Download Metabolism and Glycolysis

Ferchmin 2016
Introduction to metabolism in
general and of carbohydrates
in particular
It would have been advantageous if you had reviewed or studied
the structure of carbohydrates before the upcoming lectures.
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Note
I will appreciate if you bring to my attention any errors you may find in my handouts
and lectures (not all at once, please). Suggestions for improvement are always
welcomed.
The best way to communicate with me is at [email protected]
I never check [email protected]
My “home made” web page is www.ferchmin.org
I post all my handouts at http://www.ferchmin.org/MedStudents.html
I must assume that you are familiar with carbohydrate structures. You can study
this topic either using the SIM or my Carbohydrate Structure pptx file.
My handouts are not meant to replace books authored by a team of specialist and
edited by multimillion dollar companies. The objective of my handouts is to help
me to present the subject and to clarify doubts you may have. The final goal is to
prepare you to understand the courses that follow in the next semesters and to
pass the external exams.
The scope of National Board Exams is wrapped in the mist of the American
“burrocracy” (misspelling intended), so I can only use my previous experience and
intuition to guess what they will ask you in the NBE.
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METABOLISM
Summary of this handout:
1) General overview of metabolism. What is life?
2) Metabolic pathways. Anabolic and catabolic pathways.
3) Fate of glucose in: a) whole organism. b) specific types of cells.
4) The glycolytic pathway.
5) Regulatory enzymes of glycolysis.
6) Closer look at selected glycolytic enzymes.
7) Alcoholic fermentation and brief description of the metabolism of
ethanol.
8) Pathway of carbon in glycolysis. Use of 14C in biochemistry.
9) Metabolism of fructose and entry into glycolysis.
10) Entry of other sugars into glycolysis.
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Metabolism is the set of chemical reactions that takes place in an organisms. It provides
energy (from food or other sources), synthesizes and degrades the molecules that form
the organism.
Life could be defined as a system of steady state reactions that take place in an open
system and is endowed with the potential capability of producing similar systems.
For the sake of didactics, metabolism is divided into more or less arbitrarily
defined pathways. Beware, however, that different pathways often share
metabolites.
For every pathway you ought to know:
1) Purpose of the pathway. (Adaptive value for the organism).
2) Molecules going in and coming out? (The starting metabolites and the final products).
3) Place where it happens (organs, types of cell, subcellular compartments).
4) Regulatory enzymes. (Metabolic conditions that stimulate or inhibit the pathway).
5) Organization of the pathway and the formulas of the compounds involved. (The map
of the pathway).
6) Relationship with other pathways. (Shared metabolites, enzymes and regulations).
7) Later, you will have to visualize each pathway interacting with other pathways in
normal and in pathological conditions.
Anabolic reactions consume energy and nutrients to synthesize cell components
like proteins.
Catabolic reactions break down complex molecules and release the energy which
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is conserved in the form of ATP.
Complex
molecules are
in equilibrium
with its
precursors
The precursors can be
incorporated into large
macromolecules or
catabolized.
Many metabolites
are converted into
acetyl-CoA which
is the “fuel” of the
TCA.
The TCA together with
the respiratory chain
produce most of the
ATP.
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What happens when somebody consumes a load of glucose?
Glucose tolerance test
Blood Glucose mg/100 ml
300
Severe diabetes
250
200
Why was the
glucose high after
an overnight fast?
Moderate diabetes
150
100
Where did the
glucose go?
Normal
50
0
1
2
3
Hours after 100 g of oral glucose
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METABOLISM OF GLUCOSE
The metabolism of glucose is different in different cells. Red blood cells, neurons, liver
parenchymal cells, intestinal mucosa, kidney tubular cells, eye lens cells, cornea, testis,
and leucocytes are among those that do not require insulin for glucose uptake. However,
even for those cells insulin is important for other reasons.
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Adipocytes and skeletal muscle are mayor
players in the uptake of glucose from blood.
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It seems that astrocytes use glucose and produce lactate which is used as
energy source by neurons. There is still some disagreement about that.
