2nd Phase of Glycolysis
January 24, 2003
Bryant Miles
In the preparatory phase of glycolysis two molecules of ATP have been invested. The hexose chain has
been cleaved into two triose phosphates. The second phase contains the last five reactions of glycolysis
and is called the payoff phase. It is called the payoff phase because in these five reactions two high energy
phosphate bonds are produced. These 2 high energy phosphoryl groups are transferred to ADP to
generate 2 molecules of ATP. It is important to remember that two molecules of glyceraldehydes 3phosphate is generated per 1 molecule of glucose. The conversion of two molecules of glyceraldehydes
3-phosphate into two molecules of pyruvate is accompanied with the generation of 4 ATP molecules and
2 molecules of NADH. The net reaction for glycolysis is the production of 2 ATP.
Reaction 6 Oxidation and phosphorylation of glyceraldehydes 3-phosphate
O
The first step of the second phase of glycolysis is the oxidation and phosphorylation of
glyceraldehydes 3-phosphate to form 1, 3-bisphosphoglycerate.
CH
O
HC OH
P O-
H 2C O
ONAD+ + Pi
NADH + H+
O
O
C
The oxidation of an aldehyde to the carboxylic acid is an exergonic process. This
enzyme uses the free energy of aldehyde oxidation to synthesize a high energy acyl
phosphate, 1, 3-bisphosphoglycerate.
P O-
O
O-
HC OH
The enzyme is glyceraldehyde 3-phosphate dehydrogenase.
∆Go’ = 6.3 kJ/mol
In erythrocytes ∆G = -1.29 kJ/mol
Another reaction near equilibrium.
O
P O-
H2C O
OENZ
C
H2
O
Fun facts about glyceraldehyde 3-phosphate dehydrogenase.
1. Iodoacetate, a reagent that
GAPDH
SH + ICH2CO2
ENZ
C
S
CH CO
H
alkylates cysteine residues,
inactivates GAPDH. Iodoacetate
HI
reacts stiochiometrically with
GAPDH. The confirmed
O
presence of a
H
OPO
C
3
GAPDH
+
carboxymethylcysteine residue
+ NAD H
+ NAD + Pi
OH
O
H
C
OH
O
demonstrates an active site
cysteine residue that is essential
P
O
O
P
O
H C
O
for enzymatic activity.
O
O
2
2
2
2-
3
3
C
H
C
-
H2C
-
2
-
O
HO
32
P
O-
-
O
O
O-
+
H3C
C
O
O
O-
P
-
O
O
O
GAPDH
HO
O-
P
-
O
+
H3C
C
O
32
-
O
P
-
O
2. GAPDH quantitatively transfer
3
H from the C1 carbon of
glyceraldehydes 3-phosphate to
NAD+, establishing a direct
hydride transfer.
3. GAPDH catalyzes 32P exchange between inorganic phosphate and acetyl phosphate. Such isotope
exchange is indicative of an acyl-enzyme intermediate.
Mechanism of GAPDH
First the substrate G-3P binds to the
enzyme.
The essential cysteine residue acts as
a nucleophilic and attacks the
aldehyde carbonyl to form a
thiohemiacetal intermediate.
Next the thiohemiacetal transfer a
hydride (2e- + H+) to the active site
bound NAD+ to form NADH and a
thioester intermediate.
This NADH molecule dissociates
from the active site and another
NAD+ molecule is bound.
The enzyme binds a molecule of
inorganic phosphate which is the
nucleophile that attacks the thioester
To regenerate the active enzyme’s
sulfhydryl and form 1, 3bisphosphoglycerate.
7th Reaction: Phosphoryl transfer from 1,3-bisphosphoglycerate.
O
O
P O- The enzyme phosphoglycerate kinase catalyzes the transfer of the high energy
acyl phosphate to ADP. The result is the synthesis of two molecules of ATP.
OHC OH
This enzyme pays of the ATP debt of the first phase of glycolysis.
O
C
O
P O-
H2C O
OADP
ATP
O
C
O-
HC OH
H2C O
O
P OO-
∆Go’ = -18.9 kJ/mol
In erythrocytes ∆G = 0.1 kJ/mol
Another reaction near equilibrium.
Substrate level phosphorylation occurs when ADP is phosphorylated to form
ATP at the expense of the substrate which is 1,3-bisphosphoglycerate in this
case.
