitamin' are usually
applied to organic compounds of this kind; when metals are needed,
they are called 'trace elements'. We have seen (p. 44) that enzymes
are known which cannot act except in the presence of a metal ion;
a fact which gives a clue to the nature of many of the vitamins-they
are component parts of co-enzymes. An animal cell, for instance,
cannot manufacture nicotinamide, the active portion of DPN and
TPN, nor can it make isoalloxazine. But such molecules are well
within the synthetic capacity of plants, and to some extent of microorganisms also,l hence the animal world depends upon the plant
world not only for its primary source of free energy, but for some
of the essential compounds required in making that energy available.
These topics belong to the field of nutrition, and must not now detain
us. Instead, our attention will be directed to the breakdown of
carbohydrate in plant and animal cells, and to the exploitation of the
free energy-deriving ultimately from sunlight-that is stored up in
the form of sugars and polysaccharides.
1 The nutritional requirements of the different kinds of micro-organism are so
varied that any general statement about them is hazardous; It will be obvious,
however, that both plants and micro-orgamsms depend on an extraneous source
of trace-elements.
76
VI
CARBOHYDRA TE METABOLISM
Polysaccharide molecules like starch and cellulose are too large to
pass through cell walls: an organism needing an external supply of
carbohydrate from polysaccharides must therefore elaborate enzymes
capable of breaking them down to smaller units. Such enzymes are
of wide distribution, and they can be regarded as extracellular
glycosidases-eithe_[ secreted by cells, or attached to the cell surface
-whose function is to hydrolyse the glycosidic links in polysaccharides. For instance, the salivary and pancreatic secretions of
animals contain a number of enzymes for this purpose. Similar
enzymes occur in the seeds of plants, to make reserve carbohydrate
available for the embryo, and some bacteria are even capable of
attacking cellulose. We can single out seven kinds of glycosidase as
being of special importance, though many more have been discovered.
1. a-Amylases (1: 4-a-glycosidases). Here the amylose molecule is
attacked in a random manner at any point in the chain, the 1 : 4ex-glycosidic link being hydrolysed. Smaller units called dextrins are
formed at first, and finally the disaccharide maltose
cn.
Fo~
~o~
H~O~H
OH
OH
(glucose-I: 4-ct-glycoside)
(1)
2. ~-Amylases (1: 4-a-glycosidases). These enzymes attack amyloses
from the non-reducing end of the chains, and not at random; hence
dextrins are not formed; but the product is maltose. 1 Both ex- and
I It is important to realize that the amylase prefixes ct- and /3- have no stereochemical significance, since both are ct-glycosidases; the prefixes are merely used
to distinguish the mode of action of the enzymes. A nomenclature of the kmd
proposed by S. Peat for R- and Z-enzymes (below) would be preferable, as
leading to less confusIOn.
77
CARBOHYDRA TE METABOLISM
p-amylases attack the amylopectin type of molecule in their characteristic ways; but they cannot act on the branching points, a function
reserved to
3. R-enzymes. These enzymes hydrolyse the 1: 6-a-linkages occurring in amylopectins. They do not, however, attack a branching point
until the side-chain has been removed by the previous action of
amylases.
4. MaItases. Also 1:4-ct-glycosidases, hydrolysing maltose to two
molecules of glucose. Hence under the influence of the four enzymes
just considered all the a-links in amyloses and amylopectins can be
disrupted. We must not, however, overlook the occasional p-links
that occur in natural amyloses (p. 74), which require the attention of
5. Z-enzymes. These enzymes are ,B-glycosidases, specific to the
,B-linkage. Thus the complete hydrolysis of starch to glucose requires
five enzymes in all.
6. Cellulases. These are also ,B-glycosidases; they are found only in
certain bacteria and in creatures like snails and wood-boring insects.
The importance of bacterial ce11ulases would be hard to overrate,
since all the ruminant animals-cows, sheep, etc.-depend on cellulose
for a considerable part of their energy supply. By supporting a large
bacterial flora in the gut, such animals are enabled to make use of
cellulose, which is first broken down by the bacteria to cellobiose.
7. CeIJobiases. Also ,B-glycosidases, hydrolysing cellobiose to glucose. Enzymes of this kind are also confined to bacteria and invertebrates, and do not appear to occur in the higher animals. 1
.
As an aide-memoire Table II may be useful. For all practical
purposes the ac:ion of these hydrolytic enzymes, considered as
digestive enzymes, is irreversible; in this respect they are in sharp
contrast to the intracellular phosphorylases mentioned in the previous
chapter.
1 The further fate of glucose obtained from cellulose by these bacterial enzymes
does not now concern us; much of it is converted by the bacteria jnto fatty acids,
which are then absorbed by the ruminant.
78
CARBOHYDRATE METABOLISM
Table II
Enzymes
ct-Amylases
Type
1 :4-ct-Glycosidases
Product
Substrate
Amyloses and amyloDextnns and
pectms (random
maltose
attack)
,B-Amylases
1: 4-ct-Glycosidases
Amyloses and amyloMaltose!
pectins (endwise
attack)
1: 6-ct-Glycosidases
1 : 6-0;-Li nks in.
R-enzymes
Maltose
amylopectins
Maltases
1 :4-ct-Glycosidases
Glucose
Maltose
"Z-enzymes
,B-Glycosldases
,B-Lmks in amyloses
Maltose
Cellulases
,B-G lycosldases
Cellulose
CellobIOse
CeJloblases
,B-Glycosldases
CellobIose
Glucose
1 And, from arnylopectins, what are known as •,B-limit dextrins', i.e. large
dextrins WIth the same number of branches as the origmal amylopectm. The
p-amylases cease to act near the branchmg point.
The hexokinase reaction
We have now arrived at glucose, the principal sugar concerned in
carbohydrate metabolism. Biochemically speaking, the glucose
molecule is rather inert, and although it diffuses easily through cell
walls, a cell will often require more sugar than can be supplied by
diffusion alone. The turning of glucose into a biochemically reactive
compound, and the transport of glucose into the cell, are among the
many functions of ATP. From the work of o. Meyerhof, H. von
Euler, T. Mann and C. Lutwak-Mann, C. F. and G. T. Cori, and
others, it has been found that a group of enzymes, called hexokinases, are widely distributed in living matter; they catalyse the
reaction
glucose+ATP --.. glucose-6-phosphate+ADP.
We have already seen (p. 72) how glucose-6-phosphate arises during
photosynthesis; we shall soon find that it occupies a central position
in the metabolism of carbohydrate within the cell. Meanwhile, a
word or two may be said about the bearing of the hexokinase reaction
on the transfer of glucose from the outside to the inside of cells,
according to a scheme proposed by M. Dixon.
Although glucose (G) is freely diffusible, glucose-6-phosphate
(G-6- ®) is not, neither is ATP (II). A cell furnished with hexokinase
(II) and ATP can therefore trap glucose molecules as fast as they
move inwards, by turniJ;lg them into glucose-6-phosphate which is
unable to escape. But the hexokinase reaction lowers the concen79
CARBOHYDRATE METABOLISM
tration of {ree glucose within the cell, hence more of the sugar passes
through the cell wall. to be trapped in its turn; and in this way A TP
performs osmotic work, effectively pulling in glucose from the external
environment. The ester link of glucose-6-phosphate is energy poor,
worth about 3000 cal./mole on hydrolysis. Since the terminal
energy-rich bond of ATP is worth about 8000 cal./mole, the standard
(outside)
G
f
G
~
G-6-®
(inSIde)
cell wall
(II)
free energy change for the hexokinase reaction, -/),FO, is about
5000 cal./mole, which means that the equilibrium is far to the right,
and the reverse reaction does not occur to an appreciable extent.
It must be borne in mind, however, that the transport of glucose
through some kinds of cell wall is a very complex process, ATP and
hexokinase being by no means the only factors involved_
Just as glucose-6-phosphate can be turned into starch by plants,
via glucose-I-phosphate, so in animals it can be transformed by the
same route into glycogen. In a series of studies from 1937 onwards
C. F. and O. T_ Cori showed that glycogen resembles amylopectin;
they were able to isolate from muscle, in addition to a phosphoglucomutase, both a 1: 4-phosphorylase and a 1: 6-phosphorylase,
the latter (like the Q-enzymes of plants) responsible for branching
the chains. Thus in animal cells we have the following relationships:
ATP
glucose
phosphophosglucose-6- ~ glucose-l- ~
gJycogen+
hexo- phosphate gluco- phosphate phorylases inorganic
kinase
mutase
phosphate
-+-
The hexokinase reaction not only draws glucose into the cell, but,
by turning it into a more reactive compound (glucose-6-phosphate), .
enables a storage mechanism to come into play. In the ordinary
way of things, an animal cell will usually have a reserve of polysaccharide, in the insoluble form of glycogen, upon which it can
draw should the external supply of glucose be cut off.
Breakdown of glucose-6-phosphate
I
In both plants and animals the extraction of free energy can be
accomplished in two ways, one of which bears considerable likeness
80
BREAKDOWN OF GLUCOSE-6-PHOSPHATE
to the dark reactions of photosynthesis,
but in reverse. From the
/
investigations of o. Warburg in)93l, and of F. Dickens in 1938,
which led to much subsequent work, this pathway is usually called
by their names. It is also sometimes known as the pentose phosphate
pathway.
The first step in the degradation of glucose-6-phosphate by the
Warburg-Dickens route is a dehydrogenation (III). This glucose6-phosphate dehydrogenase is TPN-linked, and the immediate product
0-
0
6·phosphogluconolactone
CH 0
®
OH
kt;
2
~H
H
H
OH
COOH
OH
6-phosphogluconic acid
am
of its action is a lactone, which then hydrolyses spontaneously to
6-phosphogluconic acid. At the next stage, 6-phosphogluconic dehydrogenase, also TPN-linked, produces ribulose-5-phosphate and
CO 2 (IV).
In sum, then,
glucose-6-phosphate + 2TPN + H 20-+
nbulose-5-phosphate+ 2TPNH2 + C02.
By oxidation of each TPNH 2 molecule through the flavoproteincytochrome system 3 molecules of ATP are produced (p. 60); hence
81
CARBOHYDRATE METABOLISM
6 molecules of ATP appear when the terminal carbon of glucose6-phosphate is removed as CO 2 ,
COOH
I
HCOH
CH 20H
to
-2H
HOtH
HtOH
I
HCOH
~
HtOH
I
HCOH
+C0 2
tH 2O®
tH 20®
6-pbospboglucomc acid
ribulose-5-pbospbate
(IV)
From this point, ribulose-S-phosphate regenerates glucose-6phosphate through a series of reactions,
6 pentose phosphate - 5 hexosephosphate,
which can be represented as a carbon flow-sheet (V). It will be
observed that this scheme is symmetrical in arrangement, and each
half can be divided into the following steps:
(a) C S+CS-C7 +C3 ;
(b) C 7 +C3 .... C4 +C 6 ;
(c) C 4 +Cs ....
C6+C3.
The C 3 (triosephosphate) molecules from each half then yield
another C 6 molecule.
Reaction (a) is preceded by two transformations: in the first,
a molecule of ribulose-S-phosphate is acted on by an epimerase to
form xylulose-S-phosphate (p. 69); in the second, another molecule
of ribulose-S-phosphate, under the influence of pentose phosphate
isomerase, becomes ribose-S-phosphate. The products then react
together (VI). The enzyme responsible is transketolase (p. 68), and
3-phosphoglyceraldehyde and sedoheptulose-7 -phosphate are formed.
Reaction (b) then takes place between these products, catalysed
by a transaldolase (VII). In this reaction an aldol condensation has
taken place, but at the same time CH 2 0H. CO. CHOH- and a
hydrogen from the sedoheptulose-7-phosphate have been transferred
to 3-phosphoglyceraldehyde, yielding fructose-6-phosphate; hence
the name transaldolase for the enzyme. The fate of the fructose6-phosphate will appear presently.
82
BREAKDOWN OF GLUCOSE-6-PHOSPHATE
Reaction Cc) is also preceded by the conversion of a molecule of
ribulose-5-phosphate to xylulose-5-phosphate, whictl then reacts
,/
with erythrose-4-phosphate:
3-phosphoglyceraldehyde
xylulose-5-phosphate
+
erythrose-4-phosphate
""
+
fructose-6-phosphate
C6
(V)
This reaction has already been discussed on pp. 68-9; the enzyme is
a transketolase. Thus the reactions of both halves of the flow-sheet
add up to:
6 pentose phosphate
"'" 4 fructose-6-phosphate
+ 2 3-phosphoglyceraldehyde
83
CARBOHYDRATE METABOLISM
CHzO®
HboH
HOtH
to
3-phosphoglyceraldehyde
t H20H
+
CHzOH
xylulose-5-phosphate
toI
+
HOCH
CHO
I
HCOH
I
HCOH
I
HCOH
HtOH
HtOH
HboH
I
CHzO®
tHzO®
ribose-5-phosphate
sedoheptulose-7-phosphate
(VI)
CHzO®
CHzO®
I
HCOH
HtOH
I
HCOH
HtOH
HtOH
tHO
HOtH
erythrose-4-phosphate
to
~
+
CH20H
to
t H20H
+
CHO
HOtH
HtOH
HtOH
I
HCOH
tH 0 ®
2
(YIn
tHzO®
fructose-6-phosphate
As to the fructose-6-phosphate, phosphohexo-isomerase converts it
to glucose-6-phosphate (p. 72). Now triosephosphate isomerase
(p. 67) establishes a triosephosphate pool, from which fructoseI : 6-diphosphate can be formed. The latter, by the action of a
specific phosphatase, loses its phosphate at position 1, and the fifth
84
Cs®
Cs®
C s®
!l
tE
Cs®
Cs®
Cs®
tl
!E
tE
tE
Cs ®
Cs ®
Cs ®
C s®
C s®
C s®
(xylulose)
(nbose)
(xylulose)
(xylulose)
(ribose)
(xylulose)
TK
TK
(C2l
(C 2l
C 3®
C7 ®
C7 ®
all rIbulose
C 3®
~TA
TA1
[C 3l
[C 3l
C4®
C4 ®
TK
TK
[C 2l
[Cll
C 3®
C 3®
Y
pF®®OH
C6 ®
C6 ®
II II
c6 ®
c6 ®
c6 ®
c6®
II
II
c6 ®
C6®
(VIII)
ExplanatIOn of symbols
TK = transketolase
I = isomerase
T A = transaldolase
E=epimerase
A=aldolase
P = phosphatase
85
C 6®
all fructose
II
c6 ®
all glucose
CARBOHYDRA TE METABOLISM
molecule of fructose-6-phosphate is isomerized to glucose-6phosphate.
The full carbon flow-sheet now becomes that shown in (VIII).
Writing the whole process in the form of equations, we have:
(I)
(ii)
6 glucose-6-phosphate+ 12TPN + 6H20 ......
6 nbulose-5-phosphate+ 12TPNH2 + 6C02.
6 ribulose-5-phosphate ->- 5 glucose-6-phosphate+ I®OH.
And in sum
(iii)
glucosc-6-phosphate+ 12TPN +6H20
->-
6C0 2+ 12TPNH2+ l®OH.
The complete oxidation of a glucose molecule by the WarburgDickens pathway therefore produces 12 molecules of TPNH2 ,
equivalent to 36 molecules of ATP. But a molecule of ATP was
consumed in the hexokinase reaction, by which the glucose was
originally phosphorylated. Hence the oxidation of glucose in the
cell has given a net yield of 35 molecules of ATP, roughly equivalent
to 35 x 8000 = 280,000 cal. of free energy available for chemical
work in the cell. This figure represents about 40 % of the total free
energy content of a glucose molecule-the balance being lost as heat
and in entropy changes.
