Lecture #11
Glycolysis and Gluconeogenesis
Slide 1. Glycolysis is a metabolic pathway in which glucose is converted to
pyruvate or lactate. The Gluconeogenesis pathway achieves the reversal of
glycolysis—that is in the gluconeogenesis pathway lactate or pyruvate are
converted back to glucose.
Slide 2. The combined pathways of glycolysis and the citric acid cycle
form the core of energy yielding metabolism in most organisms. A
number of carbohydrates, amino acids, and glycerol are substrates for the
glycolysis pathway. Under aerobic conditions, the product of glycolysis is
pyruvate. The pyruvate can be converted to acetylCoA and carbon dioxide
by the pyruvate dehydrogenase complex. The acetylCoA is then oxidized to
two molecules of carbon dioxide in the citric acid cycle.
Slide 3. Before they can serve as nutrients, ingested disaccharides and
polysaccharides are first converted to monosaccharides by the action of
hydrolase enzymes. Common monosaccharide products of these enzymes
include glucose, galactose and fructose. These monosaccharides can then be
converted to pyruvate by the glycolysis pathway.
Slide 4. This photograph shows the Olympic champion sprinter, Michael
Johnson who won gold medals in the 200m and 400m races. At these
distances most of the energy comes from aerobic and anaerobic glycolysis.
Slide 5. This diagram highlights the divergence in glucose metabolism
under aerobic and anaerobic conditions. During a long relatively slow race
such as a marathon, aerobic glycolysis is most prevalent. Glucose is
converted into pyruvate, which in turn is oxidized to acetylCoA. The
acetylCoA is then oxidized in the TCA cycle.
Under more stringent conditions, such as those that would prevail in a sprint
(or semi-sprint) such as a 400m race, glucose utilization surpasses the
availability of oxygen and anaerobic conditions predominate. Under
anaerobic conditions much of the glucose is converted into lactate.
Slide 6. This diagram shows that there is another possible end product for
glycolysis in addition to pyruvate and lactate. In yeast and other
fermentative organisms, anaerobic conditions result in the conversion of
glucose into ethanol and carbon dioxide.
Slide 7. An overview of glycolysis shows the following:
-the six carbon hexose, glycose is transformed into two molecules of the
three carbon triose, pyruvate.
-at the hexose level a single intermediate is phosphorylated at two sites and
two ATP's (2 sites X 1 ATP per site) are cleaved to two ADP's
-at the triose level two compounds transfer phosphate to ADP at two sites, so
four ADP's (2 sites X 2 ATP's per site) are phosphorylated to four ATP's
-if glycolysis proceeds through to pyruvate (aerobic metabolism) two
NADH’s are produced
The net balance sheet for the aerobic glycolysis is that the metabolism of
one glucose molecule results in the production of two pyruvates, two
ATP’s and two NADH’s.
Slide 8. Glycolysis Part I. This slide shows the top half of the glycolysis
pathway. (The division of glycolysis into two parts is arbitrary, serving only
to improve visualization of the overall process.) There are five steps in this
part of the pathway, catalyzed by five different enzymes. (The names of the
enzymes are shown in blue, and the nature of the reactions catalyzed is
indicated in green.) In this part of glycolysis, one molecule of the six carbon
compound glucose is converted into two molecules of the three carbon
intermediate glyceraldehyde 3-phosphate. There are two kinase reactions in
which intermediates are phosphorylated. These reactions utilize ATP and
produce ADP. This means that the initial stages of glycolysis costs the cell
two ATP equivalents. This cost must be taken into account when we
calculate the overall energy balance of glycolysis. The last reaction show
interconverts dihydroxyacetone phosphate and glyceraldehyde 3-phosphate
effectively producing two molecules of glyceraldehyde 3-phosphate. This
intermediate continues on down the second half of the pathway. So from
here on down there are two three carbon molecules in the pathway. This is
an important consideration when we calculate the energy balance for
glycolysis.
Slide 9. Glycolysis Part II. Part two of glycolysis also has five steps and
five different enzymes, making a delightful symmetry with the part one.
This second part starts and ends with two three carbon intermediates. The
first reaction shown, the conversion of glyceraldehyde 3-phosphate to 1-3bisphosphoglycerate, is the key to energy production by the overall pathway.
This oxidation-reduction reaction not only introduces additional phosphate
residues into the pathway, but also results in the production of two
molecules of NADH. Under oxidative conditions, those two molecules of
NADH can enter the oxidative phosphorylation scheme which ultimately
results in the net production of four molecules of ATP. The addition of an
extra phosphate residue produces a bis-phosphate product, 1-3bisphosphoglycerate. The two 1-3-bisphosphoglycerate molecules are
immediately converted to two 3-phosphoglycerates with the concomitant
production of two molecules of ATP. This is an example of the coupling of
an intermediate with a high phosphate hydrolysis potential to the production
of ATP. At this point the two ATP’s used in part I of glycolysis have been
regenerated so that initial energy price has been repaid. Two more ATP’s
are produced in the last reaction of the pathway, which is catalyzed by
pyruvate kinase. This means that glycolysis gives a net yield of two ATP
molecules directly with the potential for four more ATP’s from the oxidation
of the two NADH’s.
