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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)
Triose phosphate isomerase (TIM)
C=O
H-C-O-
CH2-OH
H
PO3-2H-C=O
H-C-OH
CH2-O- PO3-2
1(3)
2
3(1)
5 (2)
4 (1)
6 (3)
Dihydroxyacetone phosphate(DHAP)
Glyceraldehyde-3-phosphate(GAP)
H-C-OH
H-C-OH
CH2-O- PO3-2
enediol intermediate
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.
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6th reaction of glycolysis (Gº’ = +1.5 kcal/mol)
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
H-C=O
H-C-OH
CH2-O- PO3-2
2
1
3
Glyceraldehyde-3-phosphate(GAP)
-PO3-2 C-O
H-C-OH
CH2-O- PO3-2
2
3
O1,3-Bisphosphoglycerate (1,3-BPG)
NAD+ + Pi
NADH + H+
1
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
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7th reaction of glycolysis (Gº’ = -4.5 kcal/mol)
3-Phosphogylcerate kinase (PGK)Mg2+
-PO3-2 C-O
H-C-OH
CH2-O- PO3-2
2
3
O1,3-Bisphosphoglycerate (1,3-BPG)
ADP
ATP
1
3-Phosphoglycerate (3-PG) C-O-
H-C-OH
CH2-O- PO3-2
O
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.
Gº’ = +1.6GAP + Pi + NAD+ 1,3-BPG + NADH
3PG + ATP Gº’ = -4.5
Net reaction
Gº = -2.9
1,3-BPG + ADP
GAP + Pi + NAD+ + ADP 3PG + NADH + ATP
in kcal/mol
8th reaction of glycolysis (Gº’ = +1.06 kcal/mol)
phosphoglycerate mutase (PGM)
3-Phosphoglycerate (3-PG) C-O-
H-C-OH
CH2-O- PO3-2
O
C-O-
H-C-O-CH2-OH
PO3-2
O
2-Phosphoglycerate (2-PG)
Phosphogylcerate mutase (PGM)
Catalyzes the transfer of the high energy phosphoryl group on phosphoglycerate.
Requires catalytic amounts of 2,3-bisphosphoglycerate (2,3-BPG) -acts as the reaction primer.
Requires a phosphorylated His in the active site
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Glycolysis1. Hexokinase (HK): (Glucose G6P), req.Mg-ATP2. Phosphoglucoisomerase (PGI): (G6P F6P)3. Phosphofructokinase (PFK): (F6P FBP),req.Mg-
ATP4. 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.
Glycolysis6. 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)
EnolaseMg2+
C-O-
H-C-O-CH2
PO3-2
O
2-Phosphoglycerate (2-PG) C-O-
H-C-O-CH2-OH
PO3-2
O
Phosphoenoylpyruvate (PEP)
H2O
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.
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10th reaction of glycolysis (Gº’ = -7.5 kcal/mol)
Pyruvate kinase (PK)K +, Mg2+
C-O-
C=O
CH3
O
Pyruvate
C-O-
H-C-O-CH2
PO3-2
O
Phosphoenoylpyruvate (PEP)
ADP
ATP
Pyruvate kinase (PK)
Couples free energy of PEP hydrolysis to ATP formation resulting in the formation of pyruvate.
Requires both K+ and Mg2+
Allosteric enzyme-
multiple 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.
Figure 17-22 Mechanism of the reaction catalyzed by pyruvate kinase.
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Overall glycolysis
2NAD+ + 2 Pi 2 NADH
2 ATP 2 ADP
Glucose + 2 ADP + 2 Pi + 2 NAD+ 2 Pyruvate + 2 NADH + 2 ATP
Glucose 2 pyruvate
4 ATP4 ADP
Need to regenerate NAD+
1. Via O2/electron transport chain (respiration).2. Anaerobically (fermentation)
Homolactic fermentation (muscle, heart)
Lactate dehyrogenase Gº’ = -6.0 kcal/mol
C-O-
C=O
CH3
O
Pyruvate
Lactate C-O-
H-C-O-CH2
H
O
NADH, H+
NAD+
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.
Figure 17-24Reaction mechanism of lactate dehydrogenase.
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Alcoholic fermentation (yeast don't have Lactate DH)
2. alchohol dehydrogenase
C-O-
C=O
CH3
O
Pyruvate
H-C-O-H
H
NADH, H+
NAD+
1. Pyruvate decarboxylase (TPP) Mg2+
, thiamine pyrophosphate
CO2 CH3
H-C=OAcetaldehyde
Ethanol
CH3
Figure 17-26 Thiamine pyrophosphate (TPP).
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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.
3. Protonation of the carbanion
4. Elimination of the TPP ylid to form acetaldehyde and regenerate the active enzyme.
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Figure 17-25 The two reactions of alcoholic
fermentation.
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Figure 17-30The reaction mechanism of alcohol dehydrogenase involves direct hydride transfer of the
pro-R hydrogen of NADH to the re face of acetaldehyde.
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Alcoholic fermentation
2ADP + 2 Pi
2 ATP
Glucose 2 Ethanol + 2 CO2
Pyruvate decarboxylase is present in brewer's yeast but absent in muscle / lactic acid bacteria
Other types of fermentations also exist…
CoASH
pyruvate acetyl-CoA + acetyl-P acetate
acetaldehyde
ethanol
Mixed acid: (2 lactate + acetate + ethanol)so, in addition to lactate production…
NADH, H+
NAD+
ADP ATP
NADH, H+
NAD+
lactate
Butanediol fermentation
C-O-
C=O
CH3
O
2 Pyruvate
NADH, H+
NAD+
CO2
Acetolactic acid
C-O-
C-C-O-CH3
H
O
O
CH3
CO2
C=O
CH3
CH3
HC-OH
Acetoin
CH3
CH3
HC-OH
HC-OH
2,3-butanediol
Other fermentations (Clostridium)
CoAH2
CH3-C-COOH
CO2
O
CH3-C-CoA
O
Acetyl-CoA
CoA
CH3-C-CH2-C-CoA
O O
CoA
Acetic acid
CO2
CoA
CH3-C-CH3
O
acetone
CH3-C-CH3
OH
isopropanol
NADH
Other fermentations (Clostridium)
H2O
NADHNAD
CH3-C-CH2-C-CoA
O O
CH3-CH=CH-C-CoA
O
CH3-CH2CH2-C-CoA
O
CH3-CH2CH2-C-OH
O H2O
2 NADH 2 NAD
CH3-CH2CH2-CH2-OHbutanol
butyric acid
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 liverIn 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
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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-1-phosphate 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.
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