Breakdown of glucose-6-pbospbate II
The second main pathway of glucose-6-phosphate breakdown, owing
muchJQJh~ioneer work of G. Embden and of o. Meyerhof in the
nineteen-thirties,is usually named after them. Whereas the WarburgDickens route depends on a supply of oxygen to the cell, the
Embden-Meyerhof route has the advantage that a portion of the
free energy can be tapped off even in the absence of oxygen. The first
stages are shown in (IX). Fructose-6-phosphate, formed by the
action of phosphohexo-isomerase (p. 72), is attacked by a phosphokinase and ATP, the product being fructose-I: 6-diphosphate, a
reaction which is not reversible to any extent. Thus, starting from
the glucose originally entering the cell, two molecules of ATP have
been consumed in the manufacture of fructose-I: 6-diphospate, which
then under the influence of aldolase is converted to triosephosphate
(X). The equilibrium of this system is determined by triosephosphate
isomerase, and has already been discussed (p. 67). In subsequent
86
BREAKDOWN OF GLUCOSE-6-PHOSPHATE
reactions of the Embden-Meyerhof pathway only the 3-phosphoglyceraldehyde is directly concerned, but as fast as it is removed the
isomerase regenerates a further supply from dihydroxyacetone
phosphate, so that in the end all six carbons of the original glucose
molecule enter into the process.
ATP+]
[ glucose hexokinase~
glucose-6-phosphate
1L isomerase
fructose-6-phosphate
t A TP + phosphokinase
fructose-l . 6-dlphosphate
1l aldolase
triosephosphate
(IX)
CHzO®
CHzO®
1
HCOH
60
·1
1
CHO
CHzOH
dlhydroxyacetone phosphate
3-phosphoglyceraldehyde
(X)
The next step-oxidation of 3-phosphoglyceraldehyde to 3-phosphoglyceric acid-is conducted by a DPN-linked triosephosphate
dehydrogenase. This step is of great interest for a variety of reasons;
we have already seen it working in reverse (p. 67), and the mechanism
must now be considered in detail.
The active centre of triosephosphate dehydrogenase appears to
contain an -SH group, and in 1951 E. Racker proposed the folH
lowing reaction scheme:
(i)
CHzO®.CHOH.CHO+HS®
~
1
CHzO®.CHOH.C-S-®
(where HS® represents the enzyme)
+ H20
6H
H
eli) CHzO®.CHOH.Ls-®
1
+DPN
OH
~
CHzO®.CHOH.C~S-®
~
(iii)
CH20®.CHOH.C~S-®
+ DPNH2
II
o
CHzO®.CHOH
II
0
C-O~®
II
o
1 : 3-diphosphoglycenc acid
+HS®
(iv)
CH20®.CHOH.C-O~®
+ADP
~
II
CHzO®. CHOH. COOH
3-phosphoglyceric acid
+ATP
0
87
CARBOHYDRATE METABOLISM
[n the first reaction, 3-phosphoglyceraldehyde combines with the
-SH group at the active centre, forming a compound with the
enzyme through an energy-poor -C-S- linkage. Then, (ii) , the
enzyme-substrate compound is dehydrogenated, the hydrogens
passing to DPN, and at the same time an energy-rich acyl mercaptide bond is formed (cf. p. 16). At stage (iii) a reaction with
inorganic phosphate regenerates the enzyme, and produces I: 3diphosphoglyceric acid, with an energy-rich bond of the acyl
phosphate type (p. 15). Last of all, (iv), a phosphoglyceric phosphokinase brings about the transfer of '" ® to ADP, yielding ATP.
Thus the oxidation of 3-phosphoglyceraldehyde is coupled with
ATP synthesis-a fact which was first recognized in 1937 by D. M.
Needham and by O. Meyerhof, and has led to all subsequent work
on oxidative phosphorylation. Such a process is known as oxidative
phosphorylation at the substrate level, in order to distinguish it from
respiratory chain phosphorylation (p. 60). And the difference
between these types of reaction amounts to this: in the former,
reduction of a pyridine nucleotide is coupled with ATP synthesis, in
the latter A TP arises only by the oxidation of reduced pyridine
nucleotide through the flavoprotein-cytochrome pathway.
3 CH20®
I
2CHOH
I
1 COOH
3- phosphoglyceric
aCid
3 CH20H
oe===
I
2CHO®
I
1 COOH
2-phosphoglyceric
acid
CH2
-HzO
~
+HzO
II
C-O~®
I
COOH
phosphoenolpyruvIc
acid
(xD
We must now pursue 3-phosphoglyceric acid to its destination
(XI). A phosphoglyceromutase accomplishes the transfer of phosphate from position 3 to position 2 of the glyceric acid. The product,
2-phosphoglyceric acid, contains an energy-poor ester bond, but
under the influence of an enzyme called enolase (which needs Mg++
as co-enzyme) loses water to form phosphoenolpyruvic acid. By this
reaction an energy-rich phosphate bond is formed, worth about
12,000 cal./mole. It is interesting to notice that whereas the total
free energy content of 2-phosphoglyceric and phosphoenolpyruvic
acids is nearly the same, by removal of water the available free energy
which can be tapped off for chemical work is greatly increased.
88
BREAKDOWN OF GLUCOSE-6-PHOSPHATE
Phosphoenolpyruvic acid then reacts with ADP, in the presence of
a phosphokinase, to give ATP and pyruvic acid (XII). This type of
transfer reaction is reversible, and the rearrangement of the enol to the
keto form of pyruvic acid goes on spontaneously, requiring no enzyme.
CHz
CH2
II
C-O-®+ADP
II
;=='
.
ATP+C-OH
;=='
tOOH
tOOH
(XIn
enol form
keto form
pyruvic acid
Starting with glucose, the eight steps leading to pyruvic acid can
be briefly summarized:
(i) glucose+ATP -+ glucose-6-phosphate+ADP,
(ii) glucose-6-phosphate .= fructose-6-phosphate,
(Iii) fructose-6-phosphate+ATP -+ fructose-l :6-diphosphate+ADP,
(IV) fructose-l : 6-diphosphate .= 2 3-phosphoglyceraJdehyde,'
(v) 2 3-phosphogJyceraldehyde+ 2DPN + 2ADP+ 2®OH .=
2 3-phosphogJycenc acid+2DPNHz+2ATP+2H20,
(VI) 2 3-phosphoglyceric aCId .= 2 2-phosphogJyceric acid,
- 2H20
(vii) 2 2-phosphoglyceric acid
2 phosphoenolpyruvic acid,
+ 2H 20
(viii) 2 phosphoenolpyruvic acid+2ADP .= 2 pyruvic acid+2ATP.
1 The dihydroxyacetone phosphate formed by aldolase being converted to
3-phosphoglyceraldehyde as fast as It is requIred.
-===
And in all,
glucose+2DPN+2ADP+2®OH -+ 2 pyruvicacid+2DPNH z +2ATP+2H20.
The two molecules of ATP used up in reactions (i) and (iii) are
recovered in the triosephosphate dehydrogenase reaction (v), two
more are formed by reaction (viii). Hence there has been a net gain
of 2 ATP molecules in the procedure, irrespective of any A TP that
could arise from the oxidation of DPNH2 through the flavoproteincytochrome chain.
It often happens that a cell is deprived of molecular oxygenperhaps for a fraction of a second, perhaps for a matter of hours or
even days. To take an example, in animals a sudden jump or burst
of speed calls for more oxygen than the circulation can immediately
supply, or again, organisms like yeast may find themselves in an
anaerobic environment for considerable periods of time. Under such
4
89
HB
,
CARBOHYDRATE METABOLISM
circumstances the flavoprotein-cytochrome chain is blocked, and
reduced pyridine nucleotide cannot be oxidized-unless some other
means are found. In muscle and other tissues, when oxygen is short,
lactic dehydrogenase provides the answer (XIII). Adding this reaction
to the over-all glucose-pyruvic acid equation
glucose+2DPN+2ADP+2®OH
-+
2 pyruvic acid + 2DPNH2
~
2 pyruvic acid+ 2DPNH 2
+2ATP+2H 2 0
21actlc acid+2DPN,
we have
glucose+2ADP+2®OH
CH 3
2 lactic acid + 2ATP+ 2H20.
CH3
toI
tHOR
I
COOH
pyruvic acid
+DPN
COOH
lactic acid
(XIII)
Thus, in the absence of oxygen, glucose (or glycogen) can be broken
down to yield a small supply of ATP; the process is called anaerobic
glycolysis, and is in the nature of a temporary expedient, enabling
the cell to tide over awkward moments. The formation of lactic acid
is undesirable in itself, as leading to changes in pH, but the blood
of animals acts as a buffer to lactic acid diffusing into it, and within
limits the pH can be kept fairly constant. In yeast, however, the
pH problem is disposed of in another way (XIV). Two enzymes are
CH 3
I
carboxylase
CO
l~OOiH
+
DPNH 2
I .......... J
pyruvic acid
DPN
acetaldehyde
ethyl alcohol
(XIV)
concerned in this process. Carboxylase, which was discovered by
C. Neuberg in 1911. requires Mg++ and thiamine pyrophosphate
(p. 91) as co-enzymes, and its action is virtually irreversible. The
second enzyme, alcohol dehydrogenase, is here working in reverse.
Anaerobic breakdown of glucose to alcohol is called fermentation,
and that word has been extended to cover a great variety of anaerobic
reactions conducted by micro-organisms.
90
CARBOHYDRATE METABOLISM
Decarboxylation of pyruvic acid
Most creatures are provided with a plentiful supply of oxygen for
most of the time, and we must now follow the fate of pyruvic acid
under aerobic conditions. Briefly, what happens at first is this:
CH 3 ·CO 1COO lH.
The carboxyl group is decomposed to CO 2 and hydrogen (which
reduces DPN), and the remaining acetyl group, CH 3 . CO-, becomes
attached to a substance called co-enzyme A. This compound, discovered by F. Lipmann in 1950, and whose structure was worked out
by J. Baddiley soon afterwards, is an adenine nucleotide; its full
constitution is given in the Appendix (p. 139); but we are now
interested only in the -SH group that the molecule contains, and
therefore symbolize it ®SH.
In the nineteen-twenties Sir R. A. Peters had observed that
pyruvate accumulates in the blood of animals and birds suffering
from vitamin Bl deficiency; the vitamin itself, known as thiamine
(or aneurin) was synthesized in 1936 by R. R. Williams; its complicated structure is given in the Appendix (p. 142), and for short
we can write R-CH2. CH2 0H. A year later K. Lohmann and
P. Schuster showed that the co-enzyme of yeast carboxylase is the
pyrophosphate of vitamin Bl, presumably formed by reaction with
ATP:
R.CH 2 .CH20H+ATP
-')0
R.CH 2 .CH2.O-®,..., ®+AMP.
Thiamine pyrophosphate, written TPP, acts also as a co-enzyme for
pyruvate decarboxylation in vertebrates; lipoic acid is thought to be
involved too; and the process falls into four stages:
(i) CH 3 . CO. COOH + TPP -+ ' acetaldehyde' - TPP
complex + CO2 .
The complex contains the elements of acetaldehyde united in some
way with TPP; and the reaction appears to be irreversible.
(ii) Then the complex is decomposed by lipoic acid (XV). The
product contains an acyl mercaptide type of energy-rich bond, and,
it will be observed, the elements of acetaldehyde.
(iii) Next, a transfer reaction takes place (XVI). The destination
of acetyl co-enzyme A will be discussed in a moment.
91
CARBOHYDRATE METABOLISM
(iv) Meanwhile, reaction (XVII) proceeds. Hence pyruvic acid
has been oxidized (or rather, dehydrogenated) as well as decarboxylated, and the process, known as oxidative decarboxylation, can
be summarized
CH3. CO. COOH + HS@+DPN -+ CH3. CO ~ S@+DPNH2+ co •.
Complex
+
rTR
5--5
lipOIC acid
TPP
('YR
HS@
SH
SH
reduced lipoic acid
acetyl lipoic acid
+CH 3 ·CO-S®
(acetyl co-enzyme A)
(XVI)
(y
SH
R
SH
+DPN
(YR
--
+DPNHz
S-S
reduced lipOiC aCId
lipoic acid
(XVII)
Oxidative decarboxylation is an irreversible reaction since the decomposition of the TPP complex is virtually irreversible, and this fact,
we shall see, is of great importance in the metabolism of certain
organisms.
The citric acid cycle
The further breakdown of acetyl co-enzyme A takes place through
the citric acid cycle-or tricarboxylic acid cycle, as it is also calledwhich was first described in 1937 by Sir H. A. Krebs after much
92
CITRIC ACID CYCLE
experimental work by himself, A. von Szent-Gyorgyi, C. Martius,
F. Knoop, and others. This series of reactions provides a common
pathway for the final breakdown of fat and protein, besides carbohydrate; it also acts as a pool of metabolic intermediates-a kind of
market, or clearing house, in which enzymes dispose of their products
or get their appropriate substrates. With these wider aspects of
the citric acid cycle we are not for the moment concerned. In
terms of carbon, the citric acid cycle (XVIII) involves the reaction
of a C2 unit (the acetyl group attached to co-enzyme A) with a C4
unit (oxalacetic acid) to make a C 6 unit (citric acid), which then
undergoes various transformations, forming two CO 2 molecules and,
eventually, oxalacetic acid. Thus 'acetyl', CH 3 .CO-, is fed into the
cycle, and two molecules of CO 2 emerge.
acetyl co-enzyme A
cltnC__"lsocltrlc~oxaloSUCClmc
(C2)
(C6)
~C02
a-ketoglutanc
(C S )
}--c~
oxalacetlc_ fumanc __- - - - succinic
(C4 )
(C4 )
(xv
(C4)
1m
CH3 CO",S®
CH2.COOH
+
CO.COOH
HO.t.COOH
I
+HS@
I
CH 2COOH
citric acid
CH2COOH
oxalacetic acid
(XIX)
The first step is carried out by the 'condensing enzyme' (XIX).
It will be observed that' acetyl' becomes attached through its methyl
group to the oxalacetic acid. The reaction is reversible, but is driven
to the right by the energy-rich acyl mercaptide bond, - !1FO being
about 8000 cal.Jmole for the process.
Next, citric acid is isomerized to isocitric acid by the enzyme
aconitase, cisaconitic acid being the intermediate (XX). Aconitase
93
CARBOHYDRATE METABOLISM
acts, in effect, by exchanging -R and -OR groups in the positions
shown. Isocitric acid, like lactic, contains the grouping
-CHOH .COOH.
Whenever we find an ~-hydroxy acid entering into a metabolic
pattern we may be fairly sure that an enzyme exists to dehydrogenate
it and in this case the TPN-linked isocitric dehydrogenase is concerned (XXI). The product, oxalosuccinic acid, is both an ~-keto
acid and a fJ-keto acid, and, in accordance with the principles of
CHz.COOH
I
HO.C.COOH
I
CH2.COOH
citric acid
CH)COOH
-HzO
~
+HzO
tII COOH
+H 2 O
---'"
~
CHz COOH
I
HC.COOH
-HzO
CH.COOH
cisacorutlc acid
tHOH COOH
isocitnc acid
(XX)
organic chemistry, we should expect that the carbo1(yl group in the
fJ-position would easily lose CO 2 • Oxalosuccinic acid in the test-tube
does behave in this way; in cells the process is hastened not hy a
separate decarboxylase but, as shown by S. Ochoa and by M. Dixon
and J. Moyle, through the mediation of the isocitric dehydrogenase
itself. In other words, the isocitric enzyme has a double function,
being both a dehydrogenase and a decarboxylase (for the latter
purpose requiring Mn++ as co-enzyme).
CH2. COOH
CHz·COOH
I
H.C.COOH
I
+TPN
H LCOOH
CHOH COOH
iSOCItriC acid
to.COOH
oxalosucciruc acid
(XXD
CH2.COOH
CH2.COOH
I
H.C.COOH
I
tH2
boa-ketoglutaric
COOH
acid
CO.COOH
oxalosuccinic acid
(XXID
The products are a-ketoglutaric acid and a molecule of CO 2 (XXII).