Two molecules of pyruvate are the other end products of glycolysis. Under
aerobic conditions these two pyruvate molecules are converted into
acetylCoA which can then enter the TCA cycle. Under anaerobic
conditions, where oxygen is rate limiting, pyruvate is converted into lactate.
Slide 10. A glance at the standard energies of hydrolysis reveals that the
phosphorylation of hexoses such as glucose and fructose requires the
input of about 3 -5 kcal/mol whereas the dephosphorylation of the trioses
phosphoenolpyruvate and bis-phosphoglycerate release 15.8 and 11.8
kcal/mol respectively. This means that ATP can donate a phosphate to the
hexoses in an energy releasing reaction and ADP can accept phosphate from
the trioses also in an energy releasing reaction.
Slide 11. Hexokinase. When the glycolysis pathway begins with glucose,
the first step is an ATP-dependent phosphorylation reaction that produces
glucose-6-phosphate. This reaction, which is catalyzed by hexokinase, has a
very favorable net negative standard state free energy change. This is our
first reaction in our first metabolic pathway, and as luck would have it, it is
also the first example of a coupled reaction. The phosphorylation of glucose
is coupled to the hydrolysis of ATP. The standard free energy ( G) of ATP
going to ADP is high (-7.3 kcal/mol) and the standard free energy needed to
phosphorylate glucose is relatively low (+3.3 kcal/mol), so that the transfer
of a phosphoryl group from ATP to glucose has a significant negative G (4.0 kcal/mol). This makes the reaction equilibrium highly in favor of product
formation. In fact, product formation is so favored that the reverse reaction
produces a negligible amount of product. As a result, in gluconeogenesis, a
different enzyme reaction must be substituted to make the reverse reaction
feasible.
The hexokinase reaction is a good example of the power of coupling a
thermodynamically unfavorable reaction to ATP hydrolysis. If inorganic
phosphate were used instead of ATP to make glucose-6-phosphate, the G
would be a positive 3.3 kcal/mol, and the reaction would not proceed in the
direction of product formation.
Slide 12. Phosphoglucoisomerase I. The next step in glycolysis is the
isomerization of glucose 6-phosphate to fructose 6-phosphate catalyzed by
phosphoglucoisomerase. The overall reaction scheme as shown on this slide
looks rather complicated, but a step-by-step analysis reveals that it involves
a series of relatively straightforward transformations. The glucose 6phosphate begins in the pyranose ring form but the isomerization reaction
takes place with the open chain linear form of the molecule.
Slide 13. Phosphoglucoisomerase II. In order for the overall reaction to
proceed, mutarotation to the open chain form must first occur. The open
chain structure then undergoes a rearrangement to form an enediol
intermediate. (An enediol is a structure in which two adjacent hydroxylated
carbon atoms are connected by a double bond.) The net result of the
isomerization is the conversion of the C-1 carbonyl oxygen to a hydroxyl
group and the conversion of the C-2 hydroxyl group to a carbonyl oxygen
atom. Translated, that means that the aldose, glucose 6-phosphate is
converted into the ketose, fructose 6-phosphate.
Slide 14. Phosphofructokinase. In the next step fructose 6-phosphate is
phosphorylated in an ATP-dependent kinase reaction, yielding fructose 1-6bisphosphate. This is the second ATP coupled reaction in the pathway, and
again the net result is a reaction that is highly favorable in the direction of
product formation. In the gluconeogenesis pathway the reversal of this step
is accomplished by an alternate reaction catalyzed by a different enzyme.
Directly reversing this step using phosphofructokinase would be
thermodynamically unfavorable. We will come back to this reaction again
when we look at the regulation of glycolysis, because phosphofructokinase
is subject to a number of interesting regulatory processes.
Slide 15. Aldolase and Triose Phosphate Isomerase. The aldolase
reaction is a key step in glycolysis in which one six carbon compound is
converted into two three carbon compounds. Aldolase catalyzes an aldol
cleavage reaction in which the substrate is split between adjacent alcohol
groups to produce two products, one an aldehyde and the other an alcohol.
The new aldehyde group is found in glyceraldehyde 3-phosphate and the
alcohol group occurs in dihydroxyacetone phosphate.
The triose phosphate isomerase reaction converts dihydroxyacetone
phosphate into a second molecule of glyceraldehyde 3-phosphate. The
mechanism of this isomerization is the same as that of
phosphoglucoisomerase, with the same type of enediol intermediate. At this
point in glycolysis one molecule of glucose has been converted into two
molecules of glyceraldehyde 3-phosphate. From here on, each step in the
glycolysis pathway involves two identical three carbon molecules.