Glucose (insulin independent)
Glucose (insulin independent)
Glucose-6P
Glucose-6P
Pentose
Phosphate
Pyruvate
Shunt
Glycogen
Lactate
CO2
Acetyl-CoA
TCA
Neurons
Pyruvate
CO2
Acetyl-CoA
CO2
TCA
CO2
Astrocytes
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Glycolysis is an universal metabolic pathway. It occurs, with minor
variations, in nearly all organisms, both aerobic and anaerobic. The
wide occurrence of glycolysis indicates that it is one of the most
ancient known metabolic pathways. In 1860 Louis Pasteur
discovered that microorganisms are responsible for alcoholic
fermentation which is a minor variation of mammalian glycolysis.
In 1897 Eduard Buchner found that cell free extracts from yeast
can sustain fermentation. This opened the possibility of in vitro
study of biological processes and the beginning of biochemistry.
In 1905 it was found that a heat-sensitive high-molecular-weight
molecules (enzymes) and a heat-insensitive low-molecular-weight
(cofactors) are required for fermentation to proceed.
The main aspects of the pathway were determined by 1940,
however, new features are being discovered as we speak .
The following slides are a detailed presentation of
glycolysis.
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GLYCOLYSIS
First stage, priming.
G-6-P is not
committed to
glycolysis
Glycolysis has two stages.
The first primes (prepares) glucose
“wasting” 2 ATP in the process.
Glycolysis is “supposed to”
produce ATP.
Committed
step of
glycolysis
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Continuation of the priming stage of glycolysis
The equilibrium constant for triose
phosphate isomerase favors
dihydroxyacetone phosphate, but since
only glyceraldehyde-3-phosphate is
capable to enter glycolysis by
subsequent reactions all the carbons
from glucose eventually become
glyceraldehyde 3-phosphate.
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Phosphofructokinase and
triosephosphate isomerase cause
a % distribution of fructose-1,6di-P (30%), dihydroxyacetone-P
(67%) and glyceraldehyde-P (3%).
The inactive metabolite
accumulates as a “reserve”
while the active one is present
in lower concentration.
(67%)
The active metabolite
glyceraldehyde-3-P is present in
lower concentration. It is more
manageable to control a small pool
than a huge one. This “strategy” is
used by other pathways. That is
the active metabolite is scarce.
(3%)
(30%)
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Second stage, ATP production from glucose.
High energy
phosphate
What happens
if NAD+ is
exhausted?
2 moles of ATP per mole of
glucose. This kinase is
reversible
This is the first
substrate level
phosphorylation
"site".
ΔG°'= - 4.5
Kcal/mole
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Continuation of second stage, ATP production from glucose.
Another high
energy
phosphate
The second substrate level
phosphorylation is done by
pyruvate kinase
Glycolysis can be totally
anaerobic. In that case, to
continue glycolysis NAD+
must be regenerated at the
expense of pyruvate that
becomes lactate.
Lactate is transferred from
peripheral tissues to liver
Lactate accumulates in muscle
during exercise and in milk
during fermentation like in
yoghurt production.
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We will consider in detail, some aspects, of
several steps of glycolysis
Hexokinase is not the committed
Committed and rate limiting
step of glycolysis!!
There is a phosphofructokinase II
that is not a glycolytic enzyme.
Phosphofructokinase II synthesizes
fructose-2,6-P
step because glucose-6-P can go
to the pentose shunt, glycogen
synthesis, and is a precursor of
inositol and glucuronic acid.
You should understand the biological
meaning of all allosteric inhibitors and
activators in all pathways. Try to
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understand not to cram!
Glucokinase is not inhibited by G-6-P,
however it is inhibited by fructose-6P.
Interestingly, the inhibition by fructose-6P
is reversed by fructose-1P. We will
discuss the dietary importance of this and
of fructose enriched corn syrup.
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Regulation of glucokinase by fructose and the role GKRP
As indicated in the table shown before, glucokinase (GK) is not inhibited by glucose-6-P. However, it
is inhibited indirectly by the next glycolytic intermediary, fructose-6P. Fructose-6P binds to the
glucokinase regulatory protein (GKRP) which is both an inhibitor and a nuclear receptor for
glucokinase. GKRP bound to fructose-6P sequesters and inhibits GK translocating it into the
nucleus. Dietary fructose (like from fructose-enriched corn syrup) is converted to fructose-1-P.