The overall reaction
isocitric + TPN
~
a-ketoglutaric acid + TPNHz + COz
is to some extent rl!versible, though the equilibrium lies far to the
right.
.
94
CITRIC ACID CYCLE
When a-ketoglutaric acid is decomposed it undergoes an irreversible oxidative decarboxylation like that of pyruvic acid (p. 91);
thia_!lline pyrophosphate (TPP), lipoic acid, DPN, and co-enzyme A
take part. The final result is shown in (XXIII). The product, succinyl
co-enzyme A, now reacts with ADP in the presence of inorganic
phosphate (XXIV). This reversible reaction, coupled with the previous one, furnishes another example of oxidative phosphorylation
at the substrate level (p. 88).
CHz.COOH
tHz
CHz COOH
I
+HS@+DPN
CH z CO-S@
succmyl co-enzyme A
+DPNH 2 +CO Z
I
CO.COOH
a-ketoglutaric acid
(XXIII)
CHz.COOH
I
succinyl
co-enzyme A
(XXIV)
CH2 COOH
succimc acid
+HS@+ATP
The metalloflavoprotein, SUCCinIC dehydrogenase (p. 52), next
produces fumaric acid (XXV), and this compound is acted on by
fumarase to form malic acid (XXVI).
Finally the DPN-linked malic dehydrogenase yields oxalacetic
acid (XXVII). The malic dehydrogenase from some tissues can use
TPN also.
CH2COOH
I
CH.COOH
II
CH COOH
fumaric acid
+FP
CH2 .COOH
succinic acid
(XXV)
CH.COOH
II
CH.COOH
fumaric acid
CHOH.COOH
+H20
tHzCOOH
malic acid
(XXVI)
CHOH.COOH
CO.COOH
tH2 COOH
+DPN
tH2COOH
malic acid
+ DPNHz
oxalacetic acid
(XXVII)
This scheme proposed by Krebs has been confirmed by isotope
studies, using 13C or I4C as tracers, and the citric acid cycle appears
to operate in most types of cell, though not perhaps in all. The cycle
95
CARBOHYDRATE METABOLISM
is in any case a good example of how a chain of reactions, controlled
and directed by enzymes. can produce a supply of free energy in a
biologically useful form; and to make this point clear we must revert
back to pyruvic acid and follow its fate by means of equations,
paying particular attention to hydrogen transfer:
(i)
(ii)
(iii)
(iv)
(v)
(vi)
(vii)
(viii)
(ix)
CH3 CO COOB+HS@ ...... CB3 CO~S@+COz +2Hto DPN,
CH3 CO~S@+oxalacetic acid ...... citric acid+HS@,
citric acid ...... cisaconitic acid ...... isocitric acid,
isocitric acid ...... oxalosuccinic acid +2H to TPN,
oxalosuccinic acid ...... et-ketoglutaric acid+CO z ,
et-ketoglutaric acid + ADP + ®OH + H20 ...... succinic acid + COz
+ATP +2H to DPN,
succinic acid ...... fumaric acid + 2H to FP,
fumaric acid+HzO ...... malic acid,
malic acid ...... oxalacetic acid +2H to DPN.
Remembering that 10 hydrogens are equivalent to 50xygens,
CH3. CO COOH
+2-!-02
......
3COz + 2HzO.
And the yield of ATP from the complete oxidation of pyruvic acid
is made up as shown in Table III. Thus 3 molecules of ATP arise
from the oxidative decarboxylation of a molecule of pyruvic acid,
and 12 from the operation of the citric acid cycle, making 15 in all.
Table III
(i)
(iv)
(vi)
(vii)
(ix)
Reaction
oxidative decarboxylation of
pyruvic
isocitric ...... oxalosuccinic
oxidative decarboxylation of
et-ketogl utaric
plus ATP synthesis from
ADP+inorgaruc phosphate
succinic -+ fumaric
malic ...... oxalacetic
Hydrogen
carrier
DPNH2
ratio
3
ATP
synthesized
3
TPNH2
DPNH z
3
3
3
3
FPHz
DPNHz
2
3
2
P/O
3
Total = 15
Glucose as a source of energy
By complete oxidation of a glucose molecule through the WarburgDickens pathway, the synthesis of 35 molecules of A TP is achieved
(p. 86). This route, however, can operate as a source of ATP only
96
GLUCOSE AS A SOURCE OF ENERGY
when the cell is freely supplied with oxygen, whereas the EmbdenMeyerhof pathway is more versatile (see Table IV). Under aerobic
conditions there is little to choose between the Warburg-Dickens
and Embden-Meyerhof pathways, so far as ATP synthesis is concerned. And the figures also show that the yield of A TP by glycolysis
or fermentation is relatively small. Although both pathways seem
to operate in most, if not all, kinds of cell, and the Warburg-Dickens
scheme provides for the manufacture of pentoses from hexoses, the
Embden-Meyerhof would appear to be the more important of the
two, not only because it can yield a little energy under anaerobic
conditions, but also because it enables glucose to be made from
simpler molecules-a point to which we must now attend.
Table IV
ATP moles/mole
of glucose
Anaerobic
glucose -->- lactic acid (or ethyl alcohol)
2
Total =2
Aerobic
glucose -->- 2 pyruvic acid
plus oxidation of 2DPNH2 (from triosephosphate
dehydrogenase reaction, p. 87)
decarboxylation
2 pyruvic
C02 + H20 (2x 15)
+ CitrIC acid cycle
2
6
=30
Total = 38
Glucose synthesis
In the Embden-Meyerhof scheme two reactions are virtually
irreversible:
glucose
ATP+
glucose-6-phosphate
hexokinase
ATP+
fructose-6-phosphate
fructose-I: 6-diphospha te
phosphokinase
These reactions, driven by A TP from left to right, might seem to
preclude the possibility of glucose being made from pyruvic acid.
But all cells possess phosphatases which can hydrolyse hexose-6-phosphates; the phosphatase in plants that hydrolyses fructose-6-phosphate has already been referred to (p. 68). Since all the other steps on
the Embden-Meyerhof route, including the action of triosephosphate
97
CARBOHYDRATE METABOLISM
dehydrogenase, are reversible, through the auxiliary action of these
phosphatases pyruvic acid and glucose are interconvertible:
Embden-Meyerhof enzymes
glucose
pyruvic acid.
The same, plus specIfic phosphatases
In the leaves of plants this reverse reaction is probably of little
importance. because photosynthesis provides an ample supply of
carbohydrate, and the seedling can draw on its starch reserves. But
animals and micro-organisms often go hungry, and it may sometimes be necessary to supplement a meagre supply of glucose from
other sources of food, the oxidation of which simultaneously furnishes ATP for synthetic purposes. Two reactions are of great
consequence here. The first was discovered in 1938 by H. G. Wood
and C. H. Werkman, and takes place in micro-organisms and possibly in plants and animals as well (XXVIII). (The reaction is rather
CO.COOH
I
CH2.COOH
oxalacetic acid
~
CH3.CO.COOH+C02
(XXVIID
pyruvic acid
more complicated than is apparent from the equation shown in
XXVIII) Enzymes catalysing the decomposition of oxalacetic acid
are called oxalacetic decarboxylases; the reverse reaction involves
CO 2 fixation, for which ATP must be supplied. The second route to
pyruvic acid was found to occur in animal ti.ssues by S. Ochoa in
1948 (XXIX). The catalyst is a combined dehydrogenase and decarboxylase, usually known as the 'malic enzyme'; it requires Mn++
as co-enzyme, and apparently oxalacetic does not occur as an intermediary-so this enzyme must be carefully distinguished from malic
dehydrogenase.
CHOH.COOH
I
CH2·COOH
mahc acid
+TPN
CH3. CO. COOH + C02 + TPNH2
(XXIX)
pyruvic acid
Thus through the Wood-Werkman reaction or the Ochoa reaction
the manufacture of pyruvate, and consequently of glucose, can be
accomplished from any substance which is capable of being converted into oxalacetic or malic acids. In the next chapter the
significance of these conversions will be discussed at more length,
and in relation to the metabolism of fat.
98
VII
FAT METABOLISM
What we call' fat' in everyday speech is chiefly made up of glycerides
-esters of glycerol with fatty acids, having the general formula (I).
Such compounds are known as 'simple lipids', the term lipid being
applied to a variety of substances which, in general, are sparingly
soluble in water but readily soluble in most organic solvents. Among
the more complex lipids one group deserves to be singled out-the
phospholipids, or phosphatides, which contain phosphate and a
nitrogenous base in addition to fatty acids (II). These phosphatides
enter into the composition of cell membranes and similar structures,
and are important constituents of the blood plasma in animals, but
we must not stop here to consider them, nor the ways and means
by which lipids of all kinds are transported into and out of cells.
CH 2 .O.CO.Rl
tH O.CO.R2
I
CH2.0.CO.R3
<n
CH2.0.CO.Rl
tH.O.CO.R2
I
~
CH2.O-P-O-base
6H
on
The natural fats, whether of animal or vegetable origin, are mixtures of triglycerides, and may contain fatty acids ranging from C4
to C 30 or more; but the acids of widest distribution are:
C I6 CH3 (CH Z)14 COOH palmitic,
CIS CH3 (CH Z)16 COOH steanc,
CIS CH3.(CHzh.CH.CH.(CH2h COOH oleic.
These three compounds may together account for something like
90 % of the fatty acids present in triglycerides; palmitic acid is of
99
FAT METABOLISM
almost universal occurrence, stearic acid is seldom absent. Oleic
acid, with a double bond between carbons 9 and 10 in the chain
(symbolized 6,9), is the most abundant representative of the unsaturated acids. In general, fats containing a high proportion of
unsaturated material are liquid at room temperature, and are then
called oils. Nearly all the fatty acids of natural occurrence possess
an even number of carbons; the only exception of any importance
to this rule is propionic acid, CH 3 . CH 2 . COOH.
Triglycerides are easily hydrolysed by a group of esterases, the
lipases (III). Under the conditions existing in cells, the equilibrium
CH 2.0.CO.Rl
CH20H
tH.O.CO.R2+3H20
~2.0.CO.R3
-===
tHOH
+Rl.COOH+RZ.COOH
I
CH20H
glycerol
+R3.COOH
(Ill)
~
CHzOH
tHOH
CH20H
~
bo
~20®
tHzO®
a-glycerophosphate
""
glucose
dihydroxyacetone
phosphate
(IV)
of this reaction lies to the right. When a fat is hydrolysed, the glycerol
moiety is converted to sugar (IV), and the free fatty acids liberated
by hydrolysis with lipase are then available for oxidation. The early
work of F. Knoop in 1904, followed by G. Embden, E. Friedmann,
H. D. Dakin, and others, proved that fatty acid chains are normally
broken down two carbons at a time, and because the acids were
supposed to be first oxidized at the carbon atom fJ to the carboxyl
group the process was known as fJ-oxidation. Taking the six-carbon
hexanoic (caproic) acid as an example (V), the long-chain acids
fJ
IX
CH3.CHZ CHz.CHz.CH2 COOH
.j.
CH 3 CH 2 CH 2 co lCH 2 .COOH
fJ-keto-hexanoic acid
.j.
CH 3 CO CH 2 COOH+oxidation products
acetoacetIc acid
.j.
oxidation products
(V)
100
FAT METABOLISM
were supposed to undergo a similar stepwise cleavage to acetoacetic
acid, and this scheme found support from the fact that acetoacetic
acid and its reduction product ,8-hydroxybutyric acid
CH3. CHOH. CH2. COOH
do occur in traces in the blood of normal animals, and in larger
quantity during carbohydrate starvation and in such disturbances of
metabolism as diabetes. 1
Since 1950 our knOWledge of fatty acid oxidation bas been considerably extended by F. Lynen, F. Lipmann, D. E. Green, A. L.
Lehninger and other workers. For simplicity we follow the brea~­
down of hexanoic acid, though it is not of common occurrence.
Lynen discovered that the first step requires ATP and co-enzyme A:
CH3.CH2.CH2.CH2.CH2 COOH+HS@+ATP -+
CH3.CH2 CH2 CH2 CH2.CO-S@+AMP (adenosine monophosphate)
acyl (hexanoyl) co-enzyme A
+ PP (pyrophosphate)
The enzymes responsible are called thiokinases;2 some being specific
to short-chain acids, and others to the medium or long-chain forms.
Probably the enzyme reacts with ATP, yielding an enzyme-AMP
compound with an energy-rich bond:
®+ATP .= ®-AMP+PP.
enzyme
Mg++ ions are required for this process. Then reaction with coenzyme A takes place:
®-AMP+HS@ .= ®-S@+AMP.
followed by
®-S@+R.COOH
fatty acid
R.CO-S@+®.
acyl co-enzyme A
The next step is a dehydrogenation of the acyl co-enzyme A, between
the IX- and jl-carbons, catalysed by an acyl dehydrogenase. These
acyl dehydrogenases are fiavoproteins, and vary in specificity like
the thiokinases:
CH3.CH2·CH2.CH2.CH2.CO-S@+FP .=
CH3 CH2.CH2.CH:CH CO-S@+FPH2
ct:p-unsaturated acyl co-enzyme A
1 In some animals, notably the rabbit, fatty acids of medium chain length
(8 to 12 carbons) can also be oxidized at the terminal methyl group, e.g.
CH3 (CH2)S COOH _,. COOH (CH2)s COOH;
the process is known as w-oxidation, but seems to be of little ~i gnificance.
2 The termmology here adopted for these enzymes was put forward by a group
of workers in 1956, but has not yet come into general acceptance.
101
FAT METABOLISM
To the unsaturated compound are now added the elements of water,
under the influence of enoyl hydrase, which in action is similar to
fumarase:
CH3·CH2.CH2.CH:CH.CO-S@+H20 .=
CH 3 CH2.CH 2.CHOH.CH2.CO-S@
,B-hydroxyacyl co-enzyme A
This product is now attacked by a DPN-linked p-hydroxyacyl
dehydrogenase:
CH3.CH2.CH2.CHOH.CH2 CO~S@+DPN .=
CH3 CH z CH2 CO CH2
fI-keto-acyl co-enzyme A
CO~S@+DPNH2
Finally, p-keto-acyl co-enzyme A is made to react with another
molecule of co-enzyme A, through the influence of P-kcto thiolase:
CH3. CH2. CH2. CO. 'CH2. CO - S@
+:
;c===
CH3.CO-S@+CH3.CH2.CH2.CO-S@
@SiH
I
acyl (butyryl) co-enzyme A
Thus a molecule of acetyl co-enzyme A has been produced, shortening
the fatty acid chain (in this case 6 carbons long) by two carbons:
and at the same time the 4-carbon residue (butyryl co-enzyme A) is
in a position to go through the series of reactions again-dehydrogenation, addition of water, dehydrogenation, and cleavage (VI).
CH3.CH2.CH2.CO-S@
1~
CH 3·CH:CH.CO-S®
1~
CH3.CHOH.CH2.CO~S®
1~
CH3. CO. CH2. Co- S®
1~ +HS®
2CH3'CO-S®
(vD
No matter how long the carbon chain of a fatty acid, once it has
been' activated' by ATP and converted into an acyl co-enzyme A,
the enzymes break it down step by step to the final product, acetyl
co-enzyme A:
CH3.(CH2)n·COOH+ (tn+l) HS@ .= (tn+l) CH 3 .CO-S®,
where n is an even number.