Slide 16. Catalytic Mechanism of Triose Phosphate Isomerase. The
mechanism of triose phosphate isomerase involves the following steps.
-Glutamate 165 acts as a general base by abstracting a proton from carbon 1.
Histidine 95 acting as a general acid, donates a proton to the oxygen atom
bonded to carbon 2, forming the enediol intermediate.
-Glutamic acid now acting as a general acid donates a proton to C-2 while
histidine removes a proton from the OH of C-1.
-The product is formed, and glutamate and histidine are returned to their
ionized and neutral forms, respectively.
Slide 17. Suppression of Methyl Glyoxal Formation. In addition to
catalyzing the rapid formation of glyceraldehydes 3-phosphate, triose
phosphate isomerase prevents the decomposition of the enediol intermediate
into the undesired side product, methyl glyoxal. In solution this side
reaction is 100 times faster than the physiologically desired reaction. The
labile enediol intermediate is trapped in the active site in such a way that
methyl glyoxal formation is prevented.
Slide 18. Stage 2 of Glycolysis. The combined action of aldolase and triose
phosphate isomerase in stage 2 of glycolysis results in the conversion of a
six carbon compound into two three carbon compounds. This has critical
implications for the energy balance of glycolysis, because the remaining
portion of the pathway occurs with two identical intermediates. The bottom
line is that from this point on, each of the reactions that produce ATP and
NADH yield two molecules of product.
Slide 19. Stage 3 of Glycolysis. In the final reactions of glycolysis,
referred to as stage 3, there are two sites at which ATP is produced and one
site where NADH is the product. Since two intermediates participate in each
of these steps, the net result in this stage is the production of 4 molecules of
ATP and two molecules of NADH.
Slide 20. Glyceraldehyde 3-Phosphate Dehydrogenase. The next reaction
is responsible for making glycolysis an energy yielding process. In fact,
there are two elements of this reaction that ultimately enable glycolysis to
sequester energy in the form of ATP. Glyceraldehyde 3-phosphate
dehydrogenase catalyzes the incorporation of an additional phosphate
residue into its product, and it simultaneously carries out an oxidationreduction reaction. Lets deal with the phosphorylation first. The starting
material, glyceraldehyde 3-phosphate has a single phosphate residue, but the
product of the reaction, 1,3-bisphosphoglycerate has two phosphates. The
1,3-bisphosphoglycerate is a mixed acid anhydride which has a very high
negative G of hydrolysis. In the further steps of glycolysis this extra
phosphate group will yield ATP. So the addition of an extra phosphate at
this point allows glycolysis to gain two additional ATP’s. (Remember there
are two molecules of each three carbon intermediate at this point in the
pathway.)
The oxidation-reduction reaction uses NAD+ as the oxidant. In the reaction
an aldehyde is oxidized to an acid, and the NAD+ is reduced to NADH.
Under aerobic conditions the two molecules of NADH can enter the
oxidative phosphorylation pathway resulting in the net production of four
molecules of ATP.
Slide 21. The Overall Mechanism of Glyceraldehyde 3-Phosphate
Dehydrogenase. The catalytic mechanism of glyceraldehyde 3-phosphate
dehydrogenase involves two steps. First, the aldehyde group at C-1 is
oxidized to a carboxylic acid coupled to the reduction on NAD+ to NADH.
Second, the newly formed carboxyl group is joined to a phosphate group to
yield an acyl-phosphate product.
Slide 22. Detailed Mechanism of Glyceraldehyde 3-Phosphate
Dehydrogenase. The reaction proceeds through a thioester intermediate,
which allows the oxidation of glyceraldehydes to be coupled to the
phosphorylation of 3-phosphoglycerate.
-Cysteine reacts with the aldehyde group of the substrate, forming a
hemithioacetal.
-An oxidation takes place with the transfer of a hydride ion to NAD+,
forming a thioester. This reaction is facilitated by the transfer of a proton to
histidine.
-The reduced NADH is exchanged for a NAD+ molecule.
-Orthophosphate attacks the thioester, forming the product.
Slide 23. Structure of NAD+. For your review here is another look at the
structure of NAD+. Remember that the oxidation-reduction process involves
the nicotinamide ring system which can accept two electrons and one proton.
Slide 24. Reduction of NAD+ to NADH. Here we see the reduction of
NAD+ to NADH. The two electrons enter the nicotinamide ring, one proton
sits on the top carbon atom and the other proton is released into the solvent.
Slide 25. Phosphoglycerate Kinase. There is an immediate payoff from the
glyceraldehydes 3-phosphate dehydrogenase reaction in the next step of
glycolysis. The enzyme, phosphoglycerate kinase catalyzes the transfer of a
phosphoryl group from the mixed acid anhydride of 1,3-bisphosphoglycerate
to ADP. This is the first direct energy payback in glycolylsis, effectively
replacing the two ATP’s that were used in the earlier steps. The other
product of the reaction is 3-phosphoglycerate.