Fructose-1-P displaces fructose-6-P and allows the free active GK to leave the nucleus. By this
mechanism, fructose-1-P increases the concentration of glucose-6-P in the liver and stimulates the
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synthesis of fatty acids and of purines.
Comparison of glucose regulation of hexokinase and glucokinase
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Regulation of phosphofructokinase I
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Mechanism of action of glyceraldehyde 3-phosphate dehydrogenase: large circle
represent the enzyme, small circle, the binding site for NAD+. This is not a regulatory
enzyme.
If arsenate is substituted for phosphate then 3-phosphoglyceroyl-arsenate is formed. The latter molecule
decomposes spontaneously and no high energy bond is conserved for ATP synthesis. This, however, is23
not
the principal toxic effect of As. We will see later its acute lethal effect on lipoic acid.
The phosphoglycerate shunt and 1,2-diphosphoglycerate
As mentioned before
this is the first
substrate level
phosphorylation
"site".
ΔG°'= - 4.5
Kcal/mole
What 2,3-diphospho
glycerate does to
hemoglobin?
What is the concentration of
2,3dPglyc in erythrocytes?
What is the net yield of ATP/mole of glucose in glycolysis if 100% of
glucose goes through the diphosphoglycerate shunt?
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Do not confuse phosphoglycerate mutase with bisphosphoglycerate mutase The latter
catalyzes the conversion of 1,3-bisphosphoglycerate to 2,3-bisphosphoglycerate.
In the enzyme's initial state, the active site contains a phosphohistidine complex formed
by phoshphorylation of a specific histidine residue.
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The alternative to lactate formation is to produce alcohol
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From: http://library.med.utah.edu/NetBiochem/tabletit.htm#carbos
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Metabolism of alcohol
Are you aware of the poisoning with car coolant (ethylene glycol) and how to deal with it?
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Use this sketch to follow the movement of labeled carbons from one metabolic
pathway to another. A question you could find later in this course might be:
Starting fatty acid synthesis from glucose-3-C14. In which positions of a newly
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synthesized fatty will the C14 be found?
Low Km, saturates
at low fructose
concentration
FRUCTOSE METABOLISM
10 time faster than
glucokinase, high
Vmax
Hexokinase
muscle
low Km
Fructose
Fructose-6P
usually from sucrose
about 15-20 % of
calories
(candies?)
If missing
Essential
fructosuria
BENIGN
Liver
Fructokinase
Fructose-1-P aldolase is also
called aldolase B. It has low
capacity, saturates rapidly.
There are isozymes A,B and C .
Fructose-1P
Fructose-1-P
ALDOLASE
Fructose-1P kinase
SLOW
Glycolytic intermediate
Fructose-1,6P
Fructose-1-P accumulation
causes hypophosphatemia,
hypoglycemia and gout. Why?
If fructose-1-P
aldolase is missing
there is liver failure.
Fructose intolerance
Aldolase
isozyme
CHO
HCOH
CH2OH
CH2OH
CO
CH2O-P
ic
yt
ol P
c
y e
Gl iose eras
tr o m
is
ATP
Glyceraldehydekinase
Ratio of activity on fructose-1,6diphosphate and fructose -1phosphate
A (Glycolytic)
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B (Hepatic)
0.9
C (Hepatic)
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Fructose-1,6-DP
glycolytic intermediate
CHO
HCOH
CH2OP
Glycolytic
Aldolase
https://cheminfo2011.wikispaces.com/Anthony+Lloyd+II+-+Final+Project
The glycolytic intermediates
can go to glycolysis (FAT) or glycogen
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Entry of glycogen into glycolysis
What is the net yield of ATP in
glycolysis?
a)
When glucose comes from free glucose.
b) When it comes from glycogen.
c)
When 100% of metabolites go through the 2,3diphosphoglycerate shunt.
d) When only arsenate (not phosphate) intervenes
in the glyceraldehyde dehydrogenase reaction?
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Overview and future projection:
Notice how Glucose relates to HMP
pathway (PPP) and to glycogen.
Visualize the NADH cycle. The
formation of CH3—CO~CoA links
glycolysis to FAT synthesis and
entry to the TCA or Krebs's cycle.
In the following lectures we
will study the synthesis of
glucose from lactate and other
sources.
Study glycolysis NOW to be
able to understand the next
lecture.
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