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’ = kcal/mol) Triose phosphate isomerase (TIM) C=O H - C - O- CH 2 - OH H PO 3 -2 H - C=O H - C - OH CH 2 - O- PO (3) 2 3(1) 5 (2) 4 (1) 6 (3) Dihydroxyacetone phosphate (DHAP) Glyceraldehyde- 3-phosphate (GAP) H - C - OH CH 2 - O- PO 3 -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 CH 2 - O- PO Glyceraldehyde-3-phosphate (GAP) -PO 3 -2 C - O H - C - OH CH 2 - O- PO O 1,3-Bisphosphoglycerate (1,3-BPG) NAD + + P i 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 3 H from C1 of GAP to NAD + - this is a direct hydride transfer. 3.Catalyzes the exchange of 32 P 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) Mg 2+ -PO 3 -2 C - O H - C - OH CH 2 - O- PO O 1,3-Bisphosphoglycerate (1,3-BPG) ADP ATP 1 3-Phosphoglycerate (3-PG) C - O - H - C - OH CH 2 - O- PO 3 -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’ = +1.6 GAP + P i + NAD + 1,3-BPG + NADH 3PG + ATP  Gº’ = -4.5 Net reaction  Gº = ,3-BPG + ADP GAP + P i + NAD + + ADP 3PG + NADH + ATP in kcal/mol

8th reaction of glycolysis (  Gº’ = kcal/mol) phosphoglycerate mutase (PGM) 3-Phosphoglycerate (3-PG) C - O - H - C - OH CH 2 - O- PO 3 -2 O C - O - H-C-O-H-C-O- CH 2 - OH PO 3 -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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Glycolysis 1. Hexokinase (HK): (Glucose  G6P), req.Mg-ATP 2. Phosphoglucoisomerase (PGI): (G6P  F6P) 3. Phosphofructokinase (PFK): (F6P  FBP),req.Mg- ATP 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 + + P i 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’ = kcal/mol) Enolase Mg 2+ C - O - H-C-O-H-C-O- CH 2 PO 3 -2 O 2-Phosphoglycerate (2-PG) C - O - H-C-O-H-C-O- CH 2 - OH PO 3 -2 O Phosphoenoylpyruvate (PEP) H2OH2O

Enolase (dehydration) Catalyzes the dehydration of 2PG to phosphoenolpyruvate (PEP). Requires two divalent cations (Mg 2+ ). Enzyme can be inhibited by F - in complex with P i, 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 +, Mg 2+ C - O - C=OC=O CH 3 O Pyruvate C - O - H-C-O-H-C-O- CH 2 PO 3 -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 Mg 2+ 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 17-22Mechanism of the reaction catalyzed by pyruvate kinase. Page 602

Overall glycolysis 2NAD P i 2 NADH 2 ATP 2 ADP Glucose + 2 ADP + 2 P i + 2 NAD + 2 Pyruvate + 2 NADH + 2 ATP Glucose 2 pyruvate 4 ATP 4 ADP Need to regenerate NAD + 1.Via O 2 /electron transport chain (respiration). 2.Anaerobically (fermentation)

Homolactic fermentation (muscle, heart) Lactate dehyrogenase  Gº’ = -6.0 kcal/mol C - O - C=OC=O CH 3 O Pyruvate Lactate C - O - H-C-O-H-C-O- CH 2 H O NADH, H + NAD +

Lactate dehyrdogenase (fermentation) Tetramer that can compose 5 isozymes with  K M V m Two sets of subunits M and H can form M 4, M 3 H, M 2 H 2, MH 3, and H 4. H-type found in aerobic tissue (heart muscle) M-type found in skeletal muscle and liver. [H-type]  K M for pyruvate and  V m - used to regenerate NAD +. Allosterically inhibited by high levels of metabolite. Used to convert lactate to pyruvate for aerobic metabolism. [M-type]  K M for pyruvate and  V m - Not inhibited by substrate. Used to convert pyruvate to lactate.

Figure 17-24Reaction mechanism of lactate dehydrogenase. Page 603

Alcoholic fermentation (yeast don't have Lactate DH) 2. alchohol dehydrogenase C - O - C=OC=O CH 3 O Pyruvate H-C-O-H-C-O- H H NADH, H + NAD + 1. Pyruvate decarboxylase (TPP) Mg 2+, thiamine pyrophosphate CO 2 CH 3 H-C=OH-C=O Acetaldehyde Ethanol CH 3

Figure 17-26Thiamine pyrophosphate (TPP). Page 604 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 17-25The two reactions of alcoholic fermentation. Page 604

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. Page 606

Alcoholic fermentation 2ADP + 2 P i 2 ATP Glucose 2 Ethanol + 2 CO 2 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=OC=O CH 3 O 2 Pyruvate NADH, H + NAD + CO 2 Acetolactic acid C - O - C-C-O-C-C-O- CH 3 H O O CO 2 C=OC=O CH 3 HC - OH Acetoin CH 3 HC - OH 2,3-butanediol

Other fermentations (Clostridium) CoA H2H2 CH 3 -C-COOH CO 2 O CH 3 -C-CoA O Acetyl-CoA CoA CH 3 -C-CH 2 -C-CoA O O CoA Acetic acid CO 2 CoA CH 3 -C-CH 3 O acetone CH 3 -C-CH 3 OH isopropanol NADH

Other fermentations (Clostridium) H2OH2O NADH NAD CH 3 -C-CH 2 -C-CoA O O CH 3 -CH=CH-C-CoA O CH 3 -CH 2 CH 2 -C-CoA O CH 3 -CH 2 CH 2 -C-OH O H2OH2O 2 NADH 2 NAD CH 3 -CH 2 CH 2 -CH 2 -OH butanol 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 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

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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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