8th Reaction: Conversion of 3-phosphoglycerate into 2-phosphateglycerate.
O
C
The enzyme phosphoglycerate mutase catalyzes the reversible shift of the
phosphate ester between C2 and C3 of glycerate.
O-
HC OH
H2C O
O
A mutase is an enzyme that catalyzes the migration of a functional group within
the substrate molecule.
P OO-
Mg2+ is required for enzymatic activity.
∆Go’ = 4.4 kJ/mol
In erythrocytes ∆G = 0.83 kJ/mol
O
C
HC
OO
H2C OH
Another reaction near equilibrium.
O
P O
-
O-
This reaction is more complicated than it looks.
Phosphoglycerate mutase has a covalent phosphoenzyme intermediate and requires 2,3bisphosphoglycerate as a cofactor. (Do you remember 2,3-bisphosphoglycerate, BPG, the allosteric
effector of hemoglobin?)
The phosphoenzyme has a phosphoryl group
covalently bound to an active site histidine
residue.
The phosphoenzyme binds 3-phosphoglycerate
and transfer the phosphoryl group from the
histidine to the C2 position of 3-phosphoglycerate
to form 2,3-bisphosphoglycerate.
2,3-bisphosphoglycerate then transfers the C3
phosphoryl group to the active site histidine
residue to regenerate the active phosphoenzyme
and produce 2-phosphoglycerate.
Once in every 100 turnovers, the intermediate
2,3-bisphosphoglycerate escapes from the
enzyme, leaving an inactive dephosphorylated
enzyme.
The unphosphorylated enzyme binds 2,3bisphosphoglycerate and transfers the C3 phosphoryl group to the histidine reactivating the enzyme
and producing 2-phosphoglycerate. For this reason, the enzyme requires a small amount of 2,3-BPG
as a cofactor for maximal activity.
Reaction 9: Dehydration of 2-phosphoglycerate
Enolase is the enzyme that catalyzes the reversible elimination reaction of
water to convert 2-phosphoglycerate into phosphoenol pyruvate.
O
C
O-
HC
O
O
H2C OH
P O-
Mg2+ required for enzymatic activity.
O-
∆Go’ = 7.5 kJ/mol
In erythrocytes ∆G = 1.1 kJ/mol
Another reaction near equilibrium
Enolase dehydrates the substrate to form a high energy phosphoenol. The
standard change in free energy for the hydrolysis of phosphoenolpyruvate is a
−62 kJ/mol.
H2O
O
C
O-
C
O
This enzyme is strongly inhibited by fluoride in the presence of phosphate.
Fuoride reacts with phosphate to form fluorophosphates (FPO32- which
P O- complexes with the Mg2+ ion located in the active site of the enzyme.
OO
CH2
Reaction 10: Transfer of the phosphoryl group of phosphoenolpyruvate.
O
C
O-
C
O
O
binds MgADP or MgATP2P O- This enzyme
2+
O-
CH2
ADP
ATP
O
C
O-
C
O
CH3
The last step of glycolysis is the transfer of the phosphoryl group of
phosphoenol pyruvate to ADP to form ATP and pyruvate. The enzyme that
catalyzes this reaction is pyruvate kinase.
Ie Mg required for enzymatic activity.
∆Go’ = -31.4 kJ/mol
In erythrocytes ∆G = -23.0 kJ/mol
Far from equilibrium.
Since 2 PEP are formed for every glucose molecule that enters the glycolytic
pathway, 2 ATP molecules are formed in this step. The ATP dept generated
during phase 1 of glycolysis was paid by the formation of ATP by the substrate
level phosphorylation of ADP by 1,3-bisphosphoglycerate. The 2 ATP
molecules produced here the payoff for glycolysis.
The large negative ∆G of this reaction makes this enzyme a target for
regulation. Pyruvate kinase has binding sites for a number of allosteric
effectors.
AMP
ATP
Fructose 1,6-bisphosphate
Acetyl-CoA
Alanine
In addition to the allosteric effectors, pyruvate kinase is regulated by covalent modification.
Hormones such as glucagon activate a cAMP-dependent protein kinase which transfers the γphosphate of ATP to the pyruvate kinase. The phosphorylated pyruvate kinase is more strongly
inhibited by ATP and alanine. The Km for PEP for the phosphorylated enzyme is also increased to the
point that at the steady state physiological concentrations of PEP, the enzyme is completely inactive.
