1. Lecture 20 –Exam 2 on Monday, Quiz next Friday –Links for glycolysis –http://www.johnkyrk.com/glycolysis.htmlhttp://www.johnkyrk.com/glycolysis.html –http://www.terravivida.com/vivida/diygly/ Metabolism and thermodynamics –Glycolysis

2. In addition to energetics -must balance redox chemistry Redox chemistry Glycolysis Glucose (C 6 H 12 O 6 ) + 6 O 2 2 pyruvate + 2 (2H) Active hydrogen 2H + + 2e - 6 CO 2 + 6 H 2 O Broken down into “half pathways” Glucose Mitochondria (2H) + 1/2 O 2 H2OH2O

3. Common carrier of (H) NAD(P) Nicotinamide adenine dinculeotide (phosphate) (oxidized form) O N N N N O OH HO O- O O O OH HO CH 2 -O-P-O-P-CH 2 N C-N-H 2 (+) PiPi NAD + + 2e - NADH + H +

4. Common carrier of (H) NAD(P) Nicotinamide adenine dinculeotide (phosphate) (reduced form) O N N N N O OH HO O- O O O OH HO CH 2 -O-P-O-P-CH 2 N C-N-H 2 PiPi H H NADH + H + NAD + + 2e -  Eº ‘ = 0.31 volt

5. Thermodynamically  Eº’ = +0.82 volt 2e - + 2H + + 1/2 O 2 H2OH2O Ease at which molecule donates electron(s) aka electromotive force  Eº’ = +0.31 volt NADH + H + NAD + + 2H + + 2e - NADH + H + + 1/2 O 2 NAD + + H 2 O  Eº’ = +1.13 volt Convert using the Nernst Equation  Gº ‘ = -n F  Eº‘ F = faraday= 23,086 cal mol  e -  volt  Gº ‘ = -2( )  131 volt) 23,086 cal mol  e -  volt  Gº ‘ = -56 kcal/mol n=mol e -

6. ATP and NAD(P)H So in metabolism, ATP formed in reaction sequences where  Gº‘ >  Gº‘ hydrolysis of ATP (catabolism) Used to drive reaction with  Gº‘ <  Gº‘ hydrolysis (<0) NAD(P)H production and ATP production are usually coupled ATP and NAD(P)H are coenzymes and therefore need to be recycled.

7. Thermodynamics and Metabolism Standard free energy A + B C + D  G o’ =-RT ln([C][D]/[A][B])  G o’ = -RT ln K eq  G o’ 1.0) Spontaneous forward rxn  G o’ = 0 (K eq =1.0) Equilibrium  G o’ > 0 (K eq <1.0) Rxn requires input of energy

8. Example The  G ’ for hydrolysis of sugar phosphate (sugar-P) R-OPO 3 2- + H 2 O R-OH + P sugar-P free sugar is -6.2 kcal/mol in a hypothetical, cell in which steady-state conc of sugar-P, free sugar, and Pi are 10 -3 M, 2 X 10 -4 M, and 5 X 10 -2 M, respectively. What is  G° ’ for the reaction? Steady-state is a nonequilibrium situation that prevails because of a balance between reactions that supply and remove these substances. The initial conditions are not at equilibrium so we can assume the reaction will proceed until it reaches equilibrium  G ’ =  G° ’ + RT ln ([sugar][Pi]/[sugar-P]) -6.2 kcal/mol =  G° ’ + (1.98 X 10 -3 kcal/deg mol)(298 deg)(2.3)log ([2 X 10 -4 M][5 X 10 - 2 M]/[10 -3 M])  G° ’ = - 6.2 kcal/mol + 2.7 kcal/mol = -3.5 kcal/mol

9. Metabolic Pathways are not at Equilibrium Metabolic pathways are not at equilibrium A B Instead pathways are at steady state. A -> B -> C The rate of formation of B = rate of utilization of B. Maintains concentration of B at constant level. All pathway intermediates are in steady state. Concentration of intermediates remains constant even as flux changes.

11. Glycolysis (Embden-Meyerhof-Parnas Pathway) Central pathway in glucose metabolism Present in al plants, animals, and bacteria Source of ATP, reducing equivalents Source of sugars In the catabolic pathway... 2 ATP to activate 4 ATP + 2 NADH Glucose 2 pyruvate 2NAD + + 2ADP 2NADH + 2ATP Lactate anaerobic NAD + Acetyl-CoA CO 2 4 CO 2 Citric acid (Krebs) cycle O2O2 NADH + ADP NAD + ATP Respiratory chain Ethanol + CO 2 anaerobic fermentation NAD +

12. Key reactions of glycolysis 1. Phosphoryl transfer. A phosphoryl group is transferred from ATP to a glycolytic intermediate or vice versa. R-OH + ATP R-O-P-O - + ADP + H + O O-O-

13. Key reactions of glycolysis 2. Phosphoryl shift. A phosphoryl group is shifted within a molecule from one oxygen atom to another. R-C-CH 2 -O-P-O - O O-O- H OH R-C-CH 2 -OH H O - O-P-O - O

14. Key reactions of glycolysis 3. Isomerization. A ketose is converted to an aldose or vice versa. C=O O R CH 2 OH H-C-OH R C-H

