Download 1 Glucose: evolution`s favorite flavor… In any metabolism course

Glucose: evolution’s favorite flavor…
In any metabolism course, you will hear a GREAT deal about glucose. It is in many ways
a universal participant in much metabolism, and connected in some way or another with
nearly all metabolism (* another thing you will hear a lot in metabolism are very reliable
rules of thumb that are not always entirely true. This is not because your professors are
lacking in informational integrity. Rather, it is because there are many versions of
metabolism out in the wide and widely varying world of evolving organisms, so
exceptions abound. What is amazing is that there are so many cases where an idea,
concept or rule is indeed almost always true). Thus, we will talk about glucose many
times in many ways (mer-ry Christ-mas…to yooouuuu (couldn’t resist)). We will discuss
its structure, its metabolism, its synthesis, its participation in building other things, its
regulation, its mis-regulation, and a little bit about it’s evolutionary ascendency to the top
of the metabolic heap. After this initial section, we will find ourselves coming back to it
for some of those topics. To some extent, perhaps we are being a bit “specieist”, that is,
perhaps we are being mammalian chauvinists, because we and other mammals are
absolutely dependent on maintaining glucose levels in our bloodstreams above certain
critical levels for our survival, and below certain levels for our long-term health. But
microbiologists, ichthyologists, herpetologists, and phytologists don’t despair: almost
without exception, every participant in life’s rich panoply of organisms loves, wants,
craves and preferably uses glucose for energy whenever possible.
Energy? Who said anything about energy? I just did, in a brilliant literary trick called
“foreshadowing” which in other situations might also be called going off half-cocked,
blowing the punch line, or getting ahead of myself. But not for long, because one of the
main uses of glucose in all organisms is to provide chemical energy. That is, energy that
is obtained by chemical reactions involving the glucose itself, or molecule derived from
it. Since chemistry is such a central part of why glucose is important, and really, how we
think about metabolism, let’s formalize our definition of chemistry. What is it? Chemistry
is the study of how atoms form molecular structures, the properties that result from those
structures, and how bond making and bond breaking occurs in forming or altering those
molecules. Included in these considerations are the rate of such processes, the energy
involved in such processes occurring, and the rules that govern which things can and can
not happen. Since a lot of metabolism involves carbon-containing molecules, much of
what we will discuss is somehow connected to organic chemistry, that is the rules and
pathways by which carbon-containing molecules are formed, altered and dismantled. This
is why organic chemistry (and the chemistry needed before it) is a prerequisite for any
reasonable metabolism course. Now, I know that sounds like an oxymoron (reasonable
metabolism), but one of the goals of these writings is to bring some semblance of reason,
or at least reasonable information density, to this notoriously dense field. And our use of
organic chemistry will reveal some of that reasonableness, because there is good news,
my brothers and sisters: we only need a little organic chemistry to understand a LOT of
metabolism.
So if chemistry is the study of bond making and bond breaking, then how does chemical
energy fit into this idea? It is fairly simple (beware of the professor that says something is
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fairly simple: it’s the fairly part that will fill up your nights with flashcards and studying).
Glucose has a molecular structure, that is, a covalent arrangement of atoms, that can be
altered by new bond making and bond breaking to give up energy. You actually already
know this, whether you know it or not. What do I mean by that? Well, think about the last
time you sat in front of a fire (I hear that each month there is a wild bonfire at Black’s
Beach…). As you know, the burning of the logs in a fire gives off serious heat energy,
and you can even harness that energy to cook things, or if you are really interested in
destroying the mood, running a steam turbine or something similarly new-fangled with
the bonfire. The reason I bring up a burning log in a discussion of glucose’s chemical
energy is that a burning log is essentially many, many molecules of glucose undergoing
oxidation, that is, bond making and bond breaking with oxygen to produce new smaller,
more stabile molecules and… heat. This is precisely the chemistry that is used by
biochemical systems to capitalize on glucose, but they do it in a much more dignified,
and much less mood-setting, way. We will go into some serious detail about how glucose
undergoes chemical changes that give up lots of biological energy, but first we need to
talk a little about the glucose molecule, and its structure. The goal being to not ever give
you a piece of information that is not needed to understand aspects of metabolism (unless
that information it TOTALLY BOSS!)
The structure(s) of molecules. I have often said that the big problem with molecular
biology and biochemistry is that while the questions aren’t that hard to understand, they
are very hard to answer because everything is so darn small. After all, we deal with
biological molecules, nature’s nanotech, which are on the nanometer scale. Because of
that feature, we always have to represent things in models or cartoons that simulate that
which we can not see. When you think about it, it is amazing how much we know about
molecule and how detailed our knowledge of their shapes and interactions in physical
space. Nevertheless, a big part of biological chemistry involves how we represent that
which we can not directly see. You know this well, since O-chem is almost entirely done
by scribbling and altering little cartoons of C’s H’s, O’s, a few fancier atoms like Mg and
Br, and various lines to represent more or fewer electrons. Since there are a number of
ways of representing molecules to emphasize different structural or chemical aspects, we
have to be clear about we do this. One of the great things about this course (IMHBO (in
my humble, biased opinion) is that the student finally gets to see a lot of examples of Ochem that you learned in class, and that directly pertain to how we are living things. Cool.
But much of it depends on representing structures, and the various ways of doing so
warrant a little review. Taking an example from pure O-chem, here are three ways of
representing butane, C4H10, the volatile gas found in cigarette lighters. The most
information-rich
version has all
the bonds to each
atom, and is what
you might first
draw on the first day of O-chem, or in your first chemistry class, because it shows every
sigma bond. The middle one has a somewhat abbreviated look but still includes all the
atoms, since you can see all 4 C’s and all 10 H’s, and how they are associated with each
atom. This is often the style that is used in day-to-day O-chem. The one of the left is the
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most abbreviated version in which each carbon is an end or a bend (catchy) and the
hydrogens are simply not drawn. Although it looks like this structure is incomplete,
organic chemists use it all the time because the convention is that sufficient H’s are
present to complete all the carbons with the indicated number of sigma bonds. For
example, one of the two “middle vertices” (a bend) has one sigma bond to each of two
neighboring carbons (so two C-C sigma bonds total) and so the assumption
is that the remaining electrons that these normal Cs need to be in a stable
molecule (8 in various bonds total) are provided by Hs. This is a very
common abbreviation used all the time in O-chem and in biochem as well. It works
because carbon-containing compounds are so well behaved. When there are double of
triple bonds, the rules still apply. Just to be thorough, let’s do one more “abbreviation
series”, and there will be a few examples in the problems. The picture shows various
renditions of 2-butene,
and to be very specific,
trans 2-butene. Again,
all three structures are of
the identical molecule
and the “no hydrogen”
version (on the left) is unambiguous because the use of Hs in fulfilling the valence
requirements of carbon in these organic molecules is so predictable. Organic chemists use
either the middle representation, or the “no hydrogen” versions a lot, or some hybrid of
those two to get their points across. But sometimes the full “every bond and atom”
version has its uses, as you will see below). Use of these structures will become second
nature, but it is good to start with everyone on the same page.
Most of the time, we will not be dealing with simple hydrocarbons, but rather organic
molecules that include oxygen, nitrogen, etc. When there are such “heteroatoms” (as they
are called to distinguish them from boring C and H) then there are still choices in
representation that are similar to those shown above,
but the heteroatoms and their associated hydrogens
are always depicted. We will use two examples:
good old ethanol, C2H5OH (top of figure), molecular
friend of mankind for many millennia and one of the
simplest oxygen-containing organic molecules we
know, and a simple neurotransmitter (and nutritional
supplement) gamma-amino butyric acid (GABA;
bottom of figure). To save a little space we have left out the anal-retentive version with
all the bonds and atoms, and just show the two common versions to get the point across.
Notice that the GABA has two heteroatom arrangements that are very important in
biochemistry: the amino group and the carboxylic acid group. At physiological pH these
will have the familiar charges by picking up or losing H+ hydrogen
ions, and so the actual in-cell representation of GABA would
include these additional details, which we will encounter many
times in our descriptions of various metabolites such as things from
the Krebs cycle or amino acids.
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Finally we can start looking at the structure of glucose. You may think that this has been
an over-detailed diversion, but especially in these early writingt, we are trying to serve
many masters, from those who feel Tin Man-rusty with their O-chem all the way to those
to write 20 step organic syntheses on dining hall napkins to challenge their Chem major
friends. To the denizens of these poles I say: “remember now?” and “hang in there, you’ll
get your chance for new challenges soon enough”, respectively.
Glucose, Lord of the Rings- Glucose has an empirical formula C6H12O6, which is the
same as many sugars (galactose, mannose, arabinose, etc.) that are very different
biochemically (they even taste different). This tells us that the detailed structure is the key
to understanding glucose’s important biochemical features. The way one most often sees
glucose is as a six-membered ring, as
shown here. I want you to notice
several things. First, this molecule is
fancy enough that we assign numbers to
each carbon, as shown in the picture.
From 1 to 6. This particular ring form is
known as !-glucose. Which probably
gives you some worry because that must mean there is at least one more form. True that!
Please notice that the six-membered ring is not composed of the six glucose carbons, but
rather has five and an oxygen that reaches from the 5 carbon over to the 1 carbon. Look
at the next structure, called "-glucose. Which carbon is different? It is the 1-carbon
which now has an OH facing up instead of down. That’s it. Why does
this happen? It turns out that in solution, alpha and beta rings of
glucose are in equilibrium, and are constantly oscillating from one
form to the other. Oscillating! Already! Our first structure is actually
two. Ahhh! Don’t freak out. In fact, this is a very reasonable
occurrence based on simple organic chemistry of glucose, which is,
when you look at it correctly, an aldehyde. That fact is revealed by a
third way to represent glucose, in its linear or open form, as shown. In
this form, the aldehyde nature of glucose is obvious, since the 1-carbon
is… an aldehyde. That is, it is of the form R-COH in which there is a
carbonyl carbon at the end of a hydrocarbon chain. Remember? If
there is a carbonyl in the middle of a chain, it is a ketone and we will
encounter many ketones in this course.
