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.

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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