15. Key reactions of glycolysis 4. Dehydration. A molecule of water is eliminated. H-C-OH H H-C-OPO 3 2- H-C COO - C-OPO 3 2- + H 2 O H COO -

16. Key reactions of glycolysis 5. Aldol cleavage. A carbon-carbon bond is split in a reversal of an aldol condensation. HO-C-H H C=O R H-C-OH R’ HO-C-H C=O R C R’ H O +

17. 1st reaction of glycolysis (  Gº’ = -4 kcal/mol) OH 1 O HO OH HO OH   * 2 3 4 5 6 Glucose OH 1 O -2 O 3 P-O OH HO OH   * 2 3 4 5 6 ATP ADP Glucose-6-phosphate (G6P) Hexokinase (HK) Mg 2+ First ATP utilization

18. Figure 17-5aConformation changes in yeast hexokinase on binding glucose. (a) Space-filling model of a subunit of free hexokinase. Page 586

19. Figure 17-5bConformation changes in yeast hexokinase on binding glucose. (b) Space-filling model of a subunit of free hexokinase in complex with glucose (purple). Page 586

20. Mechanism by induced fit The two lobes that form the active site cleft move to engulf the glucose and exclude water from the active site. This also causes catalysis by proximity. Needs Mg 2+ ATP complex for activity (free ATP is an inhibitor of the reaction)

21. 2nd reaction of glycolysis (  Gº’ = +0.4 kcal/mol) OH 1 O -2 O 3 P-O OH HO OH   * 2 3 4 5 6 Fructose-6-phosphate (F6P) Phosphoglucoisomerase (PGI) Glucose-6-phosphate (G6P) CH 2 - OH O 5 OH 1 2 3 4 6 -2 O 3 P-O isomerization of an aldose (G6P) to a ketose (F6P).

22. Phosphoglucoisomerase: mechanism Reaction 2 is the isomerization of an aldose (G6P) to a ketose (F6P). Step 1: substrate binding Step 2: an acid (Lys side chain) catalyzes ring opening Step 3: A base (imidazole portion of His-Glu dyad, removes the acidic proton from C2 to form the cis-enediolate intermediate. The proton is acidic because it is  to a carbon group. Step 4: Proton is transferred to C1. Step 5: Ring closure to form the product.

23. Page 587 Lys His-Glu

24. 3rd reaction of glycolysis (  Gº’ = -3.4 kcal/mol) fructose-6-phosphate (F6P) Phosphofructokinase (PFK) Mg 2+ CH 2 - OH O 5 OH 1 2 3 4 6 -2 O 3 P-O fructose-1,6-bisphosphate (FBP) CH 2 - OPO 3 -2 O 5 OH 1 2 3 4 6 -2 O 3 P-O ATP ADP 2nd ATP utilization

25. Phosphofructokinase: mechanism Reaction 3 is the phosphorylation of C1 of F6P Nucleophilic attack by the C1-OH group of F6P on Mg 2+ -ATP. PFK reaction is the rate limiting step in glycolysis. The activity is enhanced allosterically by AMP(activator) and inhibited by ATP and citrate (inhibitors).

26. 4th reaction of glycolysis (  Gº’ = +5.73 kcal/mol) Aldolase Fructose-1,6-bisphosphate (FBP) CH 2 - OPO 3 -2 O 5 OH 1 2 3 4 6 -2 O 3 P-O C=O H - C - O- CH 2 - OH H PO 3 -2 H - C=O H - C - OH CH 2 - O- PO 3 -2 1(3) 2 3(1) 5 (2) 4 (1) 6 (3) Dihydroxyacetone phosphate (DHAP) Glyceraldehyde-3-phosphate (GAP)

27. Aldolase Catalyzes the cleavage of FBP to form 2 trioses, GAP and DHAP. Reaction proceeds via an aldo cleavage (retro aldol condensation). There are two mechanistic classes of aldolases: Class I (animals and plants) and Class II (fungi, algae, bacteria) - proceeds through a Zn intermediate (p. 591 for Zn- intermediate)

28. Aldolase In the Class I enzyme the reaction occurs as follows: Step 1: substrate binidng Step 2: reaction of the FBP carbonyl group with the side chain amino group of Lys (Schiff base) Step 3: C3-C4 bond cleavage resulting in the enamine formation and release of GAP. Step 4: Protonation of the enamine to an iminium cation Step 5: hydrolysis of the iminium cation to release DHAP

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30. 5th reaction of glycolysis (  Gº’ = +1.83 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 1(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

31. 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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33. 6th reaction of glycolysis (  Gº’ = +1.5 kcal/mol) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) H - C=O H - C - OH CH 2 - O- PO 3 -2 2 1 3 Glyceraldehyde-3-phosphate (GAP) -PO 3 -2 C - O H - C - OH CH 2 - O- PO 3 -2 2 3 O 1,3-Bisphosphoglycerate (1,3-BPG) NAD + + P i NADH + H + 1

34. 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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36. 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 3 -2 2 3 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

37. Phosphoglycerate kinase (PK) First ATP generating step of glycolysis nucleophilic attack

38. 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 = -2.9 1,3-BPG + ADP GAP + P i + NAD + + ADP 3PG + NADH + ATP in kcal/mol

39. 8th reaction of glycolysis (  Gº’ = +1.06 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)

40. 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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