Swingin’ rings: the anomeric forms of glucose- The interchanging alpha and beta
forms of ring glucose are directly due to the chemistry of aldehydes. Aldehydes undergo
easy nucleophilic attack by oxygen in surrounding water, or those of
OH that occur in the other parts of the sugar molecule. When an
alcohol attacks an aldehyde (comin’ through the rye…) the product
that is formed is called a hemiacetal. Despite the fancy and
cumbersome name, it is a very simple structure. The basic reaction
occurs between alcohols and aldehydes in solution. A simplified
version of the reaction is shown in the figure, using generic R1 and
R2. In aqueous solution, an alcohol’s O will attack an aldehyde
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carbonyl to make an adduct that simply rearranges the extant atoms, as shown in the
picture of the generic reaction. That is a hemiacetal, and this adduct is in equilibrium with
the free alcohol and aldehyde; hence the double arrow. It is through this reaction that
linear glucose forms the familiar six-membered ring: the O of the glucose the 5-carbon
attacks the carbonyl of the 1-carbon to form a hemiacetal. The fancier version of the same
chemistry occurring with the glucose
molecule is shown, for the case of !glucose forming. Because in this case
the OH and the C=O are on the same
molecule, you get get a sixmembered ring. Remember from Ochem that the –COH aldehyde
structure is planar (due to SP2
hybridization), so the 5-OH can
attack the planar 1-C=O from one
either side. One of these yields the
alpha form, and the other results in the beta form. Because this hemiacetal formation
reaction is in constant equilibrium, any one glucose molecule is constantly undergoing
opening and closing (hence the double arrow again), so any solution of glucose has a
mixture of alpha and beta form, with a teeny bit of linear glucose that is transiting
between the closed ring forms that are favored. These different forms of glucose are
called anomers, and they are a special feature of glucose having a 1-carbonyl and internal
oxygens that can attack to from a cyclical hemiacetal. Although it is physically possible
that other Os in the glucose structure could also form similar structures with the 1carbonyl, the six membered ring is overwhelmingly favored and is the only form that is
discussed in polite circles.
Spaghetti and furniture… it’s all about anomers- The alpha and beta anomers of
glucose are employed in very different ways in biology. This is despite the fact that the
cartoon representations look almost exactly alike. We will mostly focus our discussions
on alpha-glucose, because it is used in nearly all of the biochemical reactions we observe
both in utilization and storage. This is because the enzymes (more forshadowing) that
recognize glucose only recognize and process the alpha form, even when abundant beta is
present in a glucose solution. This exquisite specificity is a routine feature of any
enzymatic processes, and as we will see in the enzyme section, things like hands-ingloves, or lock-and-key are appropriate analogies to describe why it is the case that
enzymes and other large molecules can act so picky. But the two glucose anomers allow
us to make a beautiful point about the remarkable structural specificity of biochemical
processes, and help explain why a house does disappear after a couple rain storms. One
of the ways that glucose is stored in both plants and animals is in long polymers made by
removing a water from the 1-OH of one
molecule and the 4-OH of another to link them
together. This is called a 1-4 glycosidic bond,
as drawn. Of course, the next 4-OH (on the
left) can be similarly linked to another
incoming glucose allowing formation of long
5
chains of glucose. Sweeeet! !-glucose polymers are called starch when found in plants
and glycogen in animals (it is no coincidence that glycogen has the archaic name “animal
starch”). They are very similar, although glycogen has more branches. We will discuss
the exact way these chains and glycogen are made later.
Organisms of all stripe can metabolize alpha-linked polymers of glucose, usually in the
form of starch, from the carbo-loading marathoner eating a pound of spaghetti to the
mold on that 6 week old bread experiment in your fridge during finals. But beta 1-4
glucose polymers are a whole different story. That type of glucose polymer is known as
cellulose, and despite its similarity it is incredibly difficult to metabolize. When we want
more fiber in our diets to improve gastric function, we increase the ingestion of leaves
full of cellulose that simply passes through the gut, providing structural assests to the gut
environment while any starch in the vicinity has long been broken back down to free
sugars and taken up by the gut. So while polymers of alpha glucose are almost
universally digested by living things, only very few creatures can handle the beta linked
cellulose. Even the voracious termite, who makes a living eating wood, requires help with
this task. It turns out they harbor a symbiotic anaerobic eukaryote in their guts that is able
to break down cellulose, and these guest are so unique and useful that the termite depends
upon their presence in the gut for its wood-eating lifestyle and survival. So alpha and beta
glucose literally determine why we can live in houses make principally of beta-linked
glucose (cellulose) but would never think of trying this with a similar composition of
alpha-linked chains, which would be metabolized to a gelatinous mess after few wettings
or a damp season. “Honey.. someone ate the porch!”.
Harry Potter and the Half-Burnt Molecule- Many of you drive to school, and know
that the combustion of hydrocarbons provides the energy that brings you here with almost
no effort. That reaction can be written as shown, where the hydrocarbon has the generic
formula CnH2n+2, which is true for any saturated hydrocarbon:
CnH2n+2 + (n+n/2 +1/2) O2
nCO2 + n+1 H2O plus heat!!
Hyrdocarbons have no oxygen, and if you look at oxidation of hydrocarbons as
proceeding by oxygen reacting with them in the making and breaking of new bonds, then
each carbon is going to gain covalent bonds with oxygen to end up as CO2. None of the
carbons of hydrocarbon have any Os bonded to them, and so they have the maximum
potential for oxidative liberation of energy. That is why hydrocarbons are such good fuels
and for the same reason as we will see later, why fats are similarly very good fuels. On
the other hand, glucose is about half way there; each of the carbons has one oxygen
linked to it in a sigma bond, but the carbons are not yet CO2. So glucose is sort of “halfburnt”. There is still a lot of energy that can be obtained from glucose by oxidation, as we
will see. But the presence of those sigma bonded OHs makes glucose readily water
soluble, easily recognizable by transporters and enzymes, and yet still able to give up lots
of energy through chemical manipulations. We will now explore who this chemical
energy of glucose is unlocked, after introducing the main playa of energy metabolism,
called ATP.
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Glycolysis: breakin’ it down with glucose
As you can guess with all this glucocentric chatter, there must be reasons for such an
emphasis on this universal molecule. As you know from burning a newspaper or a log,
there is a lot of chemical energy that can be derived from glucose. Since chemical energy
is the business of using bond making and bond breaking (chemistry) to store and release
energy in useful ways, we need to discuss another major player in Earth metabolism,
called ATP. It is safe to say that ATP is the main way that the many millions of species
on our planet safely store chemical energy to use as needed. The nature of those needs we
will discuss in more detail later, but suffice it to say that the structure of ATP has allowed
a lot of ways for biochemical systems to use the energy captured in its structure. So let’s
get to it.
ATP, the 20$ bill of the cell- ATP stands for adenosine triphosphate. It structure is
shown and two things jump out out to the casual (molecular) observer. First, it looks a
heck of a lot like the building blocks for the polynucleotides that allow the storage and
implementation of genetic information. And that is because it is
one of those building blocks: ATP is one of the four nucleotides
that is used to build RNA, and its deoxy cousin dATP is used to
build DNA. So ATP has illustrious roots. Nature is very good at
using the same thing for different purposes, and ATPs double
life as an RNA building block and the principle source of energy currency is just another
example of evolution’s tendency to make do with what is around. In this treatment of
metabolism, we will only talk about the metabolic roles for ATP, and as you will see, this
is a huge part of its molecular “life”.
The second thing that jumps out is the three
phosphoric acid groups all in a row, all
bristling with negative charge, as indicated in
the drawing. Because this molecule is always
depicted at physiological pH, the phosphorus
atoms are drawn in phosphate groups, that is,
in which the phosphoric acid groups are
appropriately ionized for pH7. (NB: it is a
general (and not formally taught) role of chemistry nomenclature that the “ic” acid forms
the “ate” ion: phosphoric acid: phosphate; acetic acid: acetate; carbonic acid: carbonate.
See?) The depicted structure is fairly rigorous, showing the oxygens, the bonds, the
phosphates all in their ungainly chain. Another common way to depict the phosphate
group is either with a Pi (for inorganic phosphate, since there are no carbons in the group)
or like this: P to just keep things tidy. You will see these used interchangeably
because usually we are not concerned with the detailed acid-base chemistry, or the details
of the phosphate’s bonds to the oxygens, but rather the transfer of these Pi (see?) groups
onto and off of the ATP molecule.
Water on, water off- Much of the chemistry, that is the bond making and bond breaking,
that we will observe in this treatment of metabolism involves the opposing processes of
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hydrolysis and dehydration. That is, the breaking of a bond by the addition of a water
molecule (hydrolysis), or the formation of a bond by the removal of a water molecule
from the components being bound together (dehydration). I know you have seen this;
some examples that may come to mind are amino acids joining together to from peptide
chains, or simple sugars (like glucose) being combined to make polysaccharides. Let’s
apply this thinking to ATP losing one of its phosphates by hydrolysis. We will then build
on thinking about this reaction in understanding the ways that ATP energy is stored and
harnessed all over biology. The release of the third phosphate from ATP by hydrolysis is
shown below. We are ONLY considering the last phosphate and the one it is attached to.
The remainder of the ATP molecule is simply called “etc.”. The usual way people
describe this is “water is added across the P-O bond being broken”. What really happens
is a bit fancier, but the net result is indeed water providing an OH to replace the Pi that
has just left its roost in the ATP molecule. Because your author is lazy, the double arrow
is used to indicate the reverse reaction, in this case dehydration synthesis. The hydrolysis
of ATP gives up a lot of energy, and that energy can be used in a variety of ways. The
reason that Pi gives up a lot of energy is that the triple phosphate in the ATP molecule is
not a very stable arrangement: all those negative charges make for a phosphate that would
be a lot more stable if it were free in solution. Furthermore, when a free phosphate group
is in aqueous solution, there are more resonance forms available than when it is locked in
ATP, so it’s a win-win situation in terms of energy release. This is the fundamental
chemistry of ATP. Almost all uses of ATP as an energy source capitalize on the energy
of hydrolysis of ATP. All else is involves the details of coupling that reaction to the
desired energy-requiring process. And the opposite is true. Essentially ALL modes of
energy acquisition (with some exceptions) involve using chemical energy to run the
energy-requiring dehydration reaction: that is, to form ATP from Pi and ADP. So what
we want to observe is how the breakdown of glucose allows the formation of ATP from
the more stable parts ADP and Pi. That is, how the breakdown of glucose allows the
reaction above to run from right to left spontaneously. It is not a mystery, and there is no
such thing as deficit spending in biology. The reason that ADP and Pi can be converted
into ATP by glucose breakdown is that the breakdown of glucose gives up more energy
than it takes to form ATP from its parts. Period. This is always the case. No exceptions.
So the reaction(s) we want to examine in our first foray into actual metabolism are those
that allow the breakdown of glucose to occur in such a way as to allow ATP to form. This
actually occurs in two very different ways. In the first, a very simple set of chemical
transitions allow direct transfer of a Pi group from a very high energy molecule directly
to ADP, to form ATP. This process is called glycolysis. In the second set of reactions, the
energy of glucose oxidation (combustion) is captured and then used to form ATP from its
components. So now, I am happy to say, we are ready to examine what happens in
glycolysis.
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The simple principle: the domino theory of thermodynamics- Glycolysis uses a very
simple way to form ATP from ADP: a molecule is generated in the course of glycolysis
that has a Pi attached to it in such a high energy state that transfer of that Pi to ADP is a
downhill process, that is, it gives up energy rather than requires it. There are plenty of
molecules with attached Pi for which this would be the case. It is important to realize that
ATP is not at some kind of “energy pinnacle”. It is comfortably in the middle of those
molecules that have attached Pi. Molecules that have an attached Pi that is even more
prone to come off will be able to transfer that Pi to ADP. So glycolysis works by forming
molecules with Pi attached that are even more prone to release the Pi by hydrolysis than
ATP. Thus, the net effect of releasing the Pi from the donor molecule to ADP as the
acceptor is the production of ATP in a “downhill” process. Let’s think about this a bit
more. Suppose there was a molecule called Y-OH that can have an attached phosphate.