Fates of Pyruvate
The two molecules of pyruvate and 2 molecules of
NADH produced from one molecule of glucose during
glycolysis have three possible metabolic fates.
Under aerobic conditions, pyruvate is oxidized, with the
loss of CO2 to produce acetyl-CoA and NADH. AcetylCoA then enters the citric acid cycle where it is
completely oxidized into CO2 and H2O. Under aerobic
conditions the NADH produced from glycolysis and
the citric acid cycle are reoxidized into NAD+ in the
mitochondrial electron transport chain.
Under anaerobic conditions NADH accumulates as a
product of GAPDH catalyzed sixth step of glycolysis.
NAD+ is required as a substrate for GADPH and soon
becomes limiting.
In order to maintain production of ATP via the
glycolytic pathway under anaerobic conditions, NAD+ has to be regenerated from NADH. There are two
possible anaerobic pathways.
1.) Homolactic acid fermentation.
O
HC
H2C
O
O
CH
Pi
OH
O
HC
OH
P
O
-
H2C
P
O
O
NAD+
H3C
O
C
C
H
-
O
GAPDH
O-
OH
O
O-
O
P
O
C
O
-
For lactate dehydrogenase
-
NADH + H+
O-
H3C
Lactate Dehydrogenase
O
O
C
C
Under anaerobic conditions, such as when muscle
cells are vigorously working, pyruvate is reduced to
lactic acid to regenerate NAD+. This process is
called homolactic fermentation.
O
-
∆Go’ = −25.1 kj/mol
In erythrocytes ∆G = −14.8 kJ/mol
Speaking of erythrocytes. Red blood cells do not
have mitochondrian. They convert glucose into 2
molecules of lactate even under aerobic conditions.
Other tissues (retina, brain) convert glucose into two molecules of lactate under aerobic conditions.
H2C
+
N
H
O
O
C
C
H3C
H
H2C
HIS195
N
H
-
H3C
O
O
H
N
H
H
O
C
C
H
The Pro-R hydrogen of NADH is
transferred to the pyruvate to form L-lactate.
CO2O-
= HO
C
H
CH3
O
The mechanism of LDH is shown to the
left.
The pro-R hydride is transferred from
NADH to C2 of pyruvate with the
concomitant transfer of a proton from the
imidazolium His-195.
L-Lactate
NH2
NH2
+
H--Pro R
H--Pro-S
N
H
N
O
Lactate dehydrogenase, LDH catalyzes the
reduction of pyruvate to lactate with
absolute stereospecificity.
HIS195
N
R
R
2.) Alcoholic fermentation
O
CH
HC
H2C
C
Pi
OH
O
O
P
O
HC
-
O
-
OH
O
GAPDH
-
-
P
O
O
O
Under anaerobic conditions some plant
tissues, invertebrates, protists, and
microorganisms (yeast) regenerate NAD+
by alcoholic fermentation. Pyruvate and
NADH are converted into ethanol, CO2
and NAD+.
Yeast under anaerobic conditions
regenerates NAD+ by alcoholic
fermentation. This is an important
reaction in making beer, wine and spirits.
In addition the CO2 produced by alcoholic
fermentation leavens bread.
O
O
H2C
P
O
O
-
ONADH + H+
NAD+
O
C
O
H
H3C
C
O
O
H
H3C
Alcohol Dehydrogenase
C
H
Pyruvate
Decarboxylase
H
H3C
O
O
C
C
O
-
The conversion of pyruvate into ethanol
is a two step process. The first step
involves the decarboxylation of pyruvate
by the enzyme pyruvate decarboxylase.
Thiamine pyrophosphate
O
H3C
H2
C
+
NH2
Aminopyramidine
Ring
O
P
O
C
N
C
H2
N
N
H3C
H2
C
S
Thiazolium Ring
O
O
-
-
P
O
This enzyme requires the coenzyme
thiamine pyrophosphate, TPP.
-
O
Pyrophosphate
This cofactor is bound tightly by the
enzyme.
H
Acidic proton
Thiamine pyrophosphate functions as an electron sink to
stabilize the build up of negative charge of the carbonyl
carbon atom in the transition state.
