Lecture 21
– Quiz on Friday-Glycolysis, Amino acids
– Bonus seminar on Friday: Prof. Candace
Haigler, 148 Baker, 3-4:30PM; use same format
for Extra Credit as previous seminars or if you
cannot make it, write a summary of the Haigler
Paper on our webpage.
– Glycolysis
– Fermentation (anaerobic metabolism)
5th reaction of glycolysis (Gº’ = +1.83 kcal/mol)
H
4 (1)
H-C=O
1(3)
H-C-O-PO3-2
5 (2)
H-C-OH
6 (3)
2
CH2-O-PO3-2
Glyceraldehyde3-phosphate
(GAP)
Triose
phosphate
isomerase
(TIM)
H-C-OH
H-C-OH
CH2-O- PO3-2
enediol intermediate
3(1)
C=O
CH2-OH
Dihydroxyacetone
phosphate
(DHAP)
Triose phosphate isomerase (TIM)
Only GAP continues on the glycolytic pathway and TIM
catalyzes the interconversion of DHAP to GAP
Mechanism is through a general acid-base catalysis
Final reaction of the first stage of glycolysis.
Invested 2 mol of ATP to yield 2 mol of GAP.
Page 593
6th reaction of glycolysis (Gº’ = +1.5 kcal/mol)
1
Glyceraldehyde-3-phosphate H-C=O
2
(GAP)
H-C-OH
CH2-O- PO3-2
3
Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH)
NAD+ + Pi
NADH + H+
O
1,3-Bisphosphoglycerate
(1,3-BPG)
1
C-O -PO3-2
2
H-C-OH
3
CH2-O-PO3-2
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Tetramer (4 subunits)
Catalyzes the oxidation and phosphorylation of GAP by NAD+
and Pi
Used several experiments to decipher the reaction mechanism
1. GAPDH inactivated by carboxymethylcysteine-suggests
that GAPDH has active site Cys
2. GAPDH quantitatively transfers 3H from C1 of GAP to
NAD+- this is a direct hydride transfer.
3. Catalyzes the exchange of 32P and an analog acetyl
phosphate-reaction proceeds through an acyl intermediate
Page 596
7th reaction of glycolysis (Gº’ = -4.5 kcal/mol)
O
1
1,3-Bisphosphoglycerate
C-O -PO3-2
2
(1,3-BPG)
H-C-OH
3
CH2-O- PO3-2
ADP
3-Phosphogylcerate kinase
(PGK)
Mg2+
ATP
O
3-Phosphoglycerate (3-PG)
C-OH-C-OH
CH2-O-PO3-2
Phosphoglycerate kinase (PK)
First ATP generating step of glycolysis
nucleophilic attack
Phosphoglycerate kinase (PK)
Although the preceeding reaction (oxidation of GAP) is
endergonic (energetically unfavorable), when coupled with
the PK catalyzed reaction, it is highly favorable.
in kcal/mol
GAP + Pi + NAD+
1,3-BPG + ADP
1,3-BPG + NADH Gº’ = +1.6
Gº’ = -4.5
3PG + ATP
GAP + Pi + NAD+ + ADP
3PG + NADH + ATP Gº = -2.9
Net reaction
8th reaction of glycolysis (Gº’ = +1.06 kcal/mol)
O
3-Phosphoglycerate (3-PG)
C-OH-C-OH
CH2-O- PO3-2
phosphoglycerate mutase
(PGM)
O
2-Phosphoglycerate (2-PG)
C-OH-C-O- PO3-2
CH2-OH
Phosphogylcerate mutase (PGM)
Catalyzes the transfer of the high energy phosphoryl group on
phosphoglycerate.
Requires catalytic amounts of 2,3-bisphosphoglycerate (2,3BPG) -acts as the reaction primer.
Requires a phosphorylated His in the active site
Page 599
Glycolysis
1. Hexokinase (HK): (Glucose  G6P), req.Mg-ATP
2. Phosphoglucoisomerase (PGI): (G6P  F6P)
3. Phosphofructokinase (PFK): (F6P  FBP),req.MgATP
4. Aldolase: (FBP  GAP and DHAP)
5. Triose posphate isomerase(TIM): (DHAP  GAP)
Called the first stage of glycolysis
• First 5 steps-require 2 mol ATP to get 2 mol GAP.