That molecule is called Y-Pi
Suppose an enzyme exists that allows the transfer of the Y-attached phosphate to ADP to
from ATP. Now you know that ATP is storing a lot of chemical energy, something like
31 kJ/mole, that can be released by going exactly the opposite way: converting ATP to
ADP and Pi. So this formation of ATP is going to cost energy. But as long as the
hydrolysis of Y-Pi into Y-OH and Pi gives off more energy than the conversion of Pi and
ADP into ATP, the process will occur in an energetically downhill manner (negative #G,
spontaneous) and ATP can be formed. How do we tell what sort of molecules can
participate in the successful production of ATP? Well, we need only look at the
hydrolysis reactions of each. For Y-Pi, the hydrolysis reaction is drawn as shown, with
the Y-Pi depicted as Y-OPO32- The more energy release upon hydrolysis, the more prone
Pi is to leave this
particular molecule
and go somewhere
else. When we make
ATP, we are doing the exact opposite reaction: a dehydration that couples ADP and Pi by
producing a water. That is, we are running a hydrolysis reaction backwards. So we can
cast the transfer of Pi from Y-Pi to ATP as a hydrolysis (Y-Pi losing its phosphate) and
hydrolysis run backwards (ADP gaining a phosphate). See?
“So why complicate this by breaking it into two reactions?” you might ask. The reason is
this: when we consider the transfer of Pi to ADP in this “two reaction” way, we can
evaluate the ability of ANY Pi-containing molecule to successfully drive formation ATP
from ADP simply by knowing the energy of
hydrolysis for the other phosphate-containing
molecule Y-Pi. If the forward hydrolysis
reaction for the Y-Pi donor gives off more energy than the identical hydrolysis reaction of
ATP to ADP and Pi, then the Y-Pi donor can be used to produce ATP. Why? Because the
total free energy (#G) for Pi being transferred from the Y-Pi donor to ADP is the sum of
the hydrolysis energy of the Y-Pi plus the energy required to run the ATP hydrolysis
reaction backwards, adding a Pi to ADP with the production of a water. If the forward YPi hydrolysis reaction produces more energy than that produced by hydrolysis of ATP,
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then the reverse reaction, formation of ATP by dehydration, can be driven by the
hydrolysis of Y-Pi. This is the true power of thermodynamics: the energy of a complex
reaction can be predicted from any combination of reactions that yield the one you want
to understand. In this case, the hydrolysis of Y-Pi to form Y-OH + Pi combined with the
condensation of Pi + ADP to form ATP is the combination of reactions we use, and the
energy of the transfer reaction is the sum of the energies of the two processes. So why,
you still ask, is this useful? Because the energy of
hydrolysis is easy to measure, and has been tabulated
for many phosphorylated compounds. This previously
obtained and available information can be used by
anyone anytime to predict the ability of a
phosphorylated compound to successfully convert
ADP into ATP. It is no more complicated than
thinking about how to lift a heavy object. To lift a
heavy object, you must use the energy of dropping a
heavier object (see picture). So long as a bigger object
(man on left) is going downward, a big object (man on
right) can go upward. In energetics, there’s no free
lunch.
Glycolysis: making phosphates restless… Glycolysis is a set of ten reactions, and we
will look at all of them. But before that, let’s be clear on the outcome. The main function
of glycolysis is to produce two distinct donor molecules that can transfer a Pi group to
ADP to make ATP in an energetically favorable way. So this pathway makes real-life
versions of the high
energy Y-Pi molecule
described above. The two
molecules are shown in
the picture, with their
names, 1,3 bisphosphoglycerate, and
phosphoenoylpyruvate. In
each case, the energy given off by hydrolysis of one of their phosphates is much greater
than the energy given off by hydrolysis of ATP. Thus, hydrolysis of either donor has
sufficient energy to drive run the ATP hydrolysis reaction backwards, driving the
conversion of ADP into ATP. These high energy phosphates are each part structures you
would not encounter that often. The first (1,3 bPG) is an “acid anhydride”, that is, a
dehydration product of two weak acids (phosphoric and carboxylic). As you may recall
from O-chem, these things are very reactive; in fact acid anhydrides are often used as
reagents in organic syntheses. The other is an enoyl phosphate, that is, the phosphate is
bound to an OH formed by a carbonyl on the 2-carbon (where the phosphate is in the
picture) making a tautomer to from a double bond and an OH. Again this is a very
reactive molecule. Both of these molecules have “anxious” phosphates that will produce
a lot of energy upon hydrolysis; enough to successfully transfer that Pi to ADP. So either
donor can successfully transfer its phosphate to ADP to produce the much-desired ATP.
So the name of the game is taking glucose and producing these two phosphate donor
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molecules, and using each to make an ATP from an ADP. So we blew the joke!...we gave
away the punchline of glycolysis by describing what happens first. It’s why the movie
Titanic was so unpopular… everyone knew that the ship sinks! But since this is not
comedy and biochemistry is no joking matter (except when funny) it is not clear why this
is a bad idea. So let’s delve into glycolysis.
Since we know the “goal” of glycolysis, at least in terms of making ATP, is to make the
two phohsphorylated donor molecules shown above from glucose, you can already
imagine a couple features of the reaction pathway. First, each only has three carbons
while glucose has six. So somewhere along the way glucose is going to get cut or
chopped up. In fact, the glucose is cut cleanly in half giving us two 3 carbon molecules
from which our two “cranked up” phosphate donors are made. That is why it is called
glyco-lysis (sugar breaking …get it?). Second, you will notice that glucose has no
phosphates, while our donors have them, and their presence is the critical feature for our
transfer scheme to work. So you can guess that somewhere along the way, phosphates are
going to be added to glucose, or its parts.
The preparatory phase: metabolism’s first great irony- It turns out that for neutral
glucose, C6H12O6, to enter glycolysis (or most other metabolism for that matter), it must
first be made into a phosphorylated form called glucose 6-phosphate, shown in the
picture. This is one of the phosphates that ends up in the 3-carbon donor compounds that
directly transfer phosphate to ADP. But here’s the irony (“an outcome of events contrary
to what was, or might have been, expected”). In order to add the phosphate to glucose,
ATP is used! So the first thing that happens in glycolysis is the consumption of ATP to
make ADP. So who
said biochemistry has
no similarity to our
economy? Don’t
worry, the situation
gets better, and
although glycolysis goes “into the red” in terms of ATP production (since so far we have
consumed it), a net profit is made, and net ADP is converted into ATP when all is said
and done. The correct aphorism in this case is “it takes money to make money”. The
enzyme that adds phosphate to things from ATP is called a “kinase”, and in this case, the
Pi from ATP is being added to the 6 carbon of glucose by “hexokinase”, which comes in
several versions. They all catalyze this reaction, but their curve shape and regulation is
different. The reaction for hexokinase is shown above.
You may have noticed that each of our two “cranked up” donor compounds 1,3 bPG and
PEP shown above are phosphorylated, so it is perhaps not surprising that a second
phosphate is added to the other end of the molecule, on the 1-carbon. But first, the
glucose is isomerized into a different sugar, called fructose, which is a familiar member
of the diet. Fructose is exactly like glucose in composition, (C6H12O6) but is a ketone
instead of an aldehyde. So first an isomoerase is used to alter the glucose-6P into
fructose-6P. This is some pretty simple chemistry, and you might guess that this reaction
has a low free energy since the two products have almost identical stability. The basic
11
conversion of glucose isomerization into fructose is shown (just the
sugars), and the “textbook” version is shown as well. “WHAT!?”
you say, those two reactions look totally different!”. Yes and no.
That sugar-only version is indeed what the enzyme
phosphoglucoisomerase does, converting the glu aldehyde into the
fru ketone. But how to the rings shown in the actual reaction fit into
this? Remember those anomers we spoke about above, the alpha
and beta forms of glucose? Well, that goes on with fructose as well,
and again, the 5-OH attacks the carbonyl to form the hemiacetal
ring but now it is a five membered ring. So looks different but not
very different at all. The glucose-6P is opening and closing as we described above, and
the isomerase catches the open chain from (that has the aldehyde structure like in the “Rgroup” depiction) and converts it into the ketone form, thus making fructose-6P. The
fructose-6P similarly forms anomeric rings, and
in fact they are the predominant form of fructose
in solution. So usually textbooks (you are free to
confirm this) show the phosphoglucoisomerase
converting six-membered ring glucose-6P into
five-membered ring fructose-6P, which makes
the enzyme reaction look a lot more complicated
than it really is. The enzyme is really just catching the rare, aldehyde open chain form
and converting that into the open chain 2-ketone, fructose-6P. Then the 5-OH attacks the
2-carbonyl to make the five membered ring form of fructose-6P, as shown.
Phosphosfructokinase (PFK-1): more deficit spending- The next reaction along the
path involves another expenditure of ATP to produce fructose 1,6 bisphosphate
(Fr1,6bP), with the 1 and 6 carbons of fructose each with an
attached phosphate, shown in the picture. Now we have used
two ATPs, and any frugal person might wonder how this
series of events is going to lead to making an ATP profit…
Although we won’t dwell on this now, it is worth noting that
this second kinase, PFK-1 is an incredibly important enzyme
due to the profound regulation is undergoes in the control of mammalian metabolism. So
we will revisit phosphofructokinase later. It is worth noting that while glucose-6P can be
used for a number of metabolic reactions, once we have produced fr1,6bP, we are
strongly committed to continuing with glycolysis; there is not an easy way out that
wouldn’t cost a lot in terms of wasted ATP. Reactions of this sort in metabolic pathways
are called “committed steps”, and they are often sites of multifaceted regulation. So now
we have doubly phosphorylated fructose, with two phosphates at each end of the
molecule. It’s time to break up…
Now we are ready for the eponymous (“of or related to the name of something”) reaction,
that is, the reaction for which glycolysis is named. The lysis step. In this reaction, the
fructose bisphosphate is cut in half, to give 2 three carbon compounds, each with a
phosphate. The chemistry of this process is actually something pretty simple that you
have encountered in O-chem (see why we have that prereq…?), and was called an aldol
12
condensation. In that
version, shown below
in “R-group” style, a
ketone and an
aldehyde (the “ald”)
combine to give an alcohol (“ol”) adduct. That is the reaction that is employed by the
enzyme aldolase (a-ha!) to cleave the fr1,6bP into to 3 carbon molecules, which are, not
surprisingly an alcohol and an aldehyde. It is really an aldol condensation in reverse since
we start with the alcohol, and end up with a ketone and an aldehyde. Namely,
dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). You may
recall that this condensation works because the carbon next to the ketone can more
readily release a proton to make a carbanion, and that carbanion can attack an aldehyde to
make the alcohol.