Slide 26. Reactions Converting 3-Phosphoglycerate to Pyruvate. The
next step in glycolysis is the conversion of 3-phosphoglycerate to 2phosphoglycerate. This reaction which is catalyzed by
phosphoglyceratemutase, involves the transfer of a phosphoryl group from
the number 3 carbon to the number 2 carbon of glycerate.
The next step is a dehydration reaction, catalyzed by enolase, in which water
is removed from 2-phosphoglycerate to produce a double bond. The product
of this reaction is phosphoenolpyruvate (PEP). This compound is a
phosphoenol derivative meaning the phosphate group is adjacent to a
carbon-carbon double bond. The proximity of the phosphate to the double
bond gives phosphoenolpyruvate a very high negative G of hydrolysis (in
fact it has the highest negative G value of any compound on our hydrolysis
table).
The final reaction in glycolysis is catalyzed by pyruvate kinase. This is the
second reaction in glycolysis that accomplishes substrate level
phosphorylation—a process in which ATP is formed by the direct transfer
of a phosphoryl group from an organic phosphate molecule to ADP. In this
reaction the substrate phosphoenolpyruvate is converted to pyruvate with the
transfer of a phosphate residue to ADP to form ATP.
The production of two molecules of ATP by pyruvate kinase is what finally
gives glycolysis a net positive ATP yield. The other two molecules of ATP
produced by the phosphoglycerate kinase reaction replaced the ATP’s use in
the early stages of glycolysis. The two ATP’s from the pyruvate kinase
reaction constitute the actual net ATP gain in the pathway.
I would like to make one final point about the energy balance in glycolysis.
In addition to the ATP’s formed directly by substrate level phosphorylation,
four extra ATP’s can be formed from the NADH produced by
glyceraldehyde 3-phosphate dehydrogenase. However this only occurs
under aerobic conditions. Under anaerobic conditions, NADH does not
participate in oxidative phosphorylation and does not yield ATP. Thus the
only net payoff of anaerobic glycolysis is the two ATP’s formed directly in
the pathway. We will discuss anaerobic glycolysis at greater length in a few
minutes.
Slide 27. The Mechanism of Pyruvate Kinase. The pyruvate kinase
reaction is the second reaction of glycolysis in which substrate level
phosphorylation occurs. The high hydrolysis potential of
phosphoenolpyruvate makes the transfer of the phosphoryl group
thermodynamically favorable, and so the reaction equilibrium is very far in
the favor of product formation. In fact, this reaction is essentially
irreversible. To convert pyruvate back to phosphoenolpyruvate in the
process of gluconeogenesis, it is necessary to replace pyruvate kinase with
two alternate energy requiring reactions.
The mechanism of the pyruvate dehydrogenase reaction occurs in two
stages. First the phosphoryl group is transferred to ADP to form ATP with
the concomitant formation of the enol form of pyruvate. This enol
intermediate is unstable and is rapidly converted to the keto form of
pyruvate in an energy releasing reaction.
Slide 28. Summary of the Reactions of Glycolysis.
-In step 1, glucose is phosphorylated to glucose 6-P
-In step 2, glucose 6-P is isomerized to Fructose 6-P
-In step 3, fructose 6-P is phosphorylated to fructose 1-6-bis-P
(Thus far two ATP's have been used.)
-In step 4, fructose 1-6-bis-P is split into two trioses, dihydroxyacetone P
and glyceraldehyde 3-P.
-In step 5, the dihydroxyacetone P is then converted to another molecule
of glyceraldehyde 3-P. For the rest of the pathway there are two molecules
of triose.
-In step 6, glyceraldehyde 3-P is oxidized and phosphorylated to give 1,3bis-phosphoglycerate . In this step 2 NADH’s (2 molecules x 1 per
reaction) are produced.
-In step 7, 1,3-bis-phosphoglycerate donates a phosphate to ATP (This is
where two of the four ATP's are produced.) producing 3-P-Glycerate.
-In step 8, 3-P-Glycerate is converted to 2-P-Glycerate in a mutase
reaction.
-In step 9, 2-P-Glycerate is transformed to phosphoenolpyruvate in a
dehydration reaction.
-In step 10, which is the end of aerobic glycolysis, phosphoenolpyruvate
donates a phosphate to ADP to form ATP. (This is where the last two of
the four ATP's are produced.)
Under aerobic conditions glycolysis has a net yield of two ATP's and
two NADH’s
Slide 29. The energetics of Glycolysis. This figure gives the standard
free energies and the net free energies for all of the reactions of
glycolysis. There are a couple of important points to be made here.
1. For any biological reaction to proceed in the forward direction it
must have a net negative free energy ( G ) of hydrolysis under the
metabolic conditions found in the cell. That is, only energy releasing
reactions will give a net yield of product.