It is instructive to compare the net yield of ATP from the complete
oxidation of the 6-carbon hexanoic acid and the 6-carbon sugar,
102
FAT METABOLISM
glucose. For the latter (p. 96) we have 35 molecules of ATP by the
Warburg-Dickens pathway, or 38 by the Embden-Meyerhof pathway. For the fatty acid, in the reaction
CHJ.(CH2)4.COOH+HS®
hexanoIc aCId
~
CHJ(CH2h.CO-S®+H20,
butyryl co-enzyme A
a molecule of FP is reduced to FPH z 7 and one of DPN to DPNH 2 •
On oxidation through the usual channels these substances yield
2 + 3 = 5 molecules of A TP; deducting 'the A TP used up in the
thiokinase reaction, the net yield is 3. 1 Then QY the changes
CH3.(CH2)Z.CO-S®+HS®
butyryl co-enzyme A
~
2CH 3·CO-S®
acetyl co-enzyme A
once again 5 molecules of ATP are produced; but thiokinase is not
involved, hence these 5 molecules are clear gain. The total for the
over-all process is thus 8 molecules. And when acetyl co-enzyme A
is oxidized through the citric acid cycle, 12 molecules of ATP are
produced (p. 96); from a molecule of hexanoic acid, therefore, the
total gain is 8 + (3 x 12) = 44 molecules ATP. Carbon for carbon,
then, the fatty acids can generate a little more energy than carbohydrates in a biologically useful form. But calculations of this kind
must be accepted with a certain amount of reserve, because doubtless
there are many cell reactions of which we are still ignorant, and in
real life the yield of A TP from various sources may not be quite the
same as it appears to be on paper. We may observe, however, that
one of the functions of A TP is to act as a • primer' for those reactions
which lead to its own synthesis; just as glucose is turned into glucose6-phosphate, so the relatively inert fatty acids are turned into reactive
acyl derivatives of co-enzyme A.
Metabolism of propionic acid
Although the overwhelming majority of fatty acids in nature contain
an even number of carbon atoms, acids with an odd number of
carbons do occur, and the step-wise breakdown of such acids, two
carbons at a time, will ultimately yield propionyl co-enzyme A (VII).
It has long been known that propionic acid can serve as a source
of glucose, and in 1955 S. Ochoa was able to isolate co-enzyme A
1 Not four, because in the thiokinase reaction two -® are lost from ATP
and in the synthesis of ATP from AMP these two - ® must be put back.
103
FAT METABOLISM
derivatives of methyl malonic and succinic acids as intermediaries
(VIII). In this curious reaction methyl malonyl co-enzyme A is
acting as a 'carrier' of the elements of carbon dioxide.
CH3·CHz·CH2·
CH 3
l.. CHz ·CH2· ..~ CHz CO~S® ~
CH2.CO~S®
+
2 acetyl co-enzyme A
propionyl co-enzyme A
(VII)
/co-s®
CH J .CH2 ·CO-S@ +C02 __",CH l -CH
~COOH
methyl malonyl co-enzyme A
~
+ proplOnyl
co-enzyme A
COOH
"T"lO
via
cltnc
cycle
elucoso
fH 2 COOH
deacylase
I
~H2
CH 2 COOH
~H2
SUCCinIC aCid
CO-S ®
+ co-enzyme A
sllccinyl co-enzyme A
+ proplOnyl co-enzyme A
(VIm
Interconversion of fat and carbohydrate
The steps leading from fatty acids to acetyl co-enzyme A are
reversible, as was demonstrated by G. Popjak in 1952. If acetic acid
is labelled with the 14C isotope in the carboxyl group and fed to
animals, it is converted, by the thiokinase reaction, to acetyl coenzyme A:
CH3.14COOH+HS@+ATP "'"
CH3.14CO~S@+AMP+PP
From the pool of labelled acetyl co-enzyme A, by reversal of the
p-keto thiolase reaction, acetoacetyl co-enzyme A is obtained,
labelled in the positIOns shown:
CH3 . 14CO . CHZ.14CO - S@
and by successive reductions and condensations, we have
CH) . 14CH z .CHz .14CH z . CHZ.14CHz. CHZ.14CO-S®
oetanoyl co-enzyme A
104
INTER CONVERSION WITH CARBOHYDRATES
Such other acyl derivatives, or free acids, as can be isolated in this
kind of experiment also possess the 14C label on alternate carbons.
The final stage of fat synthesis-formation of triglyceridesprobably occurs in sometrung like the way shown in (IX). It may
also be mentioned here that cells contain enzymes-the deacylaseswhich can hydrolyse acyl derivates to free acids and @SH (VnD.
Rl.CO~S®
CH 20H
tHOH
t
+
R2.CO~S®
CHz.O.CO,Rl
-+
R3·CO~S®
H 20H
tH.O.CO.R2
I
+
3®SH
CH2.0.CO R3
(IX)
carbohydrate
(glucose)
1~
pyruvlaCld
fat
(fatty aCIds)
oxalacetIc aCId
acetyl co-enzyme A_------'>----.,--./
(X)
With this reversibility offat breakdown in mind, and incorporating
the carbohydrate processes, diagram (X) can be drawn up. There are
two things to notice about this scheme-a fact and a consequence.
The fact is that, so far as we can judge, the oxidative decarboxylation
of pyruvic acid is irreversible. And the consequence is that whereas
carbohydrate can be turned into fat, fat cannot be turned into carbohydrate by this route. Yet plants, and certain micro-organisms, are
known to effect the latter change without difficulty. and the question
arises-how do plants manufacture carbohydrate from fat? In 1957,
Sir H. A. Krebs and H. L. Kornberg proposed a mechanism for the
process which is called the glyoxylic acid cycle. Acetyl co-enzyme A
reacts with oxalacetic acid to give, by the enzymes of the citric acid
cycle, isocitric acid:
CH3 CO-S®
+
-+
citric -+ cisaconitic -+ isocitric
oxalacetic
Then an enzyme, isocitritase, breaks up isocitric into succinic and
glyoxylic acids (Xl). This enzyme is a kind of aldolase and the reaction
is reversible. Another molecule of co-enzyme A now reacts with
105
FAT METABOLISM
glyoxylic acid, under the influence of the enzyme malic synthetase
(XII). Here the methyl group of acetyl co-enzyme A adds on to the
aldehyde group of glyoxylic acid, so the reaction is similar to that
brought about by the' condensing enzyme' (p. 93). And the cyclic
process may be represented by (XIII). Over-all, the reaction is
2 acetic + 0
->-
succinic + HzO.
Succinic acid being readily convertible to malic, oxalacetic and
thence to pyruvic, by the glyoxylic and citric acid cycles working in
conjunctIon the conversion of fat to carbohydrates is readily achieved.
CH2.COOH
CHz.COOH
succinic
I
H.C COOH
tHz.COOH
I
CRQ
CHOH.COOH
isocitric
+
glyoxylic
tOOH
(XI)
COOH.CHO+CHJ.CO",S@+H20
->-
COOH
I
CHOH
I
+HS@
CHz
tOOH
malic acid
(XI!),
CH3
cO~S@
\.
oxalfcetlc --__"~--'l"'~ isocitric
-2H
t--,"~iru,
I
malic oC
"\
glyoxylic
CH3·CO~S(t\)
(XIII)
In animal tissues, however, this glyoxylic acid cycle has not been
detected. We have noticed earlier (p. 93) that the citric acid cycle is
not merely a vehicle for the oxidation of acetyl co-enzyme A, but something more than that: a metabolic pool into which enzymes pour
their products, or draw off their substrates. From time to time, then,
the concentration of (let us say) oxalacetic acid may be lowered
because it is tapped off for other purposes, and, moreover., the cycle
cannot revolve at an unlimited rate. Although on paper a very tiny
106
INTERCONVERSION WITH CARBOHYDRATES
catalytic quantity of oxalacetic acid should suffice for the oxidation
of any amount of acetyl co-enzyme A, in practice the cycle WIll stop
turning unless a small but definite concentration of oxalacetic aCId
is present. But animal cells depend for their supply of oxalacetic, in
part at least, on pyruvic acid, from which oxalacetic can be manufactured by the Wood-Werkman or Ochoa reactions (p. 98).
For reasons that are still not clear, most kinds of cell seem to
prefer carbohydrate to any other form of foodstuff. An extreme
case is represented by the brain tissue of animals, which is very
sensitive to lack of glucose; if the blood sugar falls at a rapid rate
the brain ceases to work properly, and the animal is thrown into
convulsions, or worse. When an animal is starved of all food its cells
gradually become depleted of glycogen, and sooner or later the
major part of its energy supply must come from fat, since the cellular
proteins are only broken down for fuel as a last resort. Consequently
a large amount of acetyl co-enzyme A is being produced at the very
moment that the supply of oxalacetic acid is running low; the citric
acid cycle ca,nnot oxidize acetyl co-enzyme A fast enough, and the
p-keto thiolase reaction (p. 102) is brought into play (XIV). Acetoacetic acid, the acetone formed from it by (spontaneous) decarboxylation, and p-hydroxybutyric acid (p. 101) are together known as
2CH3'CO~S@
p-keto
thiolase
CH3
CO.CH2·CO~S@+@SH
1+H20
CH3' co. CH2. COOH + ®SH
acetoacetIc acid
(XIV)
ketone bodies, which we saw earlier in this chapter can arise not
only in starvation but in such metabolic disturbances as diabetes and
von Gierke's disease, where the breakdown of carbohydrate is impaired. It must be borne in mind, however, that the citric acid cycle
seems to operate far more effectively, as regards the disposal of
acetyl co-enzyme A, in some creatures than in others. Thus the
chicken in its egg lives almost entirely on fat, yet ketone bodies do
not accumulate.
107
FA T MET ABOLISM
The enzymes concerned in the oxidative breakdown of fatty
acids are to a large extent localized in small particles called mitochondria-bodies about 3p. long and 0'5p, in diameter-with which
the cell contents are liberally supplied. Each particle is surrounded
by a very thin pellicle or membrane, far thinner than a cell wall.
Mitochondria contain the enzymes of the citric acid cycle and those
responsible for oxidative phosphorylation, as well as enzymes
attacking fatty acids,! and since many of the energy-rich bonds
required by living systems are generated in them, they have been
termed the 'power houses' of the cell. This epithet is even more
appropriate now than when it was first coined, because the part
played in vital synthesis by acyl mercaptides is coming into prominence.
Synthetic reactions of acyl mercaptides
Among the most remarkable of the compounds classed as lipids is
the group called steroids, whose functions are many and various, and
far from being properly understood. 2 Some of them are concerned
in the calcification of teeth and bone, others affect the transport of
electrolytes across cell-membranes, still others determine the phenomena of sex, and the whimsical distribution of hair on the human
frame. Common to them all is the ring (XV), to which a variety
(XV)
of groups and side-chains may be attached. Yet K. Bloch bas shown
by isotope studies that this complex fusion of rings is manufactured
entirely from acetyl co-enzyme A, the distribution of carbons being
as shown in (XVI). The various stages of steroid synthesis have all
1 The enzymes of glycolysis, including triosephosphate dehydrogenase, are,
however, also distributed throughout the cytoplasm. In bacterial cells, which
do not contain recognizable mitochondria, many important enzymes are associated With the cell-wall.
2 The name steroid is derived from a group of alcohols, the. sterols, which
were the first sterOids to be isolated and studied by organic chemists.
108
ACYL MERCAPTIDES
been identified, but are too elaborate to set down here. As a further
example of acyl mercaptides in the synthesis of complex structures,
we may consider some reactions of succinyl co-enzyme A. A class
of compounds called porphyrins is very widely distributed in nature:
the prosthetic group of cytochrome-c has been mentioned earlier
::::::::~~,
(11
o/~(~~·
I
~
I
·~/·~o/·
(XVI)
(p. 53), and the chlorophylls (p. 62) are closely related. It was
shown by Hans Fischer that all the porphyrins are theoretically
derived from the substance porphin (XVII), which is made up of
four pyrrole residues linked together by -CH= units. By tacking
side-chains to this tetrapyrrole nucleus the different sorts of porphyrins are arrived at. Although the full story has not yet been
made out, the work of D. Shemin and of C. Rimington indicates
HC~
NH
that some of these structures can be manufactured from succinyl
co-enzyme A and the amino-acid glycine (XVIII). Two molecules of
the product, O'-amino laevulic acid, condense together to form
porphobilinogen (XIX), which is known to be a precursor of several
porphyrins (a typical example is given in the Appendix, p. 144). The
109
l<A 1 Mti 1 AlIUL11>M
enzyme responsible for the condensation, 8-amino laevulic dehydrase,
was discovered by A. Neuberger in 1954. There is every indication
that the most complex heterocyclic systems can be built up in this
way, or by similar routes, from very simple units; the spectacular
synthesis oftropinone in vitro by Sir Robert Robinson in 1917, from
succindialdehyde, methylamine and acetone dicarboxylic acid (XX),
takes on a prophetic quality. But after looking briefly at one of the
'growing points' of biochemistry it is time to return to the beaten
track.
CHz.CO~S®
I
CHz.COOH
succinyl co-enzyme A
+
®SH
+
---
CHz.COOH
bHz
NHz.CH z COOH
glycine
CO CHNHz.COOH
et-aIl1lno-p-keto-adiplc acid
,j. -COz
CHz.COOH
I
CHz.CO CHz.NHz
8-amino laevulic acid
(XVIII)
COOH
COOH
bHz
I
CHz
I
COOH
tHz tHz
t-t
CHz
II
I
OC-CH:z-NHz
HC
II
C-CHz-NHz
W
porphobIlinogen
(XIX)
CHz.COOH
CH:z-CH--CHz
I
bo
I
CHz.COOH
tropinone
(XX)
110
I
N.CH3 CO +2CO z
ICHz--CH--CH
I
I
z
VIII
PROTEIN METABOLISM
The majority of proteins are of high molecular weight, and cannot
diJIuse through cell walls, so before the constituent ammo acids can
b,? absorbed, for food or for other purposes, it is necessary that the
proteins should be broken down more or less complctely-processesr.
that are carried out by proteinases and peptidases. These Qroteolytic~
enzymes act by hydrolysing the peptide linkage:
Rl_CO-NH-R2+H zO "" RI.COOH+NH2.R2.
Such reactions are reversible, but the standard free energy change,
-!!..Fo, is round about 30QO cal./mole, and equilibrium lies far to
the right. In general, proteolytic enzymes fall into two classes. called
endopeptidases and exopeptidases; those that occur in animals have
been studied by M. Bergmann, J. S. Fruton, and others, and will
serve for purposes of illustration.
A polypeptide chain, forming part of a protein, can be attacked
in two ways-at either end, or at random points along its length 1 (I).
endopeptidases
NH 2- -
•
-- • -- •
t
I
\
-- • -- • -- • -- •
-- •
--COOH
t
exopeptldases
exopeptidases
(I)
(It will be helpful at this point to refer to the diagram of the insulin
molecule on p. 29.) The enzyme pepsin, secreted by the stomach,
is a typical endopeptidase, attacking polypeptide chains wherever
aJomatic R-groups (of L-phenylalanine or L-tyrosine) occur (II).
Trypsin, secreted by the duodenum, exhibits a dlfferent sort of
specificity (III), where Rl or R2
= -(CH 2h.CH 2 .NH 2 , as in L-Iysine
or
-(CH2)3.NH.C(:NH).NH2, as in L-arginine.
1
Compare the action of cr.- and p-amylases (p. 77).
111
.
PROTEIN METABOLISM
These differences in substrate specificity are reflected by different
pH optima; pepsin works best in the acid interior of the stomach,
about pH 2-3, and trypsin in the region pH 7-8. Reference to the
diagram of the insulin molecule will show how vulnerable it is to
attack by e~dopeptidases, and indeed this hormone must be injected,
being quite ineffective when fed by the mouth to diabetic patients.
9
elc. --NH--CH--CO - : -
NH-- CH - - co --etc.
I
HO/H
R
(II)
Rl
I
:
"
etc.-NH-CH-CO-+ NH-CH-CO-etc.
:
I
HO/H
R2
(III)
Rl
I
:
...