One of the tricks in metabolism (and much of science really) is learning what to ignore in
order to understand an idea. You will become experts at this, because in many cases the
actual chemistry of a process is fairly straightforward if you can see the “R-group”
rendition that you are used to from your O-chem or
other classes, but the actual metabolites have all sorts
of other bells and whistles that do not participate in the
chemistry, but do distract the already-overburdened
student. The aldolase reaction is a good example. To
the right is the aldolase reation drawn with the
“player” atoms written in red, and all the other “bell
and whistle” atoms in black. You can see that the aldol
reaction is just like the simple one drawn above, but
all the non-participant black atoms make it a bit hard
to see that, perhaps. Practice makes progress, if not perfect…. The top 3-carbon molecule
is DHAP (dihydroxyacetone phosphate) and the lower molecule is G3P, glyceraldehyde3-phosphate. We have split the glucose!!
Payday is finally coming- Wow. It looks like we are going from bad to worse! Now we
have doubled the number of molecules involved and spent two ATPs without getting
anything useful out. But both of these situations will change. First, only glyceraldehyde3-phosphate (G3P; bottom 3 carbon molecule in reddened figure above) is used for the
next steps. But what about the DHAP? Fortunately, each DHAP is converted into G3P in
an isomerization reaction that has identical chemistry to the one that converted glucose
into fructose above, but is accomplished by the enzyme
triose phosphate isomerase (TPI). It is really the exact
same chemistry (ketone <---> aldehyde (see figure)) with
a different enzyme, because enzymes are so picky about
which molecules they assist in reactions. Because only the
G3P is employed for subsequent steps, TPI makes the
glycolytic pathway much simpler. Both the DHAP and the G3P undergo the same payoff
reactions, after the DHAP is converted into G3P. That is why some people draw
glycolysis like a “coathanger” with the aldolase reaction causing the branch, and the TPI
13
isomerization being the bottom of the hanger (see picture). You can also represent
glycolysis in this way with an emoticon, ---<|__ which is really a get-a-life-icon, to be
precise.
It is worth taking the time to get
comfortable with the naming and numbers.
Particularly, become comfortable with
which carbons on the DHAP and G3P
correspond to the original six carbons on glucose. Before the TPI step, the identities are
completely unambiguous. Each of the six carbons in glucose is exactly one of the carbons
in these two three carbon molecules But after TPI makes the DHAP into another G3P,
then a given G3P carbon can come from either one of two positions in the original
glucose. Convince yourselves of this fact, and this idea.
Look at the G3P molecule structure (right). There is one phosphate on
that 3 carbon molecule. That is going (someday) to be added to an
ATP. But if that were the only thing to occur in the payoff phase, with
each of the two G3Ps made from a single glucose, then we would only
get back two ATPs, and we would have a break-even phase instead of a
payoff phase. So as you might predict from that consideration, another
Pi must somehow enter the picture, and that is what occurs with the
fanciest enzyme of the lot, called GAPDH, or glyceraldehye-3-phosphate dehydrogenase.
From the name, you can already tell that hydrogen is going to be removed from the
reaction; in fact and actual oxidation is going to occur. We will delve into how this is an
oxidation later, but it is one. It is a wild reaction that shows the degree to which an
enzyme can make an unlikely thing happen with great probability. And what happens
(finally) is the production of our first of two Pi donors (remember Y-Pi) that has a
phosphate sufficiently energized for spontaneous transfer to ADP. The chemical reaction
is shown below. Note that an NAD+ is required for this reaction to occur.
GAPDH, the alchemist of glycolysis- G3P is an aldehyde, and those carbonyls are
always getting attacked in way or another. The one of 3-phosphoglycerate is shown with
a little green arrow. Chemistry is a cruel world… In the case of GAPDH, the nucleophile
that attacks GAPDH is actually an –SH group that is part of the enzyme molecule,
residing in the active site where the enzyme does its catalysis. Recall that S is in the same
chemical period as O, and its chemistry is in many ways similar. So just like the 5-OH in
glucose attacks the aldehyde function to make an anomer, an SH group that is part of the
active site of GAPDH attacks the aldehyde of GAPDH to make an analogous adduct
(called a thiohemiacetal). But then the enzyme magic happens. Because enzymes are big,
they can contain a lot of molecular information. The complex active site of GAPDH has a
binding site in just the right place for the molecule NAD+. NAD+ is an electron carrier
14
that we will see often; it gets reduced to NADH, so it is an oxidizing agent. The most
natural way to view the electrons that NAD+ accepts is as the molecular species called a
hydride ion, or an H-. You might recall that in O-chem there are many times when a
powerful reducing agent is used molecules in a synthesis by providing hydrides anions,
that is H- , to attack things. The most common source of hydrides in O-chem is lithium
aluminum hydride or LiAlH4 which we often saw in those fancy multi-step syntheses in
O-chem. It turns out that the hydride ion is a very natural way that electrons are removed
from and added to metabolites in biochemical reactions, and NAD+ (and its close
anabolic cousin NADP+) is a main grabber and releaser of this form of electrons.
The G3P bound in adduct to the S of the active site presents a tempting morsel for the
NAD+ that just happens to be sitting in the right place: an H- (in the form of a sigmabonded –H on the 1-carbon) that can be abstracted, causing the oxidation of the attached
aldehyde group, and the reduction of the NAD+ to its other common form, NADH. We
will hear a lot more about NADH and its formation. For now, its job is done in the
reaction cycle of GAPDH, and it floats off as one of the products of the reaction. So now
what? Well, what we end up with is a very reactive intermediate, called a thioester, that
can participate in a reaction to make the desired phosphate donor. Amazingly, a PO43group, yes, the Pi that we usually see being spit off in reactions, serves as the nucleophile.
This is a testimony to the remarkable power of enzymes. The chances of Pi being able to
do this in solution are very low, but the enzyme allows this reaction to occur with great
alacrity (alacrity: eagerness; liveliness; enthusiasm; promptness; speed), and viola, we end up
with a the 1,3 bisphosphoglycerate (1,3bPG); the acid anhydride donor mentioned above
that can form ATP from ADP by transfer of the phosphate that is now on the 1-carbon.
So this is an example of the energy held in
the glucose structure being cleverly
liberated to create a highly active and
transferable phosphate, in the form of the 1
phosphate of the 1,3 bPG.
It is worth noting that in the course of this
reaction, we have seen the conversion of
an aldehyde (left side of picture; the 1 carbon of G3P) into a carboxylic acid, since the
form we ended up with after the NADH was produced is a thioester, that is, an adduct of SH with a carboxylic acid (right side of picture). So the reaction is an oxidation, changing
–COH to -COOH, but the change is hidden by being bound to the sulfer group of the
enzyme. Again, the chemistry is simple, but the molecules are complex. This is one way
that metabolism can be made less cryptic, by seeing through the non-involved structures
to the simple reactions that are occurring. It’s all about what we ignore.
Finally some ATP… The 1,3BPG molecule has two phosphates. The one at the 3
position has been riding along since back when it was part of the glucose structure (either
on the 1 or the 6 carbon.. we can’t tell). But the Pi attached to the 1 carbon is in a
phosphoanhydride bond that is very reactive and more than energetic enough to
successfully and efficiently transfer to ADP to make the desired ATP. That reaction is
catalyzed by the enzyme phosphoglycerate kinase, and the simple reaction is shown
15
below. The carbon from which the Pi is lost is marked with green again. But wait a
minute…phosphoglycerate KINASE!? “I thought that kinases transferred phosphates
from ATP to other things. This reaction is transferring phosphate from something (1,3
BPG) to ADP to make ATP; just the opposite. What’s the deal?” Don’t fret. The reason
this is called phosphoglycerate kinase is that the reaction will work both ways (it has to
by the definition of a catalyst). In the early days of enzymology, this enzyme was studied
backwards, by adding lots of ATP to a reaction mix with 3-phosphoglycerate (the product
of our ATP regenerating reaction) to form 1,3 BPG and ADP. The reaction will be
catalyzed in either direction, but in glycolysis we are looking at the production rather
than the use of ATP. There are a number of enzymes that are named for the backwards
reaction from the usual way the pathway runs. Sometimes it is an accident of
experimental history, and sometimes either direction can occur in different metabolic
situations. In this situation (phosphoglycerate kinase), both cases are true: the enzyme is
named for the history of how it was studied, and there are metabolic circumstances we
will study where the kinase reaction that consumes ATP is used instead of the ATPgenerating reaction we are talking about here. Anyway, now we have made an ATP, and
are left with 3-phosphoglycerate, or 3PG.
Eno-mino-minee-mo- You can already
guess that the lone remaining phosphate
in 3PG will be the next one activated
by chemistry going on in the resident
molecule. You also know, because I
gave up the punch line above, the eventual donor molecule for making the second ATP is
phosphoenoylpyruvate (PEP). This molecule will be featured again in the future when we
think about glucose synthesis. But in glycolysis, the formation of it from 3PG is a two
reaction process that makes good sense. First, since the PEP has its phosphate on the two
carbon, the 3-phosphoglycerate is isomerized to 2-phosphoglycerate, 2PG. This is
accomplished by phosphoglycerate mutase, which basically picks up the phosphate by
attachment to an active-site residue (it happens to be a histidine) and then places it back
onto the molecule at the 2 position. Now, this new resting place for the phosphate is not
very different from the old one, and so it would still not allow transfer to ADP. But 2-PG
can be converted into PEP by removal of water. This is a remarkable trick, because the
energy of water removal is not very
high, but the resulting phosphate is now
very reactive. Sort of like when Wiley
Coyote runs off a cliff in his pursuit of the Roadrunner. He is at the
same level as before, but now can give off a lot of potential energy
by falling to the desert floor far below. Meep-meep! So now we have
the second Y-Pi donor, PEP with a cranked up phosphate, rarin’ to
transfer to ADP. That last reaction is catalyzed by pyruvate kinase, to
16
form ATP and pyruvate, or pyruvic acid. And voila, another ATP is made, and the very
important molecule pyruvate…
We don’t need no stinkin’ bailouts- Now you might be going.. “Wait! We used two
ATPs up there in the preparatory phase, and we got two ATPs out in these reactions.. so
we just broke even!”. But before you shred your visa, recall that the complete glycolysis
reaction involves two G3Ps: one that is formed by the aldolase reaction, and one that is
produced by isomerizing DHAP. So we get two ATPs our for each three carbon input for
a total of four ATP made by clever phosphotrickery (I made that up!), and two consumed
to start the ball rolling. So a net profit of two ATPs. It wouldn’t surprise me if you
already knew from earlier or AP classes, or the Discovery Channel’s brilliant series The
ATP Detectives, or whatever, that the full oxidation of a single glucose yields more than
30 molecules of glucose. So you might be thinking “what’s the use… so many reactions
and so little ATP”. Many people wrongly consider glycolysis to be just a prelude to these
bigger and better oxidative things, but that would be a grave misconception. Glycolysis is
an incredibly important pathway in its own right, and has roles in normal body function,
cancer, stress, evolution, oxygen physiology and sports of all kinds.