O
O
C
-
O
O
C
C
CH3
O
O
-
:C
CH3
Mechanism of pyruvate carboxylase
O
-
C
H+
O
R
R
:C
C
H
C
S
S
CH3
CH3
+N
CH3
+N
O
R
'
+
R'
H
O
O
OC
CH3
R
CH3
+N
C
HO
H
R
H
C
C
O
H
S
CH3
C
S
CH3
R'
CH3
N+
R'
O
C
H+
O
R
R
+
N
CH3
N
HO
C
HO
C
-
CH3
C
C
S
CH3
S
CH3
R'
R'
The next step of alcoholic fermentation is the reduction of acetaldehyde by NADH to produce ethanol and
NAD+. The enzyme that does this reduction is alcohol dehyrogenase, ADH. Yeast alcohol
dehydrogenase contains an active site Zn2+ ion. This zinc polarizes the carbonyl bond of acetaldehyde
and to stabilize the negative charge that is built up in the transition state. ADH is stereospecific it
transfers the pro-R hydride of NADH to the pro-R position of ethanol.
Yeast alcohol dehyrdogenase
Zn
2+
Zn
-
O
O
H3C
C
H3C
C
H
H
H
2+
O
H
H
H+
H
H
H
O
H3C
OH
NH2
NH2
H--Pro R
N
R
H--Pro R
H--Pro-S
+
N
R
In the Mammalian liver there is ADH that catalyzes the opposite reaction. The oxidation of ethanol in to
acetaldehyde. This enzyme has two Zn2+ ions in the active site although only one of the zincs participates
directly in catalysis through the same general mechanism.
Energetics of Homolactic acid Fermentation
Glucose + 2ADP + 2Pi + 2NAD+ 2Pyruvate + 2ATP + 2NADH +2H2O + 2H+
∆Go’ = -85 kJ/mol
Glucose 2 Lactate + 2H+
2ADP + 2Pi 2ATP + +2H2O
Glucose + 2ADP + 2Pi 2Lactate + 2ATP + +2H2O + 2H+
∆Go’ = -196.0 kJ/mol
∆Go’ = 61.0 kJ/mol
∆Go’ = -135.0 kJ/mol
Effeciency of Homolactic acid Fermentation.
2 ATP’s produced 61 kJ/mol
Standard free energy change in converting glucose into two molecules of lactate ∆Go’ = -196.0 kJ/mol.
Efficiency = 100 X 61/196 = 31%
Under the steady state concentrations of the cell the efficiency is greater than 50%.
Energetics of alcoholic fermentation.
Glucose 2 ethanol + 2CO2
2ADP + 2Pi 2ATP + +2H2O
Glucose + 2ADP + 2Pi 2Ethanol + 2ATP + +2H2O + 2CO2
∆Go’ = -235.0 kJ/mol
∆Go’ = 61.0 kJ/mol
∆Go’ = -174.0 kJ/mol
Efficiency = 100 X 61/235 = 26%
Under the steady state concentrations of the cell the efficiency is greater than 50%.
Glycolysis is used for rapid ATP production.
The rate of ATP formation in anaerobic glycolysis is 100 times faster than ATP production by oxidative
phosphorylation in the mitochondria. When tissues are rapidly consuming ATP, they generate it almost
entirely by anaerobic glycolysis.
Two type of muscle fibers Fast twitch and Slow twitch.
Fast-twitch fibers are capable of short burst of rapid activity. The cells that compose these fibers are
almost completely devoid of mitochondria. They generate nearly all of their energy by anaerobic
glycolysis. They are easily identifiable as white fibers.
Slow-twitch fibers contract slowly and steadily. They are rich in mitochondria and obtain most of their
energy by oxidative phosphorylation. The high concentration of mitochondria give these muscle fibers a
red color to the heme containing cytochromes.
The flight muscles of birds are an illustrative example. The flight muscles of migratory binds such as
ducks and geese are rich in slow twitch fibers and therefore these birds have dark breast meat. In contrast
the flight muscles of the land loving birds such as chickens and turkeys are used only for short burst of
flight to escape danger. Their flight muscles are composed mainly of fast twitch fibers that give them
white breast meat.
Sprinters muscles mainly fast-twitch fibers.
Marathon runners mainly slow-twitch fibers.