NH2 -CH-CO-NH-CH-etc.
.
HO:H
I
R2
(IV)
Exopeptidases faU into three divisions. Those called aminopeptidases require a free teoninal -NH2 group to be available (IV),
and are dependent on metal ions for t elr activity (p.
e
car oxype b
s thelr name lmp les, ydrolyse peptides where
a free terminal -COOH group is exposed. Both types of e'nzyme
can therefore bite their way, as it were, down the chain, liberating
amino acids until the di- or tri-peptide stage is reached. Here, however, their action stops, but a third group of exopeptidasesdipeptidases and tripeptidases-completes the task of hydrolysis.
Thus protein fed to animals is broken down to amino acids just as
polysaccharides are broken down to their constituent sugars.
The proteolytic enzymes that we have hitherto considered are
secreted by specialized cells or glands in the animal body,' and are
112
PROT,EIN METABOLISM
therefore extracellular; such enzymes are also manufactured by a
variety of bacteria, thougb by no means all, and sometimes by
insectivorous plants-the sundew and the pitcher-plant, for instance.
But intracellular proteolytic enzymes also occur: those from animal
cells are often called cathepsins, and in their specificity resemble the
digestive enzymes. The physiological function of intracellular proteinases and peptidases during the life of a cell will be referred to in
a moment. After death, when the cell becomes disorganized, these
enzymes are responsible for au~olysis-as when meat or game is
hung in the larder for several days to make it tender before cooking.
Assuming that an organism is plentifully supplied with amino
acids of all kinds, the metabolic fate of these acids can be treated
under two heads: (1) manufacture of proteins, and of various other
substances required by the cell; (2) decomposition of any amino
acids not so required. In living matter the picture is more complicated than this simple classification would suggest, because the
cellular proteins are being constantly broken down and resynthesized;
for example, it has been estimated by isotope studies that the halflife of the proteins in animal liver is about a week or ten days, and
in muscle several months. Hence any particular amino acid molecule
may first be incorporated into protein and then-after a variable
length of tim~-the protein is hydrolysed (by the intracellular
enzymes just mentioned) and the amino acid undergoes decomposition. It will be convenient to consider now the ways in which surplus
amino acids are disposed of.
Deamination
Several routes of amino acid breakdown are known. some of which
are of limited interest because confined to a particular compound.
For example, aspartic acid can be converted to fumaric acid and
ammonia by the enzyme aspartase (V). The disposal of ammonia
COOH
COOH
tH2
I
CHNH
2
I
tH
~="
"
CH
I
+ NH3
COOH
COOH
aspartic acid
fumaric acid
(V)
113
PROTEIN METABOLisM
will be discussed later (p. 124). Again, serine is decomposed by
serine dehydrase (VI). The first product, a-amino acrylic acid,
rearranges into a-imino propionic acid, which then reacts spontaneously with water to form pyruvic acid and ammonia. Of more
CH 2 0H
tHNHz
~
tOOH
CH 2
CH3
~NH ~
to
tOOH
tOOH
LNH2 - -
tOOH
",-amino acrylic
acid
senne
CH3
",-imino propionic
pyruvic
aCid
aCid
+NH3
(VI)
general interest is a reaction that occurs in some bacteria (VIn. This
reaction was discovered by L. H. Stickland in 1934. Like the others
just described, it can take place under anaerobic conditions, and
hence is of particular value to organisms that grow in the absence
of oxygen.
Rl
I
CHNH2
tOOH
(VII)
But the chief route of amino acid decomposition in most organisms
is an oxidative process:
-2H
R.CHNH2.COOH
amino acid
---+
H 20
R.C(:NH) COOH ---..
imino acid
R.CO COOH +NH3
keto acid
the imino acid first formed by dehydrogenation reacting spontaneously with water. We have seen (p. 23) that the naturallyoccurring amino acids almost always belong to the L-series configurationally,! but members of the D-series are manufactured in
small quantity by some plants and bacteria. If any of these D-amino
acids became incorporated into the proteins of animals, especially
into the active centres of enzyme proteins, the consequences might
be unfortunate; for certain enzymes are very sensitive to the optical
configuration of their substrates (p. 39) and, conversely, a
change in the configuration of the enzyme itself could well result in
the substrate not being attacked. It is therefore not surprising to
find in the tissues of higher animals-notably liver and kidney-an
1
Except, of course, that glycine is optically inactive.
114
DEAMINATION
enzyme which seems to act as a stereochemical censor. This D-amino
acid oxidase, as it is called, was discovered by Sir H. A. Krebs; it is
an FAD flavoprotein, belonging to the class of aerobic'dehydrogenases (p. 51), and catalyses
(i)
(ii)
(iii)
In sum
R CHNH z COOH+FP ;? R.C(:NH).COOH+FPHz•
R.C(:NH).COOH+H 2 0 - R.CO.COOH+NH3.
FPHz+Oz - FP+H20 Z'
R.CHNH2 COOH+0 2+HzO - R.CO.COO:fI+NH3+H202.
Since the breakdown of the imino acid (ii) is spontaneous and
irreversible, the reaction proceeds readily from left to right; the
hydrogen peroxide being decomposed by catalase (p. 40).
A very similar enzyme, shown to be a flavoprotein by D. E. Green,
attacks L-amino acids in the same fashion. But its activity is feeble,
the turnover number (p. 34) being less than 100, as compared with
·about 2000 for the D-amino acid oxidase. Moreover, the L-amino
acid oxidase only deaminates a restricted range of acids, and has no
action-for instance-on L-glutamic acid. Clearly, then, some other
mechanism must come into play.
Transamination
An important reaction was discovered by A. E. Braunshtein and
M. G. Kritsman in 1937 (VIII). Here the amino acid exchanges the
elements of ammonia with a-ketoglutaric acid, and is converted into
the corresponding keto acid; transamination is easily reversible,
- ~Fo being about 500 cai./mole, and all amino acids are able to
participate. It seems likely that there is a separate transaminating
enzyme, or transaminase, for each amino acid; moreover, in some
organisms oxalacetic acid can behave like a-ketoglutaric acid,
yielding L-aspartic acid.
COOH
tHNH2
booH
amino acid
I
I
co
I
(CH 2h
R
+
R
~
COOH
a-ketoglutaric
aCId
I
co
booH
keto acid
(VIII)
115
COOH
+
(tH
I
2)2
C HNH 2
bOOH
L-glutamic
acid
PROTEIN METABOLISM
But the process of transamination by itself will not serve to explain
why animal tissues like liver and kidney can produce ammonia in
quantity from amino acids. The explanation lies in a DPN-linked
enzyme, L-glutamic dehydrogenase (IX). The reduced DPNH 2 is
oxidized by the flavoprotein-cytochrome chain, providing ATP in
the usual way. It appears that L-glutamic dehydrogenase is absolutely specific for L-glutamic acid. Hence the major route of oxidative
deamination first involves transamination, followed by breakdown
of L-glutamic acid (X). The transfer of the elements of ammonia in
transamination reactions is brought about by a compound, pyridoxal
phosphate, which acts as a co-enzyme for all transaminases. Its
formula (XI) can be simplified into X.CHO; and the sequence of
reactions is represented in (XII), where X.CH2.NH2 represents
pyridoxamine phosphate. This substance then reacts with a-ketoglutaric acid (XIII). We shall see later (p. 119) that pyridoxal
phosphate is a co-enzyme for another kind of decomposition that
amino acids can undergo.
COOH
COOH
(bH2h
I
CHNH2
(tH2 h
~
t:NH
bOOH
tOOH
L-glutamic acid
+
imino acid
+
DPN
COOH
H2O
---+
(tH2h
to
+ NH3
bOOH
et-ketoglutaric acid
DPNH2
(IX)
a.lDino
acid+a:-ketoglutaric~L-glutamic+keto
~
acid
NH,
(X)
The importance of the transamination system lies not only in the
breakdown of amino acids but also in their synthesis. Most proteins
are fairly rich in glutamic acid, and in the ordinary wayan organism
will get more glutamic acid in its food than is needed for making its
own proteins. The surplus acid can therefore be used for the manufacture of other amino acids which may happen to be in short supply.
Thus a shortage of alanine could be countered by
pyruvic acid + L-glutamic acid "'" L-alanine + a-ketoglutaric acid,
116
TRANSAMINATION
CHO
OH
®OH 2C
CH 3
R
R
-H2O
tHNH2
+OHC.X
~
+HzO
tOOH
I
I
CH-N=CH.X
COOH
amino acid
R
R
+H20
to
+H2N CHz X
~
-H2O
bOOH
(XII)
Jf
I
C=N-CH2X
I
COOH
COOH
I
(CHz)z
I
X CH 2.NH2+O=C
bOOH
COOH
I
(CH 2h
-H2O
.==-----"
I
I
X. CH2-N=C
+ H20
II
COOH
I
X CHO+(CHzh
I
I
-H2O
CHNHz
COOH
I
I
(CH2h
+H20
-~
COOH
X.CH
N-CH
I
COOH
COOH
L-glutamic acid
(XIm
the pyruvic acid being obtained from carbohydrate breakdown. In
th~ same way, any other amino acid can be formed from the corresponding keto acid-:-provided that the organism is able to make the
keto acid. But in this respect the different kinds of living matter
differ considerably. It has been shown by W. C. Rose that an adult
man must be supplied with the following eight L-amino acids in the
diet: leucine, isoleucine, valine, threonine, methionine, lysine, phenylalanine, tryptophan. These compounds are the so-called 'essential
117
PROTEIN METABOLISM
amino acids', and they are dietary essentials because the corresponding keto acids cannot be synthesized in the human body)
Some adult animals, such as the rat, need histidine also, and during
infancy and the period of rapid growth an extra supply of arginine
as well-indicating that the rate of arginine synthesis is limited.
Among micro-organisms the study of amino acid requirements
has thrown light on the way in which these compounds can be made
from simpler units. Let us suppose that an organism needs a particular amino acid of which an adequate supply is not present in the
medium in which it is growing. Then the acid must be synthesized
by a series of reactions which we can write A -+ B -+ C. Each step
in the chain, A -+ B, B -+ C, will be controlled by a separate
enzyme (or several enzymes), and the problem is to discover the
precursors of C, the amino acid required. Micro-organisms grow
and multiply at a rapid rate, and from time to time mutation occurs
among the genes that control enzyme synthesis. Such' spontaneous'
mutations are rare, but the number can be increased by exposing the
organisms to X-rays, and among the mutants there will be found
one which has lost the power to turn, for instance, A into B. In other
words, the gene has been lost that controls the synthesis of the
enzyme that makes B from A. (There is a good deal of evidence that
the manufacture of each enzyme in a cell is controlled by a separate
gene.) Consequently, whereas in its 'wild' state the organism can
accomplish every stage in the synthesis of C, the mutant can only
make C from B; unless it is supplied with B its growth will come to
a standstill. If the mutant is fed with likely precursors of C, one of
them will restore the power to grow-and so the nature of B is
arrived at. In principle the recognition of an intermediate is simple
enough; in practice there are difficulties that can make this branch
of biochemistry one of the most recondite of all. We must not dwell
on these' biochemical mutants', however-except to notice that sizeable books have been written on the genetic aspects of metabolism.
1 Or because the amino acids themselves cannot be made from other amino
acids; a few examples of this type of process Wlil be mentioned later.
118
PROTEIN METABOLISM
Decarboxylation
Another way in which amino acids can be broken down is represen ted
by decarboxylation. It will have been observed that in the synthesis
of porphobilinogen (p. 110) a-amino-fJ-keto adipic acid loses CO2
and is converted into the corresponding amine:
R.CHNH2.COOH -7- R.CH2.NH2+C02'
This type of reaction is not confined to the higher animals but occurs
among plants and bacteria also; and the work of E. F. Gale and of
I. C. Gunsalus with micro-organisms drew attention for the first
time to the role of pyridoxal phosphate as a co-enzyme in biological
reactions. Although it may appear strange that one ald the same
co-enzyme can operate in both transamination and decarboxylation.
a scheme proposed by E. E. Snell in 1954 affords a possible explanation. In this scheme an amino acid combines with a trivalent metal
(such as iron) and pyridoxal phosphate to form a chelate ring
system (XIV). If this complex breaks up along the lines a ... a, then
R.CH z CHNH 2 ·COOH+M+++
H
a ......
·1· .... a
R.CH 2 C
b
b · a·i··
··.
CHO
/
.. b
····c=O
r
I
··.«N,,>x~
ell "'b
"a
o
(XIV)
with the addition of the elements of water we shall have the products
shown in (XV), as already indicated (p. 117). But a break along the
lines b ... b will yield the amine (R.CH 2 .CH 2 .NH 2), CO 2 , and
pyridoxal phosphate. Hence the protein moiety of the enzyme exercises a decisive influence on the manner of breakdown of the substrate
119
PROTEIN METABOLISM
+ co-enzyme complex. Although amino acid decarboxylases are
widely distributed in living matter, and although they undoubtedly
take part in synthetic processes, much still remains to be learnt about
their functions in protein metabolism.
R.CH 2 .CO COOH+M+++
keto aCid
NH2
IH2
+
OH
pyridoxamine phosphate
(XV)
Fate of the carbon skeleton
We have seen that the chief pathway of amino acid decomposition
is an oxidative process, involving preliminary transamination; and
the fate of the keto acids and of ammonia must now be considered.
As to the former, broadly speaking the carbon skeleton is ultimately
converted into acetyl co-enzyme A and oxidized, but the paths of
conversion are often complicated. In the diagram (XVI) a few of
the simpler amino acids have been brought together with the object
of illustrating the kind of thing that can happen.
COOH CH, CH 2 CH(NH,) (OOH_a·ketoglutarlc
l
glutamiC
CH, CH(NH,) COOH
via CitrIC cycle
~
a~anme:
/
-co,
COOH CH~s~a~:I~H') COOH---__ ;~lacettC_:;ruv~lc
glucose
t'
CH, CH, CHiCH,) CH(NH,) CDOH
3 carbons
IcarbO/
I
,soleucme~a-keto-p-methyl valenc
-CO,
CO-S®
~--b-/--CH 3
2 car ons
acetyl
co-enzyme A
CHl,
/arbons
CH CH, CH(NH,) COOH -,·keto-,so-caprOlc
CH,/
11
~
~tty aCid"
leuctne
(XVI)
120
FATE OF THE CARBON SKELETON
Alanine is turned by transamination into pyruvic acid, which can
either lose CO 2 to form acetyl co-enzyme A (p. 91), or else be converted ~o carbohydrate. The formation of glucose from certain amino
acids is called gluconeogenesis, that is, the synthesis of carbohydrate
de novo from non-carbohydrate sources.
Glutamic acid goes to a-ketoglutaric (by action of the L-glutamic
dehydrogenase) and thence by the reactions of the citric acid cycle
to oxalacetic, and finally to pyruvic acid. The importance of the
citric acid cycle as a metabolic clearing house has already been
referred to (p. 106).
Aspartic acid by transamination yields oxalacetic, and then pyruvic
acid. It is therefore glucogenic, like alanine and glutamic acid.
Leucine produces only acetyl co-enzyme A, and no pyruvic acid
at all. If leucine is fed in large quantity to a diabetic animal, the
acetyl co-enzyme A cannot be quickly metabolized via the citric acid
cycle and is converted into acetoacetic acid instead (p. 107), so amino
acids like leucine are called ketogenic, to distinguish them from
glucogenic acids like alanine.
Isoleucine, however, behaves in another way. The keto acid obtained from it loses 1 carbon as CO 2 ; then three of the remaining
carbons go to pyruvic and two to acetyl co-enzyme A. Thus isoleucine is both glucogenic and ketogenic.
The amino acids histidine and tryptophan, which contain heterocyclic rings, are broken down in a variety of ways, too complicated
to discuss here. Phenylalanine and tyrosine, however, follow a
common path, and by the use of isotopes S. Gurin showed in 1949
how the several carbons are disposed of (XVII).