2. The figure shows that three reactions have net positive G values.
According to the laws of physics this is impossible. If these G values were
indeed positive the reactions would be going backwards. What it probably
means is that our best estimates of the concentrations of certain glycolysis
intermediates are wrong, and that if we had accurate values for these
compounds, all of the G values would in fact be negative. (This may seem
trivial to you, but your life depends on it.)
Slide 30. Lactate Dehydrogenase Reaction. The production of pyruvate
marks the formal end of glycolysis. Pyruvate serves as a central
intermediate in metabolism. It can be metabolized by two key pathways.
-When the supply of oxygen is adequate (aerobic conditions), pyruvate
serves as a substrate for the pyruvate dehydrogense reaction which converts
it into acetylCoA.for entry into the TCA cycle
-Under anaerobic conditions where oxygen is limiting, pyruvate is converted
to lactate by lactate deyhdrogenase. In the lactate dehydrogenase
reaction, the reduction of pyruvate to lactate is coupled to the oxidation
of NADH to NAD+.
-When oxidative conditions again prevail, such as during recovery from
intense exercise, the lactate can be converted back into pyruvate which can
then be used in the TCA cycle or converted back to glucose (and glycogen)
by the gluconeogenesis pathway.
Slide 31. Anaerobic Glycolysis with the Production of Lactate. Under
anaerobic conditions much of the pyruvate from glycolysis is siphoned off to
form lactate. The reason for this reaction becomes apparent when we look at
this overall pathway for lactate production. Under anaerobic conditions the
conversion of glucose to pyruvate results in the net production of NADH.
The NADH tends to accumulate because there is not enough oxygen to drive
the oxidative phosphorylation process which would convert the accumulated
NADH back to NAD+. The Lack of NAD+ would result in the cessation of
glycolysis. The genius of the lactate dehydrogenase reaction is that it
regenerates NAD+ under anaerobic conditions, which allows glycolysis to
continue despite the lack of sufficient oxygen. Ultimately the build up of
lactate is itself a limiting factor in the continuation of glycolysis, but it
allows the process to continue much longer than it would if pyruvate were
the final product.
Note that when glucose is converted anaerobically to lactate there is no
net production of NADH and the sole gain from the pathway is 2 ATP's
Slide 32. Three Fates of Pyruvate. The third pathway of pyruvate
metabolism occurs in fermentative microorganisms. Under anaerobic
conditions microorganisms such as yeast, rather than forming lactate,
convert pyruvate into ethanol.
Slide 33. The conversion of Pyruvate to Ethanol. Ethanol production
from pyruvate requires two enzymatic reactions. Pyruvate is first
decarboxylate to form acetaldehyde. The aldehyde is then reduced to
ethanol with the comcomitant oxidation of NADH to NAD+.
Slide 34. Anaerobic Glycolysis with the Production of Ethanol. The
conversion of pyruvate to ethanol serves the same function in yeast as lactate
formation does in humans. The oxidation of acetaldehyde to ethanol is
couple to the conversion of NADH to NAD+. The regeneration of NAD+
allows glycolysis to continue under anaerobic conditions. In this case
ethanol levels eventually build up to the point where the yeast die, but they
have a happy life in the meantime.
Anerobic glycolysis in yeast has a similar energy balance to that in
humans giving a net yield of 2 ATP's with no net production of NADH.
Slide 35. Microbial fermentation pathways. In addition to the anaerobic
production of ethanol, there are a number of other microbial fermentations
that can be used to produce ATP. The substrates and products of these
fermentations are listed here for your amusement, but I would not suggest
memorizing them unless you have a particular interest in anaerobic
metabolism.
Slide 36. Other glycolysis substrates. There are a number of
monosaccharides other than glucose which also feed into the glycolysis.
-Fructose can enter glycolysis by two different pathways.
-Galactose enters glycolysis by a four enzyme pathway.
Slide 37. Fructose Enters Glycolysis in the Liver. In the liver fructose is
converted to glycolysis intermediates by a three enzyme sequence. First
fructokinase phosphorylates fructose at the C-1 position. The product,
fructose-1-P is then converted to dihydroxyacetone phosphate and
glyceraldehyde by an aldolase. The glyceraldehyde is then phosphorylated
to give glyceraldehyde-3-phosphate.
Slide 38. Galactose Enters Glycolysis. The entry of galactose into
glycolysis is somewhat complicated. Galactose is first phosphorylated to
give galactose-1-P. The galactose-1-P is then converted to glucose-1P by an
interesting trade with UDP-glucose. The glucose-1P is transformed into
glucose-6-P by a mutase.
Slide 39. Galactose-1-P uridylyltransferase. This is a fascinating reaction
in which UDP-glucose releases glucose-1-P and accepts galactose-1-P. The
use of nucleotide sugars to activate reactions is common in living cells.