Act NADturally- The thing about glycolysis is, like a delicious cookie on a platter of
them at a party, you can’t metabolize just one glucose. A cell has to keep doing this to
reap the energetic benefits. So a key feature of finishing up our consideration of the
glycolytic pathway is ensuring that it can keep running until the energy needs of the cell
in question are met. What does continued glycolysis require? We need more glucose, and
we need ATP (which we got from the reaction) to get things started, and we need ADP to
turn into ATP. But what else do we need? Well, after each use, the enzymes are ready to
keep working, because by their nature they are restored to their original state (this is
another rule of enzymes and all catalysts). We have plenty of Pi floating around. But the
one thing that is critical and not readily available is our new friend NAD+. This reactant
(for the wonderful GAPDH reaction) is a limited resource, and must be regenerated in
order to keep glycolysis going. If there is no NAD+ available, and that would include the
case where all of it was in the form of NADH, then there will not be any glycolysis. It
would halt at the G3P/DHAP step, a dangling coat hanger… So restoration of NAD+ is
critical. This happens by the H- being transferred somewhere else to regenerate the NAD+
reactant. There are two ways of doing this. In one version called fermentation, the NAD+
is added to another metabolite, reducing it and restoring NAD+. In the other case, H- of
NADH is sent to another cellular location to be used for further energy usage. Here we
will discuss fermentation, because when glycolysis is the only way cells get energy, this
is the mode that is employed. There are many cases where glycolysis is the only energetic
game in town, and in those instances fermentation allows it to run robustly and
efficiently.
17
We will discuss two common versions of fermentation. In each, the H- that NADH has
picked up from G3P during the production of 1,3bPG (see above) is deposited onto a
metabolite, thus restoring NAD+ and allowing it once again to participate in glycolysis.
There are many other variations of this, because in the wide world of metabolism a
number of different metabolites could be used as depots for that H-, and so evolution has
found ways to do that. The key to fermentation is that it does NOT require oxygen. So in
cases where O2 is not around, or is not sufficiently available to a cell, or not available
rapidly enough, these fermentation steps are critical for allowing ongoing production of
cellular energy by glycolysis. Because if you do not have NAD+ available, glycolysis
halts at the GAPDH step where it is a substrate. We will focus on two reactions that loom
large in our social and athletic lives. What…? Check it out:
Lactic acid till we catch our breath- Remember that at the end of glycolysis we are left
with the molecule pyruvate, CH3COCOO-, a simple carboxylic acid with a carbonyl
group. Well, one way to scarf up the NADH-bound H- is to react it with a C=O to make
CH-OH. The H- is added, and an H+ from the solution (these are always obtainable at pH
7 in the cell) comes in to complete the new alcoholic carbon. That is what lactic acid is.
So the reaction is as shown, and is catalyzed by lactate dehydrogenase:
One of the places we hear about lactic acid a lot is in exercise. During sudden exercise,
the muscles are told to move whether there is sufficient oxygen or not, and whether the
participant has trained or not. There aren’t many people who would not run from a
tornado just because they hadn’t been to the gym in many months… So often, when
people undergo intense exercise, there is not sufficient oxygen to continue metabolizing
the products of glycolysis, which as we will see is a great way to get energy when
possible. In the cases where oxygen can not enter the picture, the production of lactate is
critical for ongoing glycolytic ATP production. That is why one hears about lactic acid so
much in sports circles. The production of lactic acid is a necessary result of doing
exercise that outstrips a muscle’s ability to use oxygen to get the maximum bang out of
each glucose molecule. Does this happen much? Yes! In fact, in intense sports, like when
Usain Bolt breaks another 100 mt dash world record (as of this writing he’s got to down
to 9.68s, or over 23 mph), the prodigious energy needed for that feat (or you running for
the bus suddenly) is almost entirely anaerobic (no oxygen needed), including glycolysis.
We will talk more about metabolism and exercise later, but this is a striking example of
the importance of glycolysis and other non-oxidative ways to make ATP. Over 90% of
the energy produced for a sprinting race like the 100 yard event is produced by
metabolism that requires no oxygen, such as glycolysis.
The animal world has many examples where glycolysis is a, or even the, big deal. When
a crocodile strikes its prey-and this is one of the fastest large animal actions every
observed- that burst of energy is largely due to glycolysis. So the small amount of ATP
18
made per molecule is not a limiting factor in terms of intensity or ferocity. Basically, a
human can not outrun a crocodile who has decided that they are its next meal. Worth
remembering when in the Outback, or the African subcontinent. There is a cost to this.
After an attack, a croc has to hang out for quite a while to remove the lactate and other
byproducts of this sudden metabolic burst by the more efficient but slower oxidative
metabolism that we will talk about at length below.
See the Coal A Canth- In the case of the crocodile, the metabolic demands of catching a
large and fast-reacting mammal like a gazelle involve a disparity of glycolytic rate vs.
oxygen delivery. There are also animals that live so far away from available O2 that they
use only glycolysis to get all their ATP. The most dramatic example is the coelacanth,
which is a “living fossil”. This massive, weirdly shaped fish was though to have been
exctinct for over 100 million years, due to the existence of fossils from then an long
before. Then, in the early part of the 1900s, a live one was caught in a net, causing an
uproar in the biological and icthyological communities. I distinctly remember reading a
book about the rediscovery of the living coelacanth when I was in 3rd grade; it was one of
my early inspirations. But in my jubilant retelling of the living fossil story, for years I
mispronounced it as “coal-a-canth”, where it is indeed pronounced “see-la-canth”.
Fortunately, all my young science-loving friends pronounced it the same way (ignorance
is bliss!)… Anyway, the reason these guys (coelacanths, not my friends) were thought to
be extinct was that although perfect examples were found in fossils over 100 million
years old, no one had seen a living one despite all the fishing going on all over the world.
The probable cause of this lack of encouters is that they live so very far down in the
deepest ocean that no one stumbles on them. And in those places there is almost no
oxygen to speak of. So the coelacanth lumbers along (there are now movies of them
swimming in their deep dark element) powered almost only by glycolysis. The lactic acid
they produce is simply excreted into the ocean around them, allowing glycolysis to go
merrily along. Although the case of the coelacanth is often quoted in biochemistry text, it
is a reasonable supposition that many organisms in the deepest oceans are similarly
dependent on glycolysis. And it is a good bet as well that there are other organisms that
can grab and use the lactate that is coming out of the coelacanths and anyone else who
happens to solely employ the glycolytic pathway to do the things they want to do.
There are similarly many situations of medical importance in the human body where
glycolysis is a main supplier of energy despite us being completely oxygen dependent. As
you might suspect from the above examples, these are situations where oxygen is not
available or not present.
Alcoholic fermentation: makin’ the demon rum- An entirely different but also
common way that the pesky H- on NADH is removed to restore NAD+ is by the
production of ethanol, CH3-CH2OH, perhaps our oldest molecular friend and foe. The
two carbons from the ethanol come from pyruvate, which you will notice has three. First,
the pyruvic acid is decarboxylated to form CO2 gas, and the intermediate acetaldehyde.
This is why when one makes wine, CO2 is given off. The trick in fermenting wine is to
give the CO2 a chance to escape, while preserving the low-oxygen environment so that
more efficient but less tasty oxygen dependent metabolism is not allowed to occur. This
19
can be done with a number of home made or professional bubblers. My own attempts to
do this in middle school resulted in wildly active fermentations of apple juice set up in
the warmth behind the family furnace. Wildly active but horribly flavored. So I didn’t
become an enologist… Anyway, the acetaldehyde is then reduced by the H- of NADH to
give ethanol; so again a reaction in which a carbonyl is converted into an alcohol, just
like in the production of lactic acid above.
We know that glucose can be fully oxidized into CO2 H2O and energy. So what happens
in fermentation, since the resulting molecules could still be further burned to those final
products? Alcoholic fermentation is usually an “end stage” reaction. In the cases we
encounter, the ethanol is excreted and is not used any further by the fermenting organism,
even though we know that ethanol has more energy trapped in its structure because it
burns so well. Lactic acid is a bit different. Although our friend the coelacanth just
releases it into the deep ocean, athletes and crocodiles can further metabolize lactate that
builds up in a variety of ways to get the more profitable but more-slowly obtained fully
oxidized products. We are getting a bit ahead of ourselves here; these routes of further
oxidation will be fully explored below.
In-cytes about location- One of the features of metabolism that can easily be lost in all
the talk about carbonyl groups and oxidation and ADP is: where does it happen in a cell.
We will try to maintain a constant, vigilant inventory of where things are happening. This
is because a lot of metabolism involves partitioning between different organelles, and the
sometimes surprising movements of intermediates between various compartments in the
course of a metabolic pathway. Glycolysis keeps it pretty simple. It turns out that
glycolysis happens in the cytosol. That is, the “juice” (it is more like a gel because the
protein concentration is so high) that is right inside the plasma membrane. In bacteria,
this is (usually) the only compartment, and in cells, it is a major one. Interestingly, every
type of cell in all three kingdoms uses glycolysis, and it always occurs in the cytosol. It is
very likely that this was one of the earliest ways that arose to capture energy by
metabolism. Of course, a big question is, where did the glucose come from? Right now
we won’t dwell on this, but the evolution of metabolism is one of the great and
fascinating open questions in biological sciences.
As long as we are on the subject of cellular location of metabolism, it is worth
mentioning that organellar compartmentalization of metabolic processes is a big part of
how cells sort out and co-regulate metabolic events that are ongoing, and potentially in
conflict. We will see that there are distinct sites and compartments for different metabolic
events. The regulation of metabolite flow into cells and between cellular organelles will
be an important and sometimes surprisingly complex part of how integrated metabolism
occurs and is regulated in individual cells, whole organs, and active animals.
Pyruvate and acetate
We are now about to embark on the next big part of how glucose is oxidized to CO2,
H2O, and captured energy. The result of glycolysis is pyruvic acid, more commonly
called pyruvate because it is always ionized at normal cellular pH. Take a look at this
20
molecule. Pyruvate presents a little series of carbons in three of the four major oxidation
states: hydrocarbon, carbonyl, and carboxylic acid, in order of degree of oxidation. The
carboxylic acid carbon is basically already as fully oxidized as carbon can get, since it is
really a CO2 group, with a negative charge, that is covalently bound to the end of the
molecule. This negative charge can be removed (which is an oxidation), and the other
two carbons can be further oxidized to get each to the desired CO2 state. What
metabolism does in the next phase involves oxidizing these two carbons, using a
combination of a round metabolic pathway (as opposed to a linear one like glycolysis),
and then employing a remarkable cellular process to form new ATP in a totally different
way from how it occurs in glycolysis. The round metabolic pathway is called the Krebs
cycle, or the citric acid cycle, or the tricarboxylic acid (TCA) cycle, and its sole function
in energy metabolism is to oxidize those two pyruvate carbons by removing electrons
step by step, to use them as reducing equivalents in the later energy extraction phase. But
this requires getting pyruvate “all dressed up” to go to the Krebs ball.