Phenylalanine (an essential amino acid) is converted to tyrosine
(non-essential) by the irreversible addition of an -OH group para
to the side-chain. After oxidative deamination to p-hydroxyphenyl
pyruvic acid, CO 2 is lost, and then-oddly enough-the side-chain
migrates from position I to position 2 of the ring, an -OR group
entering position 1. By cleavage between these positions the ring of
homogentisic acid is opened, and the fumaryl acetoacetic acid formed
is hydrolysed to fumaric and acetoacetic acids. The former can go
to glucose through the citric acid cycle, and hence phenylalanine and
tyrosine are both glucogenic and ketogenic, just as isoleucine is.
5
121
HII
PROTEIN METABOLISM
CH-CH
II~.
CH
I
.
C--CH2 CHNH 2.COOH
\.CH=CH
2/
•
CH-CH
II '\.
HO--C
·
lC--CH 2 CHNH 2 COOH
tyrosine
CH=CH
\.
2/
+O~-NHJ
p·hydroxy phenyl pyruvIc aCid
.
CH-CH
1/
\.
HO--C
lC--OH
\*CH=C2/
\
*
CH,COOH
homogentisIc aCid
HOOC CH CH CO eH 2 CO CH 2 COOH
I
2
fumaryl acetoacetic
•
•••
HOOC CH CH COOH+CH).CO CH 2 COOH
•
acetoacetIc aCid
fumanc aCId
(XVII)
Glycine, serine and methionine
Although glycine can be oxidatively deaminated in the usual way,
it also takes part in a reaction of an entirely different kind. In 1948
D. M. Greenberg and W. Sakami found that glycine may be converted into serine, which then undergoes deamination by the serine
dehydrase (p. 114). This reaction involves the addition of a carbon
122
GLYCINE, SERINE, METHIONINE
atom, or rather, the elements of formaldehyde, H.CHO; and the
process is of considerable interest.
The elements of formaldehyde can arise from severi!-l sources, one
of which-and probably the most important-is the essential amino
acid methionine. The terminal S-methyl group of methionine is
readily detached (XVIII). Homocysteine, although chemically an
amino acid, is never found combined in proteins; it can be converted,
by steps that do not concern us here, into cysteine-which is thus
CH3
I
S
I
CH z
I
CHz
I
CHNHz
I
SH
- CH 3
~
COOH
metluonine
I
CH z
I
CH z
I
CHNHz
I
H ZS0 4
+
SH
I
I
CHNHz
I
CHz
--+
COOH
cysteine
COOH
homocysteine
CH3
--+
I
CO
I
COOH
pyruvic acid
(XVIII)
NH z
I
1
C=NH
+
amidine group
(from arginine)
NHz
--+
I
C=NH
I
glycine
NH.CHz COOH
guanidme acetic acid
I + -CH3 (from methionine;
t
reqnires ATP)
NHz
I
I
N.CHz
I
C=NH
COOH
CH3
creatme (methyl guanidine
acetic acid)
(XIX)
a non-essential amino acid-and ultimately pyruvic acid and sulphate.
The 'labile' methyl group of methionine can enter, as such, into a
number of reactions: a single example, the synthesis of creatine
(whose functions are discussed on p. 133) will serve for illustration
(XIX).
It will be noticed that the transfer of the amidine group from
arginine to glycine is a transamidination, not a transamination; and
123
5-2
PROTEIN METABOLISM
the transfer of the methyl group from methionine is a transmethylation.
But the methyl group can also be oxidized to formaldehyde
-CH3
o
---+
-H.CHO
and it is in this form that methionine contributes to the synthesis of
serine from glycine. The formaldehyde group is 'carried' on a complicated compound called tetrahydrofolic acid, whose formula is
given in the Appendix (p. 142), but we may here disregard the
complications and write it R = NH. Then
-H CHO+R = NH
---+
R = N.CH20H
•active formaldehyde'
The substance' active formaldehyde' (i.e. in the form of hydroxymethyl tetrahydrofolic acid) can react with glycine, thus:
R
=
N.CH20H+CH2(NH2).COOH "" CH 20H CH(NH2).COOH
serine
+R=NH
This reaction is reversible,l and therefore serine as well as methionine
can serve as a source of H.CHO in the 'active formaldehyde' unit.
This unit, by which the elements of formaldehyde are made available
for synthetic purposes, should be compared with the 'acetyl' of
acetyl co-enzyme A and the 'succinyl' of succinyl co-enzyme A; all
three units, in their different ways, represent the small building stones
from which larger molecules can be constructed (pp. 108-9).
Fate of ammonia
We must next consider what happens to the ammonia arising from
the deamination of amino acids. Ammonia is an objectionable compound, highly toxic to living cells, and several methods are known of
rendering it harmless.
In the liver and kidney tissue of the higher animals, glutamic acid
and ammonia can be made to react together (XX). It was shown in
1949 by W. H. Elliott and by J. F. Speck that the synthesis of
glutamine requires ATP: a discovery which has helped to an understanding of protein synthesis (p. 129). Glutamine in the kidney can
be hydrolysed by glutaminase to glutamic acid and ammonia, the
1
It appears that the synthesis of serine from glycine requires ATP.
124
FATE OF AMMONIA
latter substance helping to regulate the pH of urine; D. D. van Slyke
has estimated that, under certain conditions, as much as two-thirds
of the urinary ammonia is derived from glutamine.
COOH
CO.NH2
I
(CH2)z
I
CHNH2
+ NH3
glutamine
synthetase
<=====
glutaminase
I
I
(CH 2h
I
CHNH2
+ H 20
I
COOH
COOH
L-glutamic acid
L-glutarnine
(XX)
Plants also store ammonia in the form of glutamine, but more
commonly they convert aspartic acid to its amide asparagine (XXI).
This reaction is of importance in those plants whose seeds contain
a good deal of protein but no very large reserve of carbohydrate.
When such a seed begins to germinate below the surface of the soil,
where sunshine cannot penetrate, the carbon skeleton of the amino
acids can be used for fuel-the ammonia arising from deamination
being stored as asparagine. Then, when the cotyledons sprout and
photosynthesis can take place, the stored nitrogen becomes available
for the manufacture of amino acids; for instance, two molecules of
aspartic acid could be formed from asparagine and fumaric acid
(XXII). By a kind of transamination the amide group of asparagine
can probably yield -NH2 to keto acids as well.
COOH
tH2
~
+ NH3
tHNH2
CO.NH2
tH2
+H20
tHNH2
tOOH
tOOH
(XXI)
/aspartic acid
asparagine(
"'-NH3 + fumarIc acid
aspartase
aspartic acid
(p. 113)
(XXII)
But such mechanisms, in the last resort, are only temporary safety
measures; they do not enable the organism to get rid of ammonia
in quantity; and although animals can excrete a limited amount of
ammonia in the form of ammonium salts, far and away the greater
125
PROTEIN MET ABOLISM
part of their surplus nitrogen is excreted as urea. The broad outlines
of urea synthesis were estabhshed 10 1932 by Sir H. A. Krebs, since
which time our knowledge has been considerably extended by
F. Lipmann, S. Ratner, P. P. Cohen, P. Reichard, and others. The
cltrulhne
CO 2 +NH 3 - {
ormthme
1--(....,......--
.....
arginine
urea
(XXIII)
urea
(XXIV)
process can be briefly represented as in (XXIII), or in summary form
as in (XXIV). And the first step appears to be
ATP
C02+NH3
---->-
NH2.COOH
carbamic acid
---->-
NH2 co O-®+ADP
carbamyl phosphate
In 1955 F. Lipmann was able to show that CO 2 and NH 3 , combined
together as carbamic acid, can be phosphorylated by ATP to
carbamyl phosphate. This compound, with its energy-rich bond, can
readily react with ornithine to give citrulline (XXV); both these
amino acids, like homocysteine (p. 123), arise only in the course
of metabolism and are not found as constituents of proteins. The
enzyme responsible for this reaction is ornithine carbamyl transferase, purified by P. Reichard in 1957. The next step involves the.
NH2.CO.O-®
NH2
to
+
k
NH2
I
I
(CH2)3
(CH2h
I
I
CHNH2
CHNH2
I
tOOH
COOH
ornithine
citrulline
(XXV)
126
+®OH
FATE OF AMMONIA
synthesis of an arginine derivative from citrulline, and takes place as
shown in (XXVI). This condensation has been shown by S. Ratner
to require ATP; the product, argininosuccinic acid, is broken down
to arginine and fumaric acid (XXVII). Finally, arginine is hydrolysed
by the enzyme arginase to ornithine and urea (:XXVIII).
NH
II
COH
k
(tH h
COOH
I
+H2N-CH
6H2
NH
COOH
II
I
C-NH-CH
+ADP+®OH
~k
2
tOOH
tHNHz
aspartic acid
tH2
(tH2)3
tOOH
tHNHz
booH
citrulline
(enol fonn)
600H
argininosuccinic acid
(XXVI)
NH
COOH
II
t
C-NH- H
(tH 2)3
tH2
NH2
t=NH
I
NH
--0-
tHNH2 tOOH
tOOH
argininosuccinic acid
+
(6H2h
tH
II
CH
I
tHNH2
COOH
fumaric acid
tOOH
arginine
(XXVID
NH2
I
COOH
NHz
C=NH
OH
NH
H
(tH2)3
.. ··1 ........ ·· ..........
~HNH2
-+
(tHzh
tHNHz
tOOH
+
/
NHz
CO
tooH
"-NHz
ornithine
urea
arginine
(XXVIII)
The process is therefore cyclic in nature, ornithine, citrulline and
arginine acting as 'carriers' for the elements of urea during its
formation. This ornithine cycle proposed by Krebs is interlocked
with the citric acid cycle also proposed by him, and with the transamination reactions discussed earlier (XXIX). It will be seen that
127
PROTEIN METABOLISM
one of the nitro gens going to form urea enters the cycle as carbamyl
phosphate, and the other comes via the aspartic-oxalacetic system;
and the significance of the citric acid cycle as a metabolic clearinghouse will once more be apparent.
Among birds and reptiles, as compared with mammals, the chief
end-product of nitrogen metabolism is uric acid (2, 4, 8-trioxypurine). In 1948 J. M. Buchanan showed by the use of isotopes how
this molecule is built up from several sources (XXX): amino groups
(or amide, as from glutamine) contribute nitrogen to positions 1, 3
and 7, 'active formaldehyde' the carbons at 2 and 8, CO 2 the carbon
-----1
NH, -
glutatruc
C
)
atruno aCids
\.... a-ketoglutanc
keto clds
+
VIa
CO 2
cltnc cycle
'NH '
_._----~----
/'~'h~Y"''''rt."\(
lATe
CarbamYI~
A
argmmoSUCCiniC
phosphate ornithine
urea
___ )
oxa:lacehc
glutamiC ,\(ammo aCids
_)
a-ketoglutanc
keto aCids
/\:CI+CYCle
argInIne
fumaric
(XXIX)
,,'
CO~."'-'NH
ammo-N-N..G
I
'active - C 0 2
formaldehyde'
.........
4
"
··..--glycine
r5h:'.?·~·
: II;
8CO-4-- 'active
: 6C:
I : / ... ~
NH
\
/
formaldehyde'
7
NH
1
ammo-N
(XXX)
at 4, and glycine the remainder. Adenine, which is 4-amino purine
(formula in the Appendix, p. 138), can be manufactured in the same
way, the nitrogen attached to position 4 being derived from amino
or amide groups; and the synthesis of adenine is readily ac~eved by
mammals also.
128
PROTEIN METABOLISM
Protein synthesis
Hitherto we have been mainly concerned with the breakdown or
synthesis of amino acids as such, but the enzymes that direct these
metabolic changes are themselves composed of amino acids, which
must somehow be joined together to yield large protein molecules.
We have seen (p. 111) that for the free energy change during the
hydrolysis of a peptide bond - tlFo = about 3000 cal./mole, so the
reverse reaction must involve a free energy change, + tlFO, of the same
magnitude. The implication of ATP in glutamine synthesis (p. 124)
is enough to suggest the likelihood of a similar mechanism in the
making of peptide linkages, and that ATP is the driving force behind
peptide synthesis was shown by K. Bloch in 1953, the enzymes concerned being called peptide synthetases (:XXXI). The tripeptide
L-glutamic+L-Cystcine
ATP
---+
y-glutamyl-cysteine
A TP
1+
glycine
glutathione
(y-glutamyl-cysteinyl-glycine)
(XXXI)
glutathione was discovered by Sir F. G. Hopkins in 1921 (its extended
formula will be found in the Appendix, p. 142); it is thought to
be the co-enzyme of triosephosphate dehydrogenase (p. 87), and it
has other functions too. C. S. Hanes has found that the y-glutamyl
residue of glutathione can enter into transpeptidation reactions;
with phenylalanine, for instance,
glutathione + phenylalanine --+ y-glutamyl-phenylalanine + cysteinyl-glycine.
It is not yet clear whether such transfer reactions are involved in
protein synthesis. But another mechanism, based on an idea put
forward by F. Lipmann some years ago, is perhaps of more general
application. In the presence of an appropriate enzyme it has been
shown that ATP can react with amino acids to form a complex,
pyrophosphate being split off:
AA+ATP+@ --+ AA-AMP-@complex+PP.
There is evidence that amino acids •activated' in this way can unite
together to form polypeptides, although the details of the process
129
PROTEIN METABOLISM
are a little obscure. But for our purposes, simplifying the matter as
far as possible, we can write the' activated' amino acid in the form
R .CHNH2 .CO-O-@.
If this reaction were to take place at adjacent spots on an enzyme
surface, we should have (XXXII), the peptide then splitting away
from the enzyme (XXXIII). By this means a considerable number
of amino acids could be strung together, forming a long polypeptide
chain.
.
--"'1CO-CHCR1)NH2 ---~_CO-CHCR2)NH2
I
CO-CHCRl)NH2
'---¥
I
"-~
0 0 0
(XXXII)
elc.--CO--CH(Rl)--NH--CO--CH(R2)NH--CO--CH(R3)NH--elc
OH
OH
OH
(XXXIII)
But the chief puzzle about protein synthesis is to understand how
particular amino acids come to be united in a particular way. For
example, the insulin manufactured by anyone kind of animal is
always the same insulin-so far as we can judge-containing the
same sequence of amino acids. We could postulate a series of active
centres on the surface of the synthetase responsible for making
insulin, each centre being specific to a definite amino acid,l but the
question remains-what controls the formation of the synthetase
that makes insulin? Many lines of evidence go to show that enzyme
formation is controlled by nucleic acids, so called because they occur .
abundantly in cell nuclei; and by degrees a picture is being built up
of how nucleic acids are involved in cell division, mutation (p. 118),
and the manufacture of enzymes. Two classes of nucleic acid have
been recognized, the ribonucleic acids (RNA) and the deoxyribonucleic acids (DNA), the latter, it seems, being mainly responsible
for the control or organization of enzyme synthesis. Without going
into details, DNA molecules are long, thread-like structu~es of very
1
Probably more than one synthetase will be needed.
130
PROTEIN SYNTHESIS
high molecular weight, built, according to D. M. Brown and Sir A. R.
Todd, on the plan of (XXXIV). About half a dozen different nitrogenous bases can enter into this kind of structure, adenine being one
of them; the sugar is 2-deoxY-D-ribose (XXXV), distinguished from
o-ribose by having two hydrogens attached to carbon 2, instead of
a hydrogen and a hydroxyl group. We have seen (p. 18) that compounds of the type base-sugar-phosphate are called nucleotides;
DNA molecules are therefore polynucleotides, differing from one
base
etc.
I
sugar
" 00I
~=o
base
base
sugar
sugar
I
" 00I
I
" 00I
1=0
OH
OH
" 0
1=0
OH
"etc.