Generally, the formation of nucleotide sugars is driven by the cleavage of
high energy phosphate intermediates. The transferase reaction that we see
here is probably fairly energy neutral, but it is driven by the preceding
energy releasing process of UDP-glucose formation (not shown).
Slide 40. UDP-galactose-4-epimerase. The UDP-galactose product of the
galactose-1-P uridylyltransferase reaction can be converted back to UDPglucose by an epimerase enzyme.
Slide 41. Summary of Anaerobic Glycolysis II. We are now going to
consider the revesal of glycolysis which is called gluconeogenesis. In times
when energy is needed, such as during vigorous exercise, glycolysis
produces pyruvate at a faster rate than it can be used in the TCA cycle. We
have seen that under such conditions the extra pyruvate is diverted to form
lactate. When exercise ceases that lactate is converted back into pyruvate
and then into glucose. That is the function of gluconeogenesis.
Before looking at gluconeogenesis let’s review what we learned about
anaerobic.glycolysis. The key points concerning anaerobic glycolysis are:
1. There are two ATP’s are used and four produced for a net yield of two
ATP’s.
2. There are three reactions in glycolysis that are essentially irreversible (the
reactions with the arrows pointing in the forward direction only). I say
“essentially” irrevesible because no reaction is totally irreversible.
However, in these three reactions the equlibrium is so far in favor of product
that for practical purposes there is little or no reverse reaction.
The essentially irreversible reactions in glycolysis involve the conversion of
glucose to glucose 6-phosphate, the conversion of fructose 6-phosphate to
fructose 1-6-bisphosphate, and the conversion of phosphoenolpyruvate to
pyruvate.
One other key point is that when the glycolysis pathway is functioning all of
the component reactions must have at least a slightly negative G. That
necessitates that the overall reaction pathway will also have a negative G.
If the conversion of glucose to lactate is to be reversed, then this same
thermodynamic reality must prevail. That is, the overall reaction pathway of
gluconeogenesis and all of its component reactions must have a negative
G. This is accomplished by substituting alternate reactions for the three
essentially irreversible reactions in glycolysis. The three new reactions
either use additional ATP equivalents or substitute phosphatase reactions for
kinase reactions. The net effect is that, whereas glycolysis produces a net of
two ATP’s, gluconeogenesis uses a net of six ATP’s, and the G of the
overall pathway and its component reactions are shifted toward the synthesis
of glucose.
Slide 42. Gluconeogenesis Summary. This slide summarizes the essential
components of gluconeogenesis:
1. The pathway involves the synthesis of glucose from pyruvate or lactate,
and it occurs during recovery from exercise.
2. It utilizes many of the same enzymes from glycolysis (the reversible
ones).
3. The essentially irreversible steps of glycolysis must be bypassed by
substituting different reactions catalyzed by different enzymes.
4. Gluconeogenesis utilizes a net of six ATP equivalents.
5. When gluconeogenesis is occurring each of the reactions and the net
overall pathway all have a negative G.
Slide 43. The Cori Cycle. The Cori Cycle describes the physiological
process that occurs when glucose is converted to lactate during intense
exercise and then the lactate is converted back to glucose during the
recovery period. The cycle is named after its discoverers, Karl and Gerty
Cori who studied glucose metabolism at the Washington University in St
Louis. The Cori Cycle is initiated by anaerobic glycolysis during exercise.
The rapid utilization of glucose results in the production of high levels of
lactate. Most of the lactate is released from the muscle and travels in the
blood to the liver where is it taken into the cells.
In the liver the lactate is first converted to pyruvate. Some of the pyruvate is
used to produce energy by oxidation in the TCA cycle. The rest is converted
back to glucose by the gluconeogenesis. The liver is unusual in that it will
release a significant portion of its glucose into the blood. A high percentage
of the released glucose finds its way back into muscle cells where it can
serve in another round of glycolysis.
Slide 44. Gluconeogenesis I: Puruvate to Glyceraldehyde 3-Phosphate.
The slide shows the first half of the gluconeogenesis pathway with two
bypass enzymes coded in red. Pyruvate carboxylase uses one ATP to
convert pyruvate to oxaloacetate. Phosphoenolpyruvate carboxykinase uses
one GTP to convert oxaloacetate to phosphoenolpyruvate. Collectively
these two bypass enzymes use two ATP equivalents to bypass pyruvate
kinase, converting pyruvate to phosphoenolpuruvate in a thermodynamically
favorable manner.
Slide 45. Gluconeogenesis II: Glyceraldehyde 3-Phosphate to Glucose.
In the second half of gluconeogenesis two essentially irreversible enzymes,
phosphofructokinase and hexokinase are each replaced with a phosphatase
enzyme (coded in red). If the kinase enzymes were used in gluconeogenesis
they would synthesize ATP, and the reactions would be thermodynamically
unfavorable (They would have a positive G). In contrast, the phosphatase
enzymes, which bypass the kinases, hydrolyze the phosphate group from
their substrates in reactions which are thermodynamically favorable (They
have a negative G).