Glycolysis
6. GAPDH (GAP  1,3-BPG), req.NAD+ + Pi
7. PGK (1,3-BPG  3-PG), ADP  ATP-1st
step to make ATP.
8. PGM (3-PG  2-PG), phosphoryl shift,
requires 2,3 BPG
Pick up at reaction 9!
9th reaction of glycolysis (Gº’ = +0.44 kcal/mol)
O
C-O-
2-Phosphoglycerate (2-PG)
H-C-O- PO3-2
CH2-OH
Enolase
Mg2+
H2O
O
Phosphoenoylpyruvate
(PEP)
C-OH-C-O- PO3-2
CH2
Enolase (dehydration)
Catalyzes the dehydration of 2PG to phosphoenolpyruvate
(PEP).
Requires two divalent cations (Mg2+).
Enzyme can be inhibited by F- in complex with Pi, causing a
buildup in 2PG and 3PG.
Mechanism: rapid formation of carbanion by removal of a proton
at C2 by Lys (general base); proton exchanges with solvent
Elimination of water (-OH of C3) to form PEP with general acid
catalysis (Glu). This is the rate limiting step.
Page 601
10th reaction of glycolysis (Gº’ = -7.5 kcal/mol)
O
C
O
Phosphoenoylpyruvate
(PEP)
H-C-O- PO3-2
CH2
ADP
Pyruvate kinase (PK)
K +, Mg2+
ATP
O
Pyruvate
C-OC=O
CH3
Pyruvate kinase (PK)
Couples free energy of PEP hydrolysis to ATP formation
resulting in the formation of pyruvate.
Requires both K+ and Mg2+
Allosteric enzymemultiple isomers in different tissues
hormonal control by insulin/glucogon
ATP - negative feedback inhibition (allosteric inhibitor)
F-1,6-bisphophate (feedforward activator) and PEP are
positive + activators.
Page 602
Figure 17-22
Mechanism of the reaction
catalyzed by pyruvate kinase.
Overall glycolysis
Glucose
2 ATP
2 pyruvate
2 ADP
2NAD+ + 2 Pi
2 NADH
4 ADP
4 ATP
Glucose + 2 ADP + 2 Pi + 2 NAD+
2 Pyruvate + 2 NADH + 2 ATP
Need to regenerate NAD+
1. Via O2/electron transport chain (respiration).
2. Anaerobically (fermentation)
Homolactic fermentation (muscle, heart)
O
C
O
Pyruvate
C=O
CH3
NADH, H+
Lactate dehyrogenase
Gº’ = -6.0 kcal/mol
NAD+
O
Lactate
C-OH-C-O- H
CH2
Lactate dehyrdogenase (fermentation)
Tetramer that can compose 5 isozymes with KMVm
Two sets of subunits M and H can form M4, M3H, M2H2, MH3,
and H4.
H-type found in aerobic tissue (heart muscle)
M-type found in skeletal muscle and liver.
[H-type] KM for pyruvate and  Vm - used to regenerate
NAD+. Allosterically inhibited by high levels of metabolite.
Used to convert lactate to pyruvate for aerobic metabolism.
[M-type]  KM for pyruvate and  Vm - Not inhibited by
substrate. Used to convert pyruvate to lactate.
Page 603
Figure 17-24 Reaction mechanism of lactate
dehydrogenase.
Alcoholic fermentation (yeast don't have Lactate DH)
O
CO2
C-OCH
3
C=O
CH3
Pyruvate
H-C=O
1. Pyruvate
decarboxylase Acetaldehyde
(TPP) Mg2+, thiamine
NADH, H+
pyrophosphate
NAD+
2. alchohol
dehydrogenase
CH3
H-C-O- H
H
Ethanol
Page 604
Figure 17-26 Thiamine
pyrophosphate (TPP).
Involved in both oxidative and non-oxidative
decarboxylation as a carrier of "active" aldehydes.
Mechanism of Pyruvate Decarboxylase using TPP
1.