Acetate the unsung fuel- In metabolism, glucose gets all the glory. But the humble two
carbon acetyl group, CH3-CO- is an incredibly important and widely used component in
many aspects of metabolism. Both the breaking down of things for energy and parts, like
we are doing here, and the building up of big molecules from smaller components, that is,
anabolism. One of the first places we see the acetyl group is in the preparation of
pyruvate for continued oxidation. The two “interesting” carbons of pyruvate, that is, the
ones with some potential for further oxidation (left two in picture), are where the acetate
carbons that comprise the acetyl group come from.
Pyruvate is a little “museum of carbon oxidation” (now
there’s a place to take your new in-laws) going from the
highly oxidized carboxyl all the way to basically a
hydrocarbon on the left. How is the still-oxidizable acetyl
group mobilized for action? A complex and enzyme
complex is responsible for the seemingly simple process
that produces an acetyl group from pyruvate. The enzyme is called PDH, or the pyruvate
dehydrogenase complex, and you can already tell that this enzyme is going to be
removing electrons from something in the process because it is a dehydrogenase.
In nearly every case that acetate is employed in metabolism, it is carried to and from
reactions by a specific molecule known as coenzyme A, or CoA, or CoA-SH. These
names are used interchangeable, and the reason they are so useful is because otherwise,
people would have to draw this monster again and again whenever discussing how
acetate moves in and out of biochemical reactions.
Like a lot of carriers or cofactors (often cofactors are
carriers of groups that allow an enzyme to do its
catalytic thing), CoA-SH has a very complex structure
with a fairly simple “business end” that allows the
chemistry that is specializes in to occur. In this case,
the “business end” is the thiol group (red circle), -SH.
This group is intimately involved in the function
because CoA-SH is in the “thioester business”. The
21
key to understanding CoA-SH chemistry is to recall that the S atom is in the same
chemical period (periodic table column) as O, and it behaves in reactions in a very similar
way. Just like the OH group can form a covalent ester with a carboxylic acid, an SH
group can form a thioester with a variety of carboxylic acids. A thioester is the identical
ester linkage between the analogous sulfer-containing thiol group and a carboxylic acid.
Remember that we ended up with a thioester during the GAPDH reaction (office hours
trick: just say yes and check later)? And in that case the thioester could drive the unlikely
nucleophilic attack by Pi? Well, that is because thioesters are not that stable, so it is pretty
easy to remove a carboxylic acid that is in such a bond. This is good news for a carrier
like CoA-SH. You don’t want a carrier to hang on to strongly to something that it is
carrying. Like your mailman: they can’t be too attached to your mail. If he is hesitant to
let your mail out of his hands, he’s not a carrier. He’s a stalker! Joking aside, the CoA-SH
carrier is ideal for carrying carboxyl groups because the adduct is stable enough to use
and ferry about, but not so stable that it won’t let go when it is time to put the acetyl
group (in this case) into metabolism. We will see that the CoA-SH group is responsible
for carrying many carboxylic acids, and the underlying thioester chemistry is identical in
all cases. So this little bit of chemistry will serve you long and well.
Acetate production: a Cofactor Conspiracy- The simple act of converting pyruvate
into CO2, some electrons and an acetyl group that is temporarily attached to CoA
involves a remarkable series of reactions that are all coordinated by the obviously named
enzyme complex called the Pyruvate Dehydrogenase Complex, or PDH. It is a complex
because it entails multiple proteins and multiple catalytic sites, each of which catalyzes
one reaction in a series of reactions needed to get this done. The net reaction of the PDH
(lifted right from
Wikipedia!) hides a lot of
actual biochemistry:
As you now know from
glycolysis, net reactions are
useful to know where we are
going to end up, chemistrywise, but they are very
cryptic in terms of any
understanding of how one gets from the starting and ending molecules. With glycolysis,
this is maybe not surprising, because the net reaction includes ten different enzyme steps.
The PDH reaction looks like a simple net reaction, but it too is hiding some pretty
remarkable biochemistry, enzymology and even biophysics.
“Why should we learn about the PDH?” you may ask. First, the cofactors that are
employed by PDH will be encountered many times, and so it serves as a module for some
fairly general ideas in metabolism. Second, PDH represents an important regulatory
branch point where the cell decides whether to break down pyruvate to get needed
energy, or to stop there and either use pyruvate or go simply with glycolysis. That is, the
decision to catabolize or anabolize. This branch point is so important that some people
have called PDH “the minister of glucose”. Which is a little geeky and grandiose.
Finally, the PDH is a beautiful example of the importance of “supramolecular”
22
organization, that is, the importance of special arrangements of multiprotein complexes in
the cellular execution of complex tasks. And finally, the PDH is just so darn boss! But
that is my bias for sure.
PDH action step 1: TPP capture and decarboxylation: The
first thing that happens is that the useless (in terms of
oxidation) “third carbon” is lost as a carbon dioxide molecule,
and the remaining two carbon portion is covalently captured
by the first cofactor, called thiamine pyrophosphate, or TPP. This cofactor also has a big
structure, but the “business end” of this one is basically a carbon that, due to the
neighborhood it lives in, is highly acidic. That means it can lose a proton to form a
carbanion (not a place that negative people change into beach clothes, but a carbon with a
negative charge due to electron resonance). That proton on the “business carbon” is
removed by a group on the enzyme, allowing the TPP to attack the pyruvate carbonyl (the
middle –C=O) to form a covalent adduct. That is what TPP does in a
variety of processes that call for chemistry in the vicinity of a carbonyl
carbon. So when the TPP attacks the pyruvate, the CO2 comes off, and
the negative charge is compensated by addition of an H+ from the
solution. We end up with an adduct called hydroxyethyl TPP, which you
can see (left) has the carbons of our acetyl group.
PDH action step II: Movement to lipoic acid, a carrier and
a oxidizing agent: The two carbon group that resulted from
decarboxylation of pyruvate has a pair of electrons that must
be removed. In hydroxyethyl TPP, these electrons are
present as the sigma bond to the –H on the carbonyl carbon
of the original pyruvate. The goal is to transfer this group off of TPP and produce a CH3CO- acetyl group, eventually to end up as AcCoA, or CoA-S-CO-CH3. This is done by
moving the CH3-CO- and –H to the lipoic acid cofactor, which has a “business end” of
and disulfide –S-S- in a five membered ring. Now this is a very special carrier. I want
you to look at the pictures of the oxidized (cyclic) and reduced (open) lipoic acid
“business end” shown in the picture. The closed oxidized form is converted into the open
form by picking up and H- and and H+ ion, or equivalently, 2e plus 2H+ (it is the same
chemistry, just a different preference in oxidative bookkeeping). The open reduced from
is basically two sulfhydryl groups, that is, two thiols. The transfer of the two carbon unit
from the hydroxyethyl TPP to lipoic acid produces an adduct
consisting of the acetyl group and the reduced lipoic acid (see left). So
the net effect is that the lipoic acid is carrying both the electrons from
the decarboxylated pyruvate, and the acetyl group that remains after
the CO2 has floated away. Up till now, the acetyl group and the
electrons have run together; chemical roaddogs. But now, they part
ways on the road to catabolism.
Acetate and electrons go their separate ways- now that the acetyl group is part of a
thioester, it is not energetically difficult to transfer it to another thioester bond; namely,
its linkage to CoA-SH. The final enzyme activity of the PDH complex accomplishes this
23
to make the acetyl-CoA we will see in many metabolic processes. A CoA-SH swoops in
and removes the CH3-CO- group from the lipoid acid, leaving the reduced form. The rest
of the PDH actions involve re-oxidizing the lipoic acid back to its useful –S-S- oxidized
ring form. This is done by transferring the electrons to the electron carrier, called FAD to
make FADH2. Another enzyme activity then re-oxidizes FADH2 by transferring those
electrons to NAD+ to make NADH. It is important to note that now the net reaction is
true, and that all of the fancy intermediates, like TPP, and lipoic acid, and FAD, have all
been restored to their original state. One would never know that a whole collection of
reactions have occurred to bring about removal of CO2, transfer of electrons and acetyl
group to a cofactor that allows their separate processing and then the separate production
of molecules that hold the acetyl group and the electrons in separate and independent
molecules. Amazing, huh? The total set of reactions that cause this fairly simple net
reaction are shown below. You can watch a movie of the PDH complex at work on the
BIBC 102 website.
The names and arrangement of the PDH complex activities- We would be remiss to
not name the PDH enzyme activities. But it is important to realize that unlike the purified
enzymes that people often studied in vitro, and that started the field of enzymology, the
enzymes of the PDH form a large “nanomachine” with a 3d configuration that is highly
conducive to the factory-like sequence that takes pyruvate through all the steps that
underlie the fairly simple net reaction shown above. The names of the enzyme activities
reflect the reaction they each catalyze. It is important to realize that the 3d structure of the
multiple proteins involved, that is, the quaternary structure of the multiple subunits
arranged in space, place the different
enzyme active sites in great position to
give a “factory like” effect for the
movement of the lipoic acid arm from
one processing station to the next.
The names are
E1: pyruvate dehydrogenase
E2: dihydrolipoyl transacetylase
E3: dihydrolipoyl dehydrogenase
One of the annoying things about
enzyme names is their length and
complexity. One of the nice things about them is that they are usually fairly obvious
about what reaction is being hastened by the named enzyme. In this case, the E1 is
processing pyruvate by removing a CO2 and placing it on the TPP holder. The E2 is
transering the resulting acetyl group (and those electrons) to the lipoic acid, and the E3 is
then moving those electrons off the lipoic acid onto the more standard carriers, FAD and
then NAD+. Pretty clear names. (Probably the E1 would be best named “pyruvate
decarboxylase, but it ain’t.) I am personally much more interested in your knowning what
an enzyme does than being able to pull one of these thorny names out of your… heads.
Some enzymes feature important ideas and so we focus on them more than others. In the
case of the PDH complex, the big take home message is that often evolution has had
24
sufficient time to hone enzyme activities into multi-protein structures that appear almost
machine-like in their organization and efficiency. They are not machines, and they use
chemical and kinetic principles that would look wildly weird and haphazard if they were
the size of factory machines, what with all the Brownian motion, rapid rotations, kinetic
wobbling and ultra-rapid querying of possible states, but the net effect is a remarkable
efficiency that come by grouping proteins in common pathways into macromolecular
complexes. Another great thing about PDH complex is that the study of this enzyme
complex that catalyzes one net reaction (converting pyruvate into acetyl-CoA with
production of CO2 and reducing equivalents) provides almost a little classroom for
understanding five major coenzymes and cofactors that are used again and again in
metabolism. Here we are front-loading information that will serve you for your entire
journey through biomedicine, biochemistry and molecular biology.