(XXXIV)
HOH'~H
OH
H
(XXXV)
another in the nature and sequence of the nitrogenous bases attached
to the chain. Somehow or other these very large DNA moleculeslarge enough to rank as macromolecules-are able to regulate protein
synthesis, and although the whole story is still far from being made
out, 'it is supposed that different sections of the DNA macromolecule
control the synthesis-or carry the information necessary for the
synthesis-of different enzymes. Consequently information is disposed along the macromolecule like words on a tape, and DNA is
to the cell what the information tape is to automation '.1 And on
tJils analogy a gene (p. 118) may be likened to a phrase or a sentence.
Thus nucleic acids control protein manufacture, and if it is askedWhat controls DNA manufacture ?-the answer seems to be that
these compounds are able to reduplicate themselves. But here, at
the extreme tip of a 'growing point' in biochemistry, we must halt.
1
E. F. Gale (1957).
131
PROTEIN METABOLISM
Muscular contraction
Several examples of ATP performing chemical work have already
been encountered; reference must now be made to its role in
mechanical work. Muscular contraction in animals, and ciliary
movement among many forms of life, depend on the free energy
liberated by hydrolysis of ATP, and although much of the detail is
obscure, it is possible to form a rough idea of what goes on.
The contraction of a muscle fibre is actuated by structures in the
cell called myofibrils, running parallel to the axis of the fibre. In
cross-section a myofibril presents roughly the appearance shown in
(XXXVI), as determined by H. E. Huxley in 1953. It appears, then,
that the myofibril is largely made up of two kinds of thread, fairly
closely packed together, the distance between the thicker threads
being about 440 Angstrom units. These thicker threads represent a
protein called myosin, the others a protein called actin. The proteins
can be extracted from the muscle fibre, and purified, and in 1939,
long before the fine structure of the myofibril had been analysed,
W. A. Engelhardt and M. N. Lyubimova discovered that myosin is
an adenosine triphosphatase (or ATP-ase)-in other words, an
enzyme that can hydrolyse ATP to ADP and inorganic phosphate.
Actin does not possess this property .
•
•
•
•
•
•
•
•
•
•
•
• myosin
•
• actin
•
(XXXVI)
Although the amount of ATP in a resting muscle fibre is extremely
small, a supply of it can quickly be generated by an easily reversible
reaction, catalysed by creatine phosphokinase, and first noticed by
K. Lohmann in 1935 (XXXVII). The energy-rich bond in creatine
phosphate is not hydrolysed by myosin, and thus what may loosely
be called a 'store of free energy' is held available until the stimulus
arrives that causes the myofibrils to contract. When contraction
occurs, creatine phosphate is converted by the Lohmann reaction to
ATP, and the free energy of ATP breakdown is converted into
132
MUSCULAR CONTRACTION
mechanical work. While the muscle goes on contracting a further
supply of ATP can be obtained by anaerobic glycolysis, or by
oxidation of foodstuffs, creatine phosphate being resynthesized when
the muscle is at rest.
NH~®
HN=C
NH2
/
/
+ADP
HN=C
I
N-CH2.COOH
I
+ATP
I
N-CH2.COOH
I
CH 3
creatme (p. 123)
CH3
creatme phosphate
(XXXVII)
A considerable amount of evidence, developed by A. F. Huxley
in 1957, suggests that contraction of the myofibril is due to the actin
and myosin threads sliding relatively to one another, in some such
manner as shown in (XXXVIII). The work of A. von Szent-Gyorgyi
and others has shown that myosin and actin c~~form a complex in
the presence of Mg++ ions, and that this com x is broken up by
ATP, with liberation of actin, and reformed wh ATP is hydrolysed
(XXXIX). It would seem, therefore, that tension arises through the
making and breaking of cross-linkages between the protein threads,
but the precise nature of these linkages, and how ATP affects them,
can only be decided by future research.
_ _ _ _ myosin
actin
relaxatIOn
~ tension
t
(XXXVIID
.~
(
ATP~
myosin-Mg++-actin
myosin-Mg++-ATP+ actin
(XXXIX)
133
\_
f
ADP
IX
CONTROL OF METABOLISM
In the previous pages we have been following chains of chemical
process to their destination, perhaps with a feeling that these reactions
do not tell us very much about life as a whole. After all, the target
of biochemical inquiry is to explain the chemical activities of com~
plete organisms: it is manifestly a far cry from the contents of this
book to a dog or a cat, or even a cabbage. And we cannot give a
satisfactory account of how the higher animals co-ordinate those
activities because metabolic control is largely in the hands of hormones and of the nervous system, the nature of whose operations at
the biochemical level is still rather obscure. But unicellular organisms,
with no hormones and no nerves, are able to maintain themselves
with vigour and success, so there must be 'primitive' mechanisms of
control, besides the more elaborate forms; and we can usefully
confine the discussion to single cells, with particular reference to the
regulation of free energy.
In the first place, an organism will try to cope as far as possible
with changes in the external environment. The supply of food will
often be intermittent, and measures must be taken to bolster the cell
against emergencies. For instance, carbohydrate is stored to some
extent, as polysaccharide, by almost all cells, and if the external
supply is cut off, energy for chemical work may still be obtained by
drawing on the stored material. And in the exceptional case of
muscle cells, and possibly some others, energy is kept readily available
in the form of creatine phosphate (p. 133).1 Another factor outside
the direct control of a cell is the amount of oxygen available to it.
Here anaerobic glycolysis along the Embden-Meyerhof pathway can
furnish ,.., ® when the oxygen pressure falls to a low level (p. 97).
But leaving these considerations on one side, let us imagine a cell
1 Among invertebrates creatine phosphate is often replaced by arginine
phosphate, with similar functions, or by other compounds With the -NH~ ®
linkage.
'
134
CONTROL OF METABOLISM
that is freely supplied with glucose, for example, and oxygen, as well
as all the fats, amino acids, growth factors, and trace elements needed
for its continued existence. How does such a cell achieve a balance
between the free energy available from carbohydrate and the free
energy required for carrying on life? Or in other words, how is the
rate of energy production controlled?
Assuming that the enzymes in a cell are more or less fixed in
quantity, the rate at which energy is produced will be decided in the
Last resort by the concentrations of the substrates available to those
enzymes. We know that raising the substrate concentration increases
the rate of reaction, until an enzyme is working as fast as it can
(p. 42), but such maximum rates are not often reached, except
perhaps among bacteria growing under very favourable conditions.
In any case the maximum rate of a process only fixes an upper limi t;
it does not enable a cell to adjust the energy output in a flexible way,
so that energy-rich bonds are made when they are wanted, and only
when they are wanted. To achieve this object, cells need metabolic
regulation of the type that engineers cell 'feedback'.
The principles of feedback control, first analysed by J. Clerk
Maxwell in 1868, have been applied to biochemical problems by
Sir H. A. Krebs. A familiar feedback system controlling the rate of
a process is represented by the governor of a steam engine. Here a
number of weights are attached by pivoted rods to a vertical shaft,
and spin outwards and upwards as the shaft rotates. If the engine
load decreases, so that the engine tends to run too fast, the weights
rise and by an arrangement of levers reduce the supply of steam;
similarly, if the load increases, and the engine slows down, the steam
supply is increased. The essence of this simple type of feedback is
that it always operates to oppose changes in the engine running
speed, and thus contributes to steady running even against variable
loads: in a manner of speaking, it constantly feeds back information
about the load.
As far as energy production is concerned-the creation of '" ® for
chemical work in the cell-it seems likely that the triosephosphate dehydrogenase reaction occupies an important place in feedback control:
3-phosphogJyceraJdehyde+ ADP + ®OH + DPN '"
3-phosphoglyceric acid + ATP + DPNH2
135
CONTROL OF METABOLISM
For the coupled reaction, -tlFO = about 4000 cal./mole in favour
of ATP synthesis. In the presence of adequate concentrations of
triosephosphate (from glucose) and DPN, the rate of the forward
reaction is determined by the concentrations of ADP and ®OH, and
hence will be influenced by any process that produces or removes
these compounds. When ATP is broken down to supply energy for
chemical (or mechanical) work, ADP and ®OH are formed, and
thus a fall in the concentration of ATP creates the conditions
necessary for its own synthesis. When ATP is synthesized, the rate
of reaction soon slows down since ADP and ®OH are being removed ;
each tenfold reduction of reactant concentration alters tlF by 1420cal./
mole at 37° C. (p. 12), so the reaction tends to become slower as
equilibrium is approached, until at equilibrium it halts because
/).F has dropped to zero. Thus energy production is regulated by the
concentrations of the reactants in the system ADP+ ®OH ~ ATP,
very much as the speed of an engine is regulated by the position of
the governor weights. And the system ADP + ®OH ~ ATP is
coupled to the oxidation of triosephosphate and the reduction of
DPN to DPNH2, so the level of ADP and ®OH in the cell fixes the
rate at which triosephosphate (and therefore glucose) will be oxidized
in the Embden-Meyerhof pathway (p. 86).
But glucose can also be oxidized by the Warburg-Dickens pathway
(p. 81), and here the ADP+ ®OH '<'" ATP regulator likewise has
a part to play. Glucose-6-phosphate dehydrogenase is TPN-linked,
and when TPNH 2 (or DPNH2) is oxidized, through the flavoproteincytochrome chain, a coupled reaction produces ATP (p. 60).
Oxidative phosphorylation of this type therefore removes ADP and
®OH, just as the triosephosphate dehydrogenase reaction does, and
by their conversion to ATP the oxidation of TPNH2 must be slowed
down, and glucose-6-phosphate in its turn will be more slowly
oxidized. Thus we see how the rates of substrate breakdown (and of
oxygen consumption) are related to the amount of free energy needed
by the cell.
The ADP + ®OH ~ ATP system is also coupled with reactions of
the citric acid cycle: the conversion of a-ketoglutarate to succinate
(p. 95), and the oxidation of cycle intermediates by DPN- and
TPN-linked dehydrogenases and by succinic dehydrogenase. Again,
136
CONTROL OF METABOIj,ISM
therefore, the rate at which the cycle operates is determined by energy
requirements. But the Achilles H~d...of the cycle in higher animals
is oxalacetic acid, for we have seen how in carbohydrate starvation,
and deranged metabolism, aceto-acetic acid tends to accumulate
(p. 107), so that animal cells are son;tetimes deprived of ATP that
would have arisen through the oxidation of 'acetyl'. Perhaps the
glyoxylic acid cycle (p. 105), or something akin to it, has been lost
in the course of evolution.
Although feedback mechanisms do not represent the only form of
, primitive' control open to a unicellular organism, it seems quite
likely that they govern the chief routes of energy supply. Among the
higher animals and plants, some hormones probably act by influencing this sort of machinery, although other hormones, naturally
enough, must be expected to act in other ways. However such ideas
may need modification-and doubtless they will-at least they
encourage the hope that the complexities of metabolic control can
be reduced to a set of straightforward principles. 'Natura,' said
Newton, 'enim simplex est.'
137
APPENDIX
COMPLEX FORMULAE
The formulae that have been put into this Appendix for the most
part represent 'carrier' molecules, or components thereof-complicated and unwieldy structures in themselves, but aiding the transport of simple entities like hydrogen, phosphate, acyl groups, etc.
They have been excluded from the text because for many biochemical
purposes it is more important to know that a compound X can enter
into reactions of the type X + y ~ XY than to know the structure
of X down to the last detail.
Adenine (I). On the system of numbering recommended (1952)
by the Chemical Society, adenine is 4-amino-purine; in the older
literature it is 6-arnino-purine.
NH2
I
./.c..........
N
~/4 sC;--- 9~
13
11
sCH
HC~ 1 6C-......... 7 /
~N/
(n
N
H
Adenosine (II). Adenosine is a nucleoside (p. 18), the nitrogen in'
position 7 of adenine being joined by a ,B-glycosidic linkage to carbon 1
of D-ribose. The pentose sugar is in the furanose configuration.
Hence adenosine is 7-,B-ribofuranosido-adenine.
Adenosine monophosphate; AMP; adenylic acid (III). Adenosine
monophosphate is a nucleotide (p. 18); the diphosphate (ADP) and
triphosphate (ATP) are derived from it as indicated on p. 20. The
older name adenylic acid is still convenient and often encountered.
138
COMPLEX FORMULAE
Adenosine
(ID
o
CH 2- o - L H
OH
I
OH
Co-enzyme A; @SH (IV). This nucleotide can be regarded as
being built up from ADP, plus an extra phosphate on carbon 3' of
the ribose, to which structure are attached residues of pantothenic
acid, CH20H.C(CH 3h.CHOH.CO.NH.(CH2h.COOH and 2mercapto-ethylamine NH2 . (CH2h. SR.
Co-enzyme I: see Diphosphopyridine nucleotide.
Co-enzyme IT: see Triphosphopyridine nucleotide.
139
0,
o
2
CH
II
o-p-O
- ,
-~-O-CH2
b
I
t-
OH
H CH 3- ,
3
CH
tHOH
lo
lH
(lH2)2
lo
LH
I
(CHzh
lH
(IV)
o-
CO. NH 2
o
R.CHz-O
-~-O-~-CH2
I
bOH
(V)
140
N
~
II
R CH2--Q-P-0-P-0-CH2
I
I
OH
OH
(VI)
'active formaldehyde'
attached here
NH
__.'IN'''-._
H2N-~
C/
I /~"'-._
II
N~
C
lH
"'-._CH 2
I
"-0-
,......CH-CHrN
NH
CO
I
IH
CH-COOH
I
(CH 2h
I
COOH
(VlII)
141
APPENDIX
Diphosphopyridine nucleotide; DPN; Co-enzyme I (V). Writing
adenosine (above) R.CH20H, the remainder of the molecule is as
shown in (V). The biochemical name diphosphopyridine nucleotide
for this molecule is unfortunate, since it does not contain diphosphopyridine. The substance is a dinucleotide, because it contains the
sequence base-sugar-phosphate twice over (cf. p. 18), and as one
of the bases is nicotinamide, a more appropriate name would be
nicotinamide adenine dinucleotide.
Flavine adenine dinucleotide; FAD (VI). Writing adenosine (above)
as R.CH20H. the remainder of the molecule is illustrated in (VI).
This compound contains a residue of the 5-carbon alcohol D-ribitol,
a reduction product of D-ribose.
Flavine mononucleotide; FMN; riboflavin phosphate (VII). Riboflavin is vitamin B2 •
Tetrahydrofolic acid: THFA (VIII). This substance is tetrahydropteroylglutamic acid, and is made up of a tetrahydro-amino-pterin,
p-amino benzoic acid (p. 43), and glutamic acid. the pterins are_a
g_roup of compounds chemically not unlike purines, and...were first
isolated by Sir F. G. Hopkins from the wings of butterflies In the
synthesis of serine from glycine the 'active formaldehyde' unit is
attached in the position shown.
Glutathione (IX). y-glutamyl-cysteinyl glycine, so called because
the link between the glutamic acid residue and the rest of the molecule
is through the ')I-carboxyl of glutamic acid and not through the
a-carboxyL Where glutamic acid enters into the composition of
proteins it appears to be always a-linked.
l'
ex,
HOOC-CH-CH2-CH2-CO-NH
I
NH2
I
I
CH-CO-NH-CH2-COOH
CH z
JH
ax)
Thiamine (X). For the formation of thiamine pyrophosphate
(TPP) see p. 91.
142
COMPLEX FORMULAE
j
NH2
I
II
CH:r--C~_/CH
"N
3
C=C--Cl-h--CH,OlI
~C'-.....