Nucleophilic attack by the dipolar cation (ylid) form of TPP
on the carbonyl carbon of pyruvate to form a covalent
adduct.
2.
Loss of carbon dioxide to generate the carbanion adduct in
which the thiazolium ring of TPP acts as an electron sink.
Protonation of the carbanion
Elimination of the TPP ylid to form acetaldehyde and
regenerate the active enzyme.
3.
4.
Page 605
Page 604
Figure 17-25 The two
reactions of alcoholic
fermentation.
Page 606
Figure 17-30 The reaction mechanism of alcohol
dehydrogenase involves direct hydride transfer of the
pro-R hydrogen of NADH to the re face of
acetaldehyde.
Alcoholic fermentation
2ADP + 2 Pi
Glucose
2 Ethanol + 2 CO2
2 ATP
Pyruvate decarboxylase is present in brewer's yeast
but absent in muscle / lactic acid bacteria
Other types of fermentations also exist…
Mixed acid: (2 lactate + acetate + ethanol)
so, in addition to lactate production…
ADP
CoASH
pyruvate
acetyl-CoA + acetyl-P
ATP
NADH, H+
NAD+
lactate
acetaldehyde
NADH, H+
NAD+
ethanol
acetate
Butanediol fermentation
O
CO2 O
C-OCH C-O3
C=O
C-C-O- H
CH3
CH3
2 Pyruvate
O
Acetolactic acid
CO2
CH3
HC-OH
C=O
CH3
Acetoin
NADH, H+
NAD+
CH3
HC-OH
HC-OH
CH3
2,3-butanediol
Other fermentations (Clostridium)
O
CH3-C-COOH
CoA
H2
CO2
CoA
O
isopropanol
OH
CH3-C-CH3
Acetic acid
CH3-C-CoA
Acetyl-CoA
CoA
NADH
O
CoA
CH3-C-CH3
acetone
O
O
CH3-C-CH2-C-CoA
CO2
Other fermentations (Clostridium)
O
O
CH3-C-CH2-C-CoA
H 2O
O
CH3-CH=CH-C-CoA
NADH
NAD
O
CH3-CH2CH2-C-OH
butyric acid
H 2O
O
CH3-CH2CH2-C-CoA
2 NADH
2 NAD
CH3-CH2CH2-CH2-OH
butanol
What about other sugars?
Fructose - fruits, table sugar (sucrose).
Galactose - hydrolysis of lactose (milk sugar)
Mannose - from the digestion of polysaccharides and
glycoproteins.
All converted to glycolytic intermediates.
Fructose metabolism
Two pathways: muscle and liver
In muscle, hexokinase also phosphorylates fructose
producing F6P.
Liver uses glucokinase (low levels of hexokinase) to
phosphorylate glucose, so for fructose it uses a different
enzyme set
Fructokinase catalyzes the phosphorylation of fructose by
ATP at C1 to form fructose-1-phosphate.
Type B aldolase (fructose-1-phosphate aldolase) found
in liver cleaves F1P to DHAP and glyceraldehyde.
Glyceraldehyde kinase converts glyceraldehyde to GAP.
Fructose metabolism
Glyceraldehyde can also be converted to glycerol by
alcohol dehydrogenase.
Glycerol is phosphorylated by glycerol kinase to form
glycerol-3-phosphate.
Glycerol-3-phosphate is oxidized to DHAP by glycerol
phosphate dehydrogenase.
DHAP is converted to GAP by TIM
Page 619
Figure 8.16c Important disaccharides formed by linking
monosaccharides with O-glycosidic bonds.
Lactose, milk sugar.
Galactose metabolism
Galactose is half the sugar in lactose.
Galactose and glucose are epimers (differ at C4)
Involves epimerization reaction after the conversion of
galactose to the uridine diphosphate (UDP) derivative.
1. Galactose is phosphorylated at C1 by ATP
(galactokinase)
2. Galactose-1-phosphate uridylyltransferase transfers
UDP-glucose’s uridylyl group to galactose-1phosphate to make glucose-1-phosphate (G1P) and
UDP-galactose.
3. UDP-galactose-4-epimerase converts UDP-galactose
back to UDP glucose.
4. G1P is converted to G6P by phosphoglucomutase.
Page 621