You say acetate-o and I say acetato – We have now found out one way that cells
produce the acetate group. This is an incredibly important and often-used functional
group in biochemistry. From the frequency of use in both catabolism and anabolism, it is
clear that however all this… biochemistry… got here, acetate was on the scene very
early. Almost always, it is found as part of the carrier molecule acetyl-CoA, or CH3-COS-CoA, or AcCoA. The most useful way to look at this “carried acetate” is as a two
carbon “bottleneck” along the metabolic highway. Meaning that this simple two carbon
unit has many uses in metabolism, as a fuel source, as a source of carbon to build bigger
molecules, and more fancy uses like serving as a tag to add to proteins to alter their
functions, or to drugs to metabolize them. Many metabolic pathways charts have acetylCoA right in the middle of the action, and usually in that center region is the circular
pathway we are going to discuss next. But it is important to realize that many molecules
are broken down, or catabolized, into acetate, and many large molecules are constructed,
or anabolized, from acetate. So in that sense it is a bottleneck. Many pathways of
breakdown converge on producing AcCoA, and many pathways of synthesis start with
AcCoA. Bottleneck. As we discuss different pathways, we will see how often this key
two carbon unit plays a role. One of the many “traces” of evolution is the tendency to use
the same parts in different ways. One way to put it is that repurposing is easier than
inventing, and acetate is a great example of the power of using the same thing over and
over again for different purposes.
The Krebs Cycle, or, How to Burn Vinegar- Ask the average person on the street who
have been even marginally exposed to biology to name a metabolic process, or perhaps to
pick one out of a list of names, and they might well say “The Krebs Cycle”. I have even
heard the Krebs cycle mentioned on The Daily Show. It is remembered with fear and
loathing, because often it is shot at the students like some kind of structural buckshot, as
they duck behind their laptops, sometimes on the first day of class. I myself had been
exposed to the Krebs Cycle at least three times before anyone bothered to explain what is
was doing in a bigger picture sense, and how it was valuable to the cell’s function, rather
than simply describing the gory details of the structures, cofactors and enzymes. Yes, we
will go into some of those details, but first we will discuss the function(s), and keep
reminding ourselves of those functions as we roll through the cycle in the pages below.
That way, even when the structures fade in your mind, the functions will hopefully
25
remain, to be rejoined by the newly reviewed structures when the urge grips you, either
for some type of exam, or prelim, or grand rounds, or because the cycle has appeared as
important in your research, or your clinical studies, or in your burgeoning biotech.
The basic chemistry of the Krebs Cycle is the process of converting the CH3-CO group
into two CO2. This is an oxidation process, and so the acetate is literally being burnt. But
as we discussed in the ideas from the section “The Slow Burn”, it is being done a little at
a time, in a series of reactions that are… ta dah!... the Krebs Cycle. As you may already
have known and as we have spoken about, when something is getting oxidized (like the
CH3-CO- group, something else is getting reduced, that is, electrons, (or H-, or e plus H+;
however we want to look at it) are being added to something else. In chemistry this is the
principle of balancing redox reactions, which is the act of ensuring that each electron
removed from some molecular or atomic species ends up being added to some other
molecular or atomic species. It is these electrons that are the key to the catabolic function
of the Krebs Cycle. They are stored up on one of two carrier molecules
and used later to make energy in a series of transfer reactions that
eventually end up making water from O2, which as you might recall, is
a reduction reaction. See, oxidation and reduction. The Krebs Cycle
takes us half way: the CH3-CO groups are oxidized to CO2, and the
electrons produced are stored up in carriers. That’s it. The picture of
Dr. Krebs used for his 1953 Nobel Prize is shown to the right. One
wonders if he also was adept at the chemistry of hair coloring…
The oxidation of acetate
CH3-CO is not the direct
conversion of this two
carbon molecule into
CO2… if only….
Rather, it is added to a
larger molecule called
oxaloacetate (OAA) to form the tricarboxylic acid called citric acid. That reaction is
shown to the right. Note that the CoA-SH is now free, and the acetate has been added by
its CH3- “handle”. Actually, this is familiar chemistry: a carbon next to a carbonyl can be
used as a nucleophile to attack another carbonyl, a lot like in the aldolase reaction of
glycolysis. A distinct name for these reactions that deprives Herr Professor Herr Doctor
Krebs of his deserved shout-out is The Citric Acid Cycle, or the Tricarboxylic Acid cycle
(TCA). These names for the cycle are due to the tricarboxylic acid citrate being
generated in that first reaction. The subsequent reactions of the TCA cycle cause citrate
to undergo a series of rearrangements and oxidations that produce CO2, electrons on
carriers, and oxaloacetate. THAT is why it is a cycle: because the final product of the
reaction pathway (oxaloacetate) is also a substrate of the first reaction. The reaction that
gets the ball, or cycle, rolling is shown here. You will notice that because the acetate
attacks the carbonyl of OAA (oxaloacetate) with the ! carbon, it produces a second -CH2CO2 group in addition to the one that is already on the OAA. Thus, citrate is a
-
symmetrical molecule, with two identical groups (-CH2-CO2 ) on a central carbon with a
third -CO2- and an OH. This is citric acid. It is worth noting that here is one of the places
26
that chemistry does not predict what biochemistry demonstrates. Look again. You will
notice that this is a symmetrical molecule in the strictest chemical sense. That is, there is
only one spacial form; the two mirror images of this molecule are identical, and can be
supermposed. This is different from molecules with chiral centers, such as the L and D
versions of amino acids, etc. Your left hand and right hand are mirror images of each
other, and can not be superimposed. So a pure chemist would say that the two -CH2-CO2groups would behave identically in a typical chemical reaction. For example, if a reagent
were added to citrate that could react with one of the -CH2-CO2- groups in some way,
there would be absolutely no expectation that one of the two identical groups would be
favored over the other. In fact, it would be weird if it were. But as we will see, the
“fresher” -CH2-CO2- group, that is, the one just added by the citrate synthase, is treated
distinctly from the one that rode in on the oxaloacetate. The newer-CH2-CO2- group is
given a free ride through the Krebs cycle before anything happens to it. A chemist would
(and they were) be shocked by this specificity. But a biochemist would understand it
without much difficulty at all. We’ll explain a bit later.
Iso-lating the first CO2- the first thing that happens is that the symmetry of this beautiful
starting molecule is broken. Specifically, the OH is moved from its central position to one
carbon over, to form an isomer of citrate.. isocitrate. This is effected (meaning brought
about; not to be mistaken with affected) by
the enzyme aconitase, in memory of the
intermediate that is generated in the course of
this isomerization, but not shown in the
pictured aconitase reaction. One of the
amazing things about this reaction is that the OH is always placed on the -CH2- of the
“old” acetate, that is, the one that came from the OAA and never the identical –CH2from the newly added one. This is not odd to a biochemist, but a chemist would find this
very surprising. Soon it will make sense to you too! Now we are ready to oxidize. The
first oxidation is an oxidative decarboxylation, that is, removal of electron accompanied
by release of CO2. This is a great strategy for making a reaction go far to the right: a
gaseous, removable product. The electrons are removed by transfer of an H- to NAD, to
produce NADH. This is the first of several “stored electrons” that are produced by the
Krebs cycle. Remember, it is not the oxidation that is useful but the reduction that occurs
in tandem, because it is these “chemically available” electrons (on NADH) that will
eventually allow the production of MUCH ATP. The resulting molecule is called !ketoglutarate, or !KG. Note that this molecule has a similar structure to our old friend
pyruvate: a carboxyl group at the end, and a ketone carbonyl in the next position and then
some other stuff.
27
Another dehydrogenase complex: The next reaction is again an oxidative
decarboxylation, in which the !-ketoglurate from the last reaction is oxidized. But it is
quite a bit fancier than the IDH reaction above. The enzyme that does this trick is called,
not surprisingly, ! ketoglutarate dehydrogenase. However, despite its greater elaboration,
it is in fact quite familiar and another example of evolution using the same thing again in
new ways. Check it
out (picture). See
anything familiar?
Wait! There is our
old friend CoA-SH,
entering the picture to make a thioester, but in this case the end product is the bigger,
dicarboxylic acid succinate, or -CO2-CH2-CH2-CO2-. So we have again lost a CO2, and
captured the electrons in NADH. Does this reaction look like any we have encountered?
Let me give you a hint by describing the other cofactors involved in the operation of the
enzyme that catalyzes this reaction: we need CoA-SH as a substrate, we need both TPP
and lipoic acid in the enzyme, we need FAD, and we need NAD+. It sounds a lot like the
PDH reactions above, which needed all those cofactors, and
performed an oxidative decarboxylation of a keto acid
(pyruvate), releasing CO2, and producing an thioester
adduct with CoA-SH and NADH. And it turns out that this
reaction is catalyzed in precisely the same way as the PDH
reaction of pyruvate, in which it lost a CO2 molecule and
had its !-keto carbon added to CoA-SH. In this case, the !ketoglutarate loses a CO2 molecule and ends up having its
!-keto carbon added to CoA-SH. It is precisely the same chemistry and the same
enzymology, but a separate enzyme complex uses the same E1, E2, and E3 strategies to
do the job. Just like in PDH, the !-ketoglutarate dehydrogenase complex (see?) activates
the substrate by TPP attack, then transfers it to lipoic acid, and from there it is
transferred to CoA-SH. The reduced lipoic acid is then reoxidized by FAD, and the
resulting FADH2 is oxidized by NAD to form NADH. The responsible enzyme complex
!KGDH is both analogous and homologous to PDH. Meaning the separate proteins
evolved from a common ancestor who probably wasn’t quite so picky and could probably
perform oxidative decarboxylations of several !-keto acids.
Another example of Nature making studying difficult… So now we have, like
AcCoA, a thioester linked carboxylic acid (succinate) attached to CoA-SH in a fairly
unstable thioester bond. Succinyl-CoA. This molecule will give off energy upon
hydrolysis, and that energy is harnessed next to make a close cousin of ATP. The enzyme
catalyzing this reaction is called succinyl-CoA synthetase. The four carbon product is
called succinate. The reaction is shown below. Again the backwards reaction is described
in the enzyme name. Sheesh biochemists, get it together! Anyway, the hydrolysis occurs
by an inorganic phosphate attacking the thioester bond, to form a phospho-anhydride,
28
similar to what we see with GAPDH in glycolysis. The normally unfavorable reaction
runs forward because the energy of hydrolysis of the S-CoA bond is much greater than
the energy needed to make the anhydride. The resulting phosphorylated succinate is
employed by the enzyme to add phosphate to GDP to make GTP, which is energetically
nearly identical to converting ADP to ATP, and the resulting GTP is used to convert ADP
to ATP with not much trouble. So we get one example of substrate level phosphorylation,
meaning a high energy phosphate compound (in this case GTP) is directly made by
transfer to GDP from an energized phosphate compound (succinyl-phosphate; a Y-Pi).
This reaction is the “exception” to the rule of the Krebs cycle. In every other case, the
energy is derived from reducing power stored in electron acceptors, which are then used
in a different system we will talk about next. The succinyl-CoA synthetase reaction is the
glaring exception to our generality that the Krebs cycle produces reducing equivalents but
no direct high energy phosphate. If only nature were as concise and organized as our
thinking!