+/
~
C-CHr-N~
J
H
I
-
-
1~CH-S
;
Cl-
(X)
CH 2 - etc., as in DPN (above)
(XI)
i
H
HC/C~N
HtT;--hCH,_o_~_o_L0~H
R . /1
I I I'o~
o
OH
OH
(Xm
Triphosphopyridine nucleotide; TP N; Co-enzyme II (XI). The distinction between TPN and DPN is that the former contains an extra
phosphate attached to carbon 2' of the ribose in the adenosine
moiety.
Uridine diphosphate glucose; U DPG (XII). Uridine is a nucleoside
composed of uracil (a pyrimidine) and D-ribose, and two phosphates
link this structure to glucose in UDPG.
143
APPENDIX
'"----l.L--CHrCHz-COOH
CH 2
I
IH2
COOH
(XIII)
""-
""-/
N-
..
/
~: Fe++ :N,
.y
.,
N-
~
/""(XIV)
Uroporphyrin (XnD. This substance, properly called uroporphyrin
III, is set out here to complete the story of porphyrin synthesis
(p. 110). The iron of the iron-porphyrin prosthetic group of cytochrome-c (p. 53) is probably bound in the following form shown
in (XIV).
144
INDEX
Actin, 132
'Active formaldehyde', 124, 128
Acyl dehydrogenases, 101
Acyl mercaptides, 16, 17
synthesis from, 108
Adenine
structure of, 138
synthesis of, 128
Adenosine, structure of, 18, 138
Adenosine diphosphate
reactions of, 18, 20
structure of, 18, 138
Adenosine monophosphate, structure
of, 18, 138
Adenosine triphosphatase, 132
Adenosine triphosphate
hydrolysis of, 18
in muscular contraction, 132
structure of, 18, 138
synthetic reactions of, 65, 79, 86,
101, 124, 127, 129
Adenylic acid, see Adenosine monophosphate
ADP, see Adenosine diphosphate
Alanine
in metabolism, 120
structure of, 25
Alcohol, formation of, 90
AIdolases
in photosynthesis, 67, 69, 72
in Warburg-Dickens pathway, 85
Amino acid oxidases, 11 5
8-Amino laevulic dehydrase, 110
Ammonia, detoxication of, 124
AMP, see Adenosine monophosphate
Amylases, 77
Amylo pectins, 74
Amyloses, 73
Aneurin, see Thiamine
Arginase, 127
Arginine
in metabolism, 123
structure of, 26
Argininosuccimc acid, 127
Arrhenius, S., 40
Asparagine, 125
Aspartase, 113, 125
Aspartic acid
10 metabolism, 113, 120, 125
structure of, 26
Astbury, W. T., 28
ATP, see Adenosine triphosphate
Bacteria, see Micro-organisms
Baddiley, J., 91
Bassham, J. A., 70
Belitser, V. A., 60
Benson, A. A., 63
Bergmann, M., 1I 1
Bloch, K., 108, 129
Braunshtein, A. E., 115
Brown, D. M., 131
Buchanan, J. M., 128
Calvin, M., 63
Carbamyl phosphate, 126
Carbon dIOxide, fixation of
in animals, 98, 107
in bacteria, etc., 98
in photosynthesis, 64
Carboxydismutase, 66
Carboxylase, 90
Catalase, actIOn of, 40, 55, 115
Cathepsins, 113
Cellobiases, 78
Cellulases, 78
Cellulose
breakdown of, 78
formatIOn of, 75
Chloroplasts, 62
Chnstian, W., 48
Citric acid cycle, 92, 104, 105, 128
136
Citrulline, 126
Co-enzyme I, see Dlphosphopyridine
nucleotide
Co-enzyme II, see Triphosphopyridine
nucleotide
145
INDEX
Co-enzyme A
in fatty acid oxidation, 101
in oXIdatIve decarboxylation, 91, 95
structure of, 139
Cohen, P. P., 126
'Condensing enzyme', 93
Cori, C. F., 79, 80
Cori, G. T., 79, 80
Creatine, synthesis of, 123
Creatine phosphate, 132
Creatine phosphokinase, 132
Cysteine
in metabolism, 123
in proteins. 27
structure of, 26
Cystine, structure of, 27
Cytochrome-c, 53
rH value, 59
Cytochrome oxidase, 53
Cytochrome reductases, 52, 54, 55
Dakin, H. D., 100
Deacylases, 104, 105, 107
Deamination, 113
Decarboxylation, of amino acids, 119
see Oxidative decarboxylation
Dehydrogenases
action of, 47
aerobic, 51
anaerobIc, 51
DPN-Iinked, 48, 56, 135
rH values, 59
TPN-linked, 48, 56, 136
Deoxyribonucleic acids, 130
Dextrins, 77
Dickens, F., 81
Dihydroxyacetone phosphate
in photosynthesis, 67
in glucose breakdown, 84, 87
6: 7-Dimethyl-isoalloxazine, in flavoproteins, 50
1: 3-Diphosphoglyceric acid
in photosynthesis, 67
in glucose breakdown, 87
Diphosphopyridine nucleotide
mechanism of action, 49
structure of, 48, 142
Dixon, M., 57, 79, 94
DPN, see Diphosphopyridine nucleotide
Eijkman, H., 76
Elliott, W. H., 124
Embden, G., 86, 100
Embden-Meyerhof pathway, 86, 103,
134, 136
Energy, activation, 9, 39
free, concept of, 8
heat, 8,10
Energy-poor bonds, 14
Energy-rich bonds, 14
Engelhardt, W. A., 132
Enol phosphates, 15
Enolase, 88
Enoyl hydrase, 102
Entropy, concept of, 10
Enzymes
active centres of, 34
compositIOn of, 22
denaturation of, 40
inhibitors of, 41
nomenclature of, 30
solubility of, 31
specificity of, 37
Epimerases, 69, 71, 85
Erythrose-4-phosphate
in photosynthesis, 68
in Warburg-Dickens pathway, 83
Euler, H. von, 48, 79
FAD, see Flavine adenine dinucleotide
Fat and carbohydrate, interconversion
of,104
Fatty acids
oxidation of, 100
saturated, 99
unsaturated, 100
Feed-back control, in metabolism,
135
Fildes, Sir Paul, 43
Fischer, H., 109
Flavine adenine dinucleotide
mechanism of actIon, 50
structure of. 50, 142
Flavine mononucleotide
mechanism of action, 50
structure of, 50, 142
Flavocytochromes, 56
Flavoproteins, 50
metallo-, 52
rH values, 59
FMN, see Flavine mononucleotide
Free energy, see Energy
Friedmann, E., 100 .
Fructose-I: 6-diphosphate
in Embden-Meyerhof pathway, 86
146
INDEX
Fructose-I: 6-diphosphate (cont.)
in photosynthesis, 68
In Warburg-Dickens pathway, 84
Fructose-6-phosphate
in Embden-Meyerhof pathway, 86
in photosynthesIs, 68
in Warburg-Dickens pathway, 82, 84
Fruton, J. S., III
Fumarase, 95
Gale, E. F., 1I9, 131
Genes, 118
von Gierke's disease, 107
Glucose, synthesis of, 97
Glucose-I-phosphate
in glycogen formation, 80
in starch formation, 73
Glucose-6-phosphate
breakdown of, 80, 86
in glycogen formation, 80
in starch formation, 72
synthesis from glucose, 79
Glucose-6-phosphate dehydrogenase,
81
Glutamic acid
in metabolism, 115, 120, 124
structure of, 26
Glutamic dehydrogenase, 116, 121
Glutamine, 124
Glutathione
structure of, 142
synthesis of, 129
Glycine
in metabolism, 122, 128
structure of, 23, 25
Glycogen, 50
Glycolysis, 90
Glycosidases, 38, 77
Glyoxylic acid cycle, 105, 137
Green, D. E., 101, 115
Greenberg, D. M., 122
Guanidine phosphates, 16
Gunsalus, I. C., 119
Gurin, S., 121
Hanes, C. S., 73, 129
Harden, Sir A., 48
Hexokinase, 79
Hill, R., 63
Histidine
in metabolism. 117
structure of, 25
Homogentisic aCId, 121
Hopkins, Sir F. G., 76, 129, 142
Horecker, B., 70
Hormones, 4, 21, 134, 137
Huxley, A. F., 133
Huxley, H. E., 132
Hydrogen ion concentration, 31
Hydrogen peroxide, 8, 51, 55
Hydroquinone, oXldation-reduction
system, 57
jJ-Hydroxy acyl dehydrogenase, 102
Hydroxyproline, structure of, 26
Imino aCIds, 114
Insulin, structure of, 29
Isocitritase, 105
Isoleucine
in metabolism, 117, 120
structure of, 25
Kalckar, H. M., 60
Kamen, M. D., 63
Karrer, P., 50
Kearney, E. B., 52
Keilin, D., 52, 56
a-Ketoglutaric acid, 93, lIS
p-Ketothiolase, 102, 104, 107
Knoop, F., 93, 100
Kornberg, H. L., lOS
Krebs, Sir H. A., 92, lOS, lIS, 126,
135
Kritsman, M. G., 115
Kuhn, R., 50
Lactic acid, oxidation of, 46
Lactic dehydrogenase
of animals, 47, 49
of yeast, 56
Lardy, H. A., 60
Lehninger, A. L., 60, 101
Leloir, L. F., 75
Leucine
in metabolism, 117, 120
structure of, 25
Lipases, 100
Lipids, 99
Lipmann, F., 14, 60, 91, 101, 126.
129
a-Lipoic acid
in oxidative decarboxylation, 91
in photosynthesis, 63
Lohmann, K., 18,91, 132
Lutwak-Mann, c., 79
Lynen, Fo, 101
147
INDEX
Lysine
in metabolism, 117
structure of, 26
Lyubimova, M. N., 132
Mahler, H. R., 52
Malic dehydrogenase, 95
'Malic enzyme', 98
Malic synthetase, 106
Maltases,78
Mann, T., 79
Martius, C., 93
Maxwell, J. c., 135
Menten, L. M., 37
Methionine
in metabolism, 117, 123
structure of, 26
Meyerhof, 0., 48, 79, 86
Michaelis, L., 37
Micro-organisms, 2, 6, 43, 76, 78, 114
Mitochondria, 2,31, 108
Morton, R. K., 56
Moyle, J., 94
Muscle, contraction of, 20, 132
Mutation, 118
Myofibrils, 132
Myosin, 132
Myrbllck, K., 48
Neuberg, C., 90
Neuberger, A., 110
Newton, Sir Isaac, 137
Nuc1eosides, 18
Nucleotides, 18,48, 50, 131
Ochoa, S., 60, 63, 98, 103
Oils, 100
Ornithine cycle, 126
Oxalacetic acid
decarboxylation of, 98
in citrIC acid cycle, 92
Oxalosuccinic acid, decarboxylation of,
94
Oxidase
cytochrome, 53
xanthine, 39, 51, 52
p-Oxidation,IOO
w-OXIdation, 101
Oxidative decarboxylation, 91, 95,
105
Oxidative phosphorylation
'respiratory chain', 60, 88
'substrate level', 88, 95
Pauling, L., 28
Peat, S., 77
P-enzymes, 74
Pentose phosphate isomerase, 70, 82
Pentose phosphate pathway, 81, 103,136
Pepsin, 36, 111
Peptidases, III
Peptide linkage, 23, 111
Peptide synthetases, 129
Peters, SIr R. A., 42, 91
pH scale, 31
Phenylalanine
in metabolIsm, 117, 121
structure of, 25
Phosphatases, 68, 69, 71, 72, 85,97
Phosphatides, 99
Phosphoenolpyruvic acid, 89
Phosphoglucomutase, 72
6-Phosphogluconic dehydrogenase, 81
3-Phosphoglyceraldehyde
in photosynthesis, 66
in Embden-Meyerhof pathway, 87
in Warburg-DIckens pathway, 82
3-Phosphoglyceric acid
in photosynthesis, 66
in Embden-Meyerhof pathway, 87
Phosphohexo-isomerase, 72, 84, 86
Phosphokinases, 65, 79, 86, 89, 97,132
Phosphorylases, 73, 80
Photolysis of water, 62
Polynucleotides, 131
Polypeptides
breakdown of, 111
structure of, 24
synthesis of, 129
Popjak, G., 104
Porphobilinogen, 109
Porphyrins, 109, 144
Proline, structure of, 25
Propionic acid, metabolism of, 103
Proteinases, 111
Proteins
metabolism of, III
structure of, 23
synthesis of, 129
Pyridoxal phosphate, 116, 119
Pyrophosphates
formation of, 15
see Adenosine diphosphate and
Adenosine triphosphate
Pyruvic acid
decarboxylation of, 91
formation of, 46, 89, 98
148
INDEX
Q-enzymes,74
Racker, E., 70
Ratner, S., 126
Reichard, P., 126
R-enzymes, 78
rH scale, 57
Ribonucleic acids, 130
Ribose-5-phosphate
in photosynthesis, 70
in Warburg-Dickens pathway, 82
Ribulose-I: 5-diphosphate, 65
Ribulose-5-phosphate
in photosynthesis, 65, 71
in Warburg-Dlckens pathway, 81
Rimington, c., 109
Robinson, Sir R., 110
Rose, W. C., 117
Ruben, S., 63
Sakami, W., 122
Sanger, F., 28
Schardinger, F., 39, 51
Schuster, P., 91
Sedoheptulose-I: 7-diphosphate, 69
Sedoheptulose-7-phosphate
in photosynthesis, 69
in Warburg-Dlckens pathway, 82
Serine
in metabolism, 114. 124
structure of, 26
Serine dehydrase, 114
Shemin. D., 109
Singer, T. P., 52
Smith, E. L., 44
Snell, E. E., 119
Speck, J. F., 124
Starch
breakdown of, 77
formation of, 72
Steroids, 108
Stickland, L. H .• 114
Stocken, L. H., 42
Straub, F. B .• 48
Succinic dehydrogenase, 52, 55, 136
Sucrose, 75
Szent-Gyorgyi, A. von, 93, 133
Tetrahydrofolic acid
in metabolism, 124
structure of. 142
Theorell, H., 50
Thiamine, structure of, 142
Thiamine pyrophosphate
as co-enzyme for aldolase, 68
as co-enzyme for decarboxyJatlons.
91,92
formation of, 92
6:8-Thioctic acid. see ex-LIpOIC acid
Tluokinases, 101, 103
Thompson, R. H. S., 42
Threorune
in metabolIsm. 117
structure of. 26
Todd, Sir A. R., 18, 131
TPN, see Triphosphopyridme nucleotide
Transaldolase. 82
Transamination, 115
Transamidination. 123
Transketolases
in photosynthesis, 68
in Warburg-Dickens pathway, 82.
83
Transmethylation, 124
Transpeptidation, 129
TncarboxylIc aCid cycle, see Citric acid
cycle
Triglycerides
structure of. 99
synthesis of. 105
Triosephosphate dehydrogenase
in Embden-Meyerhof pathway, 87
in metabolic control. 135
in photosynthesiS, 67
Triosephosphate isomerase
in Embden-Meyerhof pathway
84
in photosynthesis. 67
in Warburg-Dickens pathway, 86
Tnphosphopyridine nucleotide
mechanism of action, 49
structure, 48, 143
Tropinone. 110
Trypsin, 30, 36, 111
Tryptophan
in metabolism, 117
structure of, 25
Tyrosine
in metabolism, 121
structure of, 25
Urea, 126
Uric acid, 128
Uridine diphosphate glucose, 75
structure of, 143
149
INDEX
Valine
in metabolism, 117
structure of, 25
Vishniac, W., 63
Vitamins,76
Warburg, 0., 48, 50,81
Warburg-Dickenspathway,81,103,136
Werkman, C. H., 98
Williams, R. R., 91
Wood, H. G., 98
Woods, D. D., 43
Xanthine oxidase, 39, 51, 52, 55
Xylu]ose-5-phosphate
in photosynthesis, 68
in Warburg-Dickens pathway, 82
Yeast, see Micro-organisms
Young, W. J., 48
Z-enzymes, 78