Slide 46. Gluconeogenesis Complete Pathway. This slide gives the
complete pathway for gluconeogenesis from lactate to glucose. Notice that
this is a balanced pathway with respect to oxidation-reduction. The NADH
that is produced when lactate is oxidized to pyruvate is converted back to
NAD+ when 1,3-bisphosphoglycerate is reduced to glyceraldehyde-3phosphate. Remember that the first part of the pathway from lactate to
glyceraldehyde-3-phosphate and dihydroxyacetone phosphate involves two
C-3 compounds and that the second part of the pathway from fructose-1-6bisphosphate involves one C-6 compound. Thus the ATP use in the first
half is 2x2 = 4 and the ATP use in the second half is 1x2 = 2 for a total of 6
ATP used.
Slide 47. Conversion of pyruvate to phosphoenolpyruvate. The reversal
of pyruvate kinase requires two enzyme catalyzed reactions each of which
requires an ATP equivalent.
The first step in this bypass is catalyzed by pyruvate carboxylase, an ATPdependent reaction which uses biotin as a cofactor. The biotin cofactor is
used in many carboxylation reactions, and we will look at it in more detail
later in the course when we consider the carboxylation of acetylCoA in fatty
acid biosynthesis. The product of the reaction is oxaloacetate which we
have seen before as the “sparking” compound in the TCA cycle. If
oxaloacetate needs to be replenished in the TCA cycle this reaction can
provide it. In the sequence of gluconeogenesis, which we are looking at
now, the oxaloacetate is converted to phosphoenolpyruvate.
The second step in this bypass is catalyzed by phosphoenolpyruvate
carboxykinase. That enzyme uses GTP instead of ATP (Which is a
thermodynamically equivalent source of energy). The formation of
phosphoenolpyruvate in this reaction is energized both by GTP cleavage to
GDP and by the decarboxylation of oxaloacetate. The addition of a carboxyl
group is a transient event, the function of which is to energize the formation
of phosphoenolpyruvate. We will see that most biological decarboxylation
reactions are energetically favorable in the direction of product formation.
This same strategy of carboxylation followed by decarboxylation is used in
the biosynthesis of fatty acids to provide extra energy.
The bottom line is that it takes two ATP equivalents to convert pyruvate to
phosphoenolpyruvate, whereas the reverse reaction in glycolysis only
generates one ATP. That imbalance between the catabolic and anabolic
reactions is one of the key features in giving both glycolysis and
gluconeogenesis a net negative G.
Slide 48. Interconversion of fructose 1-6-bisphosphate and fructose 6phosphate. The second reaction that must be bypassed in gluconeogenesis
is phosphofurctokinase. The reaction catalyzed by phosphofructokinase has
a very favorable negative G, so its reversal requires a bypass. In this case
we see a second strategy for changing the energetics of a reaction. That is
the substitution of a phosphatase for a kinase. In general reactions that are
coupled to ATP hydrolyis are energetically favorable. It is also true that the
hydrolysis of an organic phosphate molecule to release inorganic phosphate
is generally favorable. So the release of the phosphate from fructose 1-6bisphosphate as inorganic phosphate gives the formation of fructose 6phosphate a negative G. This allows that bypass reaction to proceed in the
direction of product formation.
Slide 49. Glucose-6-Phosphate Phosphatase. The last reaction which
must be bypassed in gluconeogenesis is the hexokinase reaction. Again the
involvement of ATP hydrolysis in hexokinase gives the reaction a favorable
negative G, and that makes its reversal energetically unfavorable. To
compensate for that, the ATP-dependent reaction is again bypassed by a
phosphatase reaction. The same change in energetics applies to these two
reactions—that is, the substitution of a phosphatase for a kinase gives the
reverse reaction a favorable, negative G.
Slide 50. Gluconeogenesis Complete Pathway. I know that you would be
disappointed if we did not take one last look at the overall gluconeogenesis
pathway. So I am giving you that opportunity here. The net result of
gluconeogenesis in the liver is the formation of glucose. Some of that
glucose is stored in the liver as the glycogen polymer, but much of it is
released into the blood. The glucose can be carried back to muscle cells and
then taken up and stored as glycogen—ready to supply energy for another
round of exercise.
A second fate of the released glucose is to be taken up by the brain. Brain
tissue is a big user of glucose which must be supplied from the blood. In
fact at rest the brain is the biggest user of glucose. If the level of glucose in
the blood falls below a certain critical level the brain ceases to function. It is
clear from this that the liver plays a vital role in the survival of higher
organisms by helping to maintain the level of blood glucose.