Another way of reducing- most of the oxidations in this cycle involve removal of
electrons from a molecule (the oxidation) and the reduction of NAD+ to NADH (so 2e
plus H+; our standard way of transferring reducing power. You can also view it as an Hbeing transferred as well). Succinate, which is a symmetrical dicarboxylic acid, is next
oxidized in the most simple way imaginable. Two Hs are removed from the middle CH2CH2 to make CH=CH, just like you learned about in organic chemistry when alkenes are
produced from alkanes. The H’s go to FAD, to form FADH2, and the chemistry is more
accurately described as two H atoms (H-dot) being placed on separate atoms of the FAD
cofactor. In both PDH, and !KGDH (the two very similar complexes) we have seen this
reduced cofactor
converted back to FAD
by production of NADH
and H+. But here the
reduced FADH2 has its own identity as an electron carrier. The resulting molecule is
called fumarate, and notice that chemistry tells us it could be either cis or trans fumarate,
that is with the two CO2- being in cis or in trans, but the only form that is produced is the
trans form as shown. So again, we see that there is profound stereoselectivity in the
biochemistry that is not predicted or obvious from the chemistry, and the reason for this
will be obvious soon enough.
“Bearing malate to no one…” We can get one more set of electrons out of this
situation, with a little trickery. We have so far seen two types of oxidation: removal of
electrons in the form of H- simultaneously with loss of CO2, or removal of H2 from a
molecule to make a double bond.
Now we will see one more. First,
our old pal water is added to the
double bond to make malic acid, or
malate. The enzyme is called fumarase. Note that the fumarate has no asymmetric
carbons, but the final product does (the C with an CO2-, an H, and an OH is asymmetric).
And in regular old chemistry we would get either the “D or L” form, that is, some of each
mirror image. But we only see L-malate (just one of them). It’s not so important you
29
know that it is the L form in particular, but rather that it is highly specific, and not
random. Again, chemical intuition alone would predict you’d get some of each (L and D),
but the biochemical observation is that there is very high special specificity. All of these
highly specific spatial observations are not the least bit surprising when one remembers
that enzymes create an asymmetric, 3d space in which the chemistry happens. Like a left
handed surgeon in a crowded operating room. The patient will be lying in a particular
orientation, and the surgeon will be standing on a preferred side of the operating table.
An asymmetric 3d
environment can produce
asymmetric 3d results in a
highly intuitive manner.
More about that later.
So we have a dicarboxylic acid with an OH next to one of the CO2-. The final oxidation
again involves removal of H- and H+ to produce NADH and… the starting substrate
OAA. The same thing we started with, and the reason that the cycle is… a cycle. This
OAA can then be employed to take the next acetate and off we go, again.
Young group, there’s a place you can go!- In thinking about the Krebs cycle, there is an
important subtlety that we need to make sure everyone gets. “Important subtleties” are
the bane of undergraduate existence, because they are cases where some not-so-obvious
things are considered significant to know and remember. So be it. Evolution is not always
student-friendly but we will do our best to make it as accessible as possible. What is this
subtlety? It has to do with which carbons are lost to CO2 in the steps after the two are
added. Look at the structure of citrate: the molecule is symmetrical. Another way to say
that is one can find a mirror plane where one side of the molecule looks
exactly like a reflection of the other. Specifically, the middle C has two
identical --CH2-CO2- groups attached to it, and there are no chiral, or
asymmetric, carbons in the citrate. One of the --CH2-CO2- groups came
along with the OAA (black in picture), and the other (blue in picture) is
the freshly added acetate group from the first reaction (citrate synthase)
of the Krebs cycle. So blue is new. Any chemist looking at this
molecule would say that the two -CH2-CO2-groups are identical, since
the molecule is symmetric. If you added citrate to a reaction mixture that modified or
altered one of the -CH2-CO2- groups, the reasonable prediction would be that either of the
two groups has an identical chance of being modified. We learned that in O-chem. But
the fact is, as first observed in the 1940s that these two identical and symmetrically
placed groups are treated differently by the Krebs cycle. The “old one” is always the one
that first receives the moved OH group when isocitrate is formed, and always the one that
then loses the CO2 in formation of succinyl CoA. Isocitrate is
shown to the right to refresh your memory. So the two carbons
that are lost are never from the newer -CH2-CO2- group. But if the
symmetrical groups are involved, how does the cell know which
is the “old” one and which is the “new” one? What gives, or
gave…?
30
Ogston, and enzymes, to the rescue- When this was first realized, near the end of the
Big Band Era (ask your great grandparents about that music; or look up in
Grandmapedia) it caused quite a ruckus. People claimed that the chemical reactions of the
Krebs cycle must not be correctly assigned, because such chemistry would not
distinguish between these two symmetrical groups. And that is true. But in the Krebs
cycle, this distinction is clearly made. That is because, my friends, this is biochemistry,
and there are key features of biochemistry that make this sort of unobvious
stereochemistry reasonable and intuitive. It took a thoughtful biochemist by the name of
Alexander Ogston (Nature, 162: 963 (1948) to put forth the correct way of thinking about
this dilemma. Dr. Ogston pointed out that if the enzyme (aconitase) that was recognizing
isocitrate was asymmetric, then it would be able to distinguish between these two
identical groups. An enzyme is a completely asymmetric and complex catalyst, and the
citrate will always be “viewed” by the enzyme through interactions that are defined in
space. One way to think about this is to imagine that the enzyme is like a right handed
person working on a production line, grabbing a citrate in the same way each time and
moving the OH with only their right hand. This asymmetry in the enzyme casuses an
asymmetry in the resulting process.
But this leads to another subtlety. If the two acetate groups are distinguishable by spatial
features of the aconitase enzyme, then that must further mean that the synthesis of citrate
is also asymmetric, in the sense that the new acetate is always added in one of two ways
to the OAA to make the citrate molecule, and that is the case. The diagram depicts the
two possible versions of symmetric citrate, with the “new” acetate labeled in red. If you
think about OAA as a planar carbonyl that is somehow attacked by the incoming -CH2CO2- acetate group to make citrate, then two different spatial outcomes are possible.
Either the group can be added from the “top” of
the carbonyl or from the bottom, to give one of the
two forms shown. In a “chemistry” situation like
we think about in O-chem, there should not be any
bias, and we would expect attack from either side
to have identical likelihood. Even odds. That is,
the newly added -CH2-CO2- should be in either
position when oriented with the groups as shown.
But if an enzyme is catalyzing the reaction, the
expectation is that the OAA is held in place in an
asymmetric and complex active site, and the attack
on the carbonyl is spatially directed and occurs in
only one orientation; that is the case, and the new -CH2-CO2- is always distinguishable
from the old one when this biosynthetic citrate interacts with aconitase, the next enzyme
down the line. The technical term for this is that the -CH2-CO2- is added to the “siprochiral” position, because although this molecule is mirror symmetric, it does have a
“left and “right” side. Since the new acetate is always on one of these two sides, the
subsequent reaction by aconitase, always treats the two groups differently, because the
aconitase active site can tell which side is which, it knows left from right! This insight
that enzymes can specifically tell the difference between the chemically identical “left”
and “right” sides of a prochiral molecule is now almost intuitively obvious, but at one
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time it convinced many people that the correct reactions of the Krebs cycle were wrong;
until Alexander Ogston stepped up and made sense of this observation by reminding us
that an enzyme can an almost always does provide a asymmetric environment.
The Krebs cycle is carbon neutral! - So now we have made a complete trip around the
Krebs, or Citric Acid, or the TCA cycle. I like the Krebs cycle because it has a built-in
shoutout for one of the heroes of metabolism, but you will hear all three used here and
there and so it is important to realize they are the same thing. Just to keep things simple
we will use the term Krebs cycle. In this rendition of the Krebs cycle, there are some
generalities that I want you to take home. First, you can see that the net process can be
described as adding an 2-carbon acetyl group (-CH2-CO2-), to the last molecule of the
cycle (OAA) to make the first molecule of the cycle (citrate), and then running a
sequence of reactions that release 2 CO2s and regenerate the original OAA. Not
surprisingly, OAA has 4 carbons, and citrate has 6. Since acetate has two carbons this
makes good sense and any other result would be… weird. So you see that the Krebs cycle
as described is carbon neutral: it involves no net gain or loss of carbon for each turn: for
every turn, two carbons are added as acetate, and two carbons are removed as CO2. And
this has some very important metabolic consequences. Let’s look at the Krebs cycles
balance reaction. For every turn of the Krebs cycle, we get the following balanced
reaction:
AcCoA + 3NAD+ + FAD + Pi + GDP
2CO2 + NADH + FADH2 + CoA + GTP
We put in two carbons as acetate, we get out two carbons as CO2, we also generate three
NADH, one FADH2, one GTP (that can be converted into ATP with almost no cost).
That’s it. There is a very powerful idea here and that is the use of mass balance as a way
to think about metabolism. What do we mean by this? Simply that in chemistry or
biochemistry, mass is not gained or loss. You’ll have to go to nuclear physics for loss-ofmatter type action. In chemistry, atoms enter and exit molecules, they rearrange, but they
are not lost or gained. The formalism that follows from this idea is the balanced chemical
reaction, where we make sure that the numbers of atoms on each side of a chemical
reaction are the same; nothing gained or lost. The balanced reaction for the Krebs cycle is
shown above, and we can use this highly simplified description to hone some intuition
about the Krebs cycle. Ready? Suppose we had put 1000 acetates through the Krebs
cycle. How many CO2 would be produced? So after this 1000 turns of the Krebs cycle, do
we get more cycles, fewer cycles, or the same number? A way to think about this is to
imagine a single Krebs cycle operating. That is, you have a teeny beaker with one OAA
molecule, and one copy of each enzyme. So you are operating a lone Krebs cycle in a
tiny, teeny beaker. You let this Krebs cycle turn 1000 times by consuming 1000 acetates
one at a time. OK, you’re done. How many acetates remain? How many CO2s have been
given off? How many OAAs are there after the last reaction of the 1000th cycle? So you
see, there is no increase in Krebs cycle “components” after many turns of the cycle. Net
effect is conversion of CH3-CO2- groups into CO2 and reducing equivalents. Remember
that!
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A universal furnace- One of the great things about the Krebs cycle is that is can take a
very common fuel, acetate and convert it into CO2 and reducing equivalents, in the from
of reduced carriers NADH and FADH2, for subsequent use in the production of gobs of
ATP. This is great because acetate can be derived from carbohydrate metabolism like we
say with glucose, from fat breakdown like we will soon see. Also, many amino acids can
be converted into Krebs cycle intermediates that then can enter the cycle as well. Thus,
the Krebs cycle is appropriately considered a central “hub” of metabolism, since many
molecules can feed into it as a source of energy. In addition, many of the reactions of the
Krebs cycle are reversible, so there are numerous examples of the cycle being used in the
creation of new things by taking advantage of some of these conversion reactions as well.
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