Glycolysis B

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Ferchmin 2008/ metabolism /glycolysis
METABOLISM This lecture is the first of a series
of lectures in which the metabolism of sugars
will be addressed starting with glycolysis until
its catabolism to CO2 and H2O. Two special
activities will be done in the afternoons to
complement the lectures a) Discussion of several
metabolic diseases in which the pathways studied
are involved. b) Construction of a metabolic map
of all the pathways involved. 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) Entry of fructose and 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 formulae 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 is conserved in the form of ATP.
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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.
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?
Why was the glucose high after an overnight fast?
Where did the glucose go?
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We saw that glucose was cleared from blood of a
patient that took a load of glucose. What
happened to the glucose? We will address the
fate of glucose in several tissues that take up
glucose from blood. What happens to glucose in
the cells of different tissues? The biochemical
transformations of the molecule of glucose in the
body will be the subjects of my lectures during
the following weeks.
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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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Glycolysis is the universal metabolic pathway. It
occurs, with 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 fermentation which is 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 enzymes and
a heat-insensitive low-molecular-weight cofactors
are needed for fermentation to proceed. The main
features of the pathway were determined by 1940.
New features were discovered in the last few
decades.
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G-6-P is not committed to glycolysis
GLYCOLYSIS
First stage, priming.
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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The inactive metabolite accumulates as a
reserve while the active one is present in
lower concentration.
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.
Phosphofructokinase and triosephosphate isomerase
cause a distribution of fructose-1,6-di-P
(30), dihydroxyacetone-P (67) and
glyceraldehyde-P (3).
(67)
(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". ?G' - 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 of the steps of glycolysis
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
You should understand the biological meaning of
all allosteric inhibitors and activators in all
pathways. Try to understand not to cram!
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Regulation of glucokinase by fructose and the
role GKRP
As mentioned 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
then inhibits and pulls the GK into the nucleus.
Dietary fructose (like from fructose enriched
corn syrup) is converted to fructose-1-phosphate,
which displaces fructose-6-P, inhibits GKRP and
frees GK to leave the nucleus. By this mechanism
fructose-1- phosphate could stimulate lipid
synthesys.
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Regulation of phosphofructokinase I
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The phosphoglycerate shunt and 1,2-diphosphoglycer
ate
As mentioned before this is the first substrate
level phosphorylation "site". ?G' - 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 to be confuse phosphoglycerate mutase with
bisphosphoglycerate mutase which 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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Are you aware of the poisoning with car coolant
(ethylene glycol) and how to deal with it?
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Did you know that fructose and high fructose corn
syrup makes you FAT, causes gout, e.t.c.
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Low Km, saturates at low fructose concentration
10 time faster than glucokinase, high Vmax
Fructose-1-P aldolase is also called aldolase B.
It has low capacity, saturates rapidly. There are
isozymes A,B and C .
Fructose-1-P accumulation causes
hypophosphatemia, hypoglycemia and gout. Why?
Glyceraldehyde-kinase
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?
• When glucose comes from free glucose.
• When it comes from glycogen.
• When 100 of metabolites go through the
  2,3-diphosphoglycerate 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 CH3COCoA 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 following lectures.