Slide 51. Reciprocal Regulation of Glycolysis and Gluconeogenesis. We
have now looked at two pathways running between the same pair of
compounds—glucose and lactate. In glycolysis, the catabolic direction, the
process generates two ATP’s. In gluconeogenesis, the anabolic direction,
there are six ATP’s used. It does not take higher math (lower math will
suffice) to see that the uncontrolled simultaneous function of these two
pathways would burn up ATP while producing no useful work. (Heat would
be generated, but no work would be accomplished.) So it does not take a
leap of faith to conclude that these two processes are regulated to prevent
them from functioning rapidly at the same time—that glycolysis is allowed
to function when extra ATP is needed, and gluconeogenesis occurs when the
supply of ATP is sufficient.
Both glycolysis and gluconeogenesis are, in fact, subject to exquisite
regulation at many levels. This process is still being intensely investigated,
and there are a number of questions still to be answered. The “facts” about
this regulation which appear in textbooks are sometimes inaccurate and
often incomplete. For that reason I would like you to assimilate a set of
generalizations about the regulation of glycolysis and gluconeogenesis rather
than learning detailed facts about which compound does what to a particular
enzyme.
So here are some generalizations:
1. While almost every reaction in glycolysis and gluconeogenesis is subject
to some type of control, it appears as if the bypass sites are subject to the
most stringent regulation. These are the essentially irreversible reactions in
glycolysis involving the interconversion of glucose and glucose 6-phosphate,
the interconversion of fructose 6-phosphate and fructose 1-6-bisphosphate,
and the interconversion of phosphoenolpyruvate and pyruvate.
2. There is a reason that regulation is focused at the bypass steps. Because
there are distinct enzymes functioning in each direction it allows for
regulation in one direction without exerting the same type of regulation on
the other direction. For example, you can stimulate or inhibit
gluconeogenesis without effecting glycolysis. In contrast, if the reversable
steps were targeted for regulation, inhibition of glycolysis would also result
in inhibition of gluconeogenesis.
3. The regulation of glycolysis and glucneogenesis involves allosteric
control mechanisms in which metabolites bind to target enzymes at
regulatory sites and either activate or inhibit enzyme activity.
4. Glycolysis generates ATP so it is activated when the ratio of ATP/ADP
and AMP is low and inactivated when that ratio is high.
5. Gluconeogenesis uses ATP so it is activated when the ratio of ATP/ADP
and AMP is high and inactivated when that ratio is low.
6. High levels of acetylCoA and citrate tend to inhibit glycolyis and
stimulate gluconeogenesis. The buildup of these intermediates is a signal
that the TCA cycle is overloaded and does not need more acetylCoA to be
fed in from glycolysis.
7. There is a very interesting regulatory circuit involving fructose 2-6bisphosphate. Note that this is “2-6” not “1-6”. This is a side product of
glycolysis that is synthesized and broken down by a bifunctional enzyme.
The regulation of this process is very complex. The fructose 2-6bisphosphate stimulates phosphofructokinase and inhibits fructose 1-6bisphosphate phosphatase.
Slide 52. Energy Charge Formula. The “energy charge” concept was
developed by Daniel Atkinson. What the formula does is to measure the
percentage of adenylate material which carries a “high energy” phosphate.
ATP gets two points in the numerator of the formula because it has two
phosphoanhydride bonds that can be hydrolyzed to drive reactions. ADP
gets one point because it has one phosphoanhydride bond. AMP gets no
points because it has no phosphoanhydride bonds. The denominator is the
sum of all three compounds—ATP, ADP, and AMP—the total of all of the
phosphorylated adenylate material in the cell. The ½ term normalizes the
energy charge to ATP which is arbitrarily given a value of one.
In the graph the value of one would occur at the right edge when 100% of
the adenylate material was in the form of ATP, and a value of zero would
occur when it was 100% in the form of AMP.
So, when a high energy charge prevails (ATP levels are high), glycolysis is
inhibited and gluconeogenesis is stimulated. In contrast when the energy
charge is low (ATP levels are low and AMP levels are high), glycolysis is
stimulated and gluconeogenesis is inhibited.
Slide 53. Sites in Glycolysis Subject to Negative Feedback. When an
organism is resting the three essentially irreversible enzymes are subject to
negative feedback. Hexokinase is inhibited by its product, glucose 6phosphate. Phosphofructokinase and pyruvate kinase are inhibited by a high
energy charge (high ATP).
Slide 54. Sites in Glycolysis Subject to Positive Feedback. During
exercise phosphofructokinase is subject to positive feedback by a low energy
charge (high AMP). Pyruvate kinase is activated by feedforward stimulation
by fructose 1-6-bisphosphate.
Slide 55. The Effects of Energy Charge on Phosphofructokinase. A low
energy charge results in the activation of phosphofructokinase. The enzyme
exhibits a more or less hyperbolic substrate saturation response and is
relatively active. When the energy charge is high the enzyme kinetics are
sigmoidal and the enzyme activity is inhibited.