Lecture 4 (RS) (2009)

Lecture 4 (RS) (2009) - Lecture 4 Glycolysis...

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: Lecture 4 Glycolysis (Embden-Meyerhof Pathway) Cleavage of glucose and Substrate-level phosphorylation of ADP to ATP Background material Gibbs Free Energy G = H - TS G = RTlnQ RTlnKeq Go' = RTlnKeq G = Go' + RTlnQ Reduction potential E = Eoxidant Ereductant E = G/nF Q = [Preal]/[Rreal] T = 298K (25oC) Table 4 Table 3 Enzymes Amino acids and their reactions Classification Regulation The "Powertrain" of Human Metabolism (Overview) CARBOHYDRATES PROTEINS LIPIDS Glucose Amino acids Fatty acids Oxaloacetate O2 Glycogen Glucose-6-P Pyruvate Acetyl-CoA NADH ATP Glycolysis CO2 H2O Lactate Ketone bodies Ribose-5-P NADPH NADH Cholesterol p. 21 Aerobic Glycolysis (Overview) Fischer projection-open chain CHO H HO H H OH H HO CHO OH CH2OH O 1 CH2OPO322 O 1 CH2OPO322 O DHAP 1 H OH OH CH2OH 2 H OH OH CH2OPO32HO H H H OH OH CH2OPO32- 3 HO 3 4 5 6 4 H OH OH CH2OPO32H 3 CH2OH + 4 HC 5 O ATP ADP H H ATP ADP H H 5 OH G-3-P Haworth projection Ring form GLC GLC-6-P F-6-P F-1,6-bisP 6 CH OPO 22 3 CH2OH H OH OH H OH O H OH H CH2OPO32O OH H OH OH O3POH2C H O OH CH2OH O3POH2C H O OH CH2OPO32- HPO42OH 1 ATP OH H 2 H OH H OH 3 ATP ADP H OH H 6 NAD+ NADH + H+ ADP 2 Molecules -O - C O O C O -O -O OPO32C O C O 10 O OPO32CH2 9 H OPO32- 8 H OH CH2OPO32- 7 H 3,4 2,5 1,6 C O OH CH3 ATP ADP H2O CH2OH ATP ADP CH2OPO32- PYR PEP 2-PGA 3-PGA 1,3 bisPGA p. 25 Cellular Logic Why add a phosphate? CHO H HO H H OH H OH OH CH2OH H HO H H CHO OH H OH OH CH2OH Cell glucose level ~ 4 - 5 mM Blood glucose level ~ 5 mM Cellular Logic Why add a phosphate? 1. Prevents reverse diffusion through GluT. 2. Prevents diffusion through plasma membrane. 3. Maintains the Glucose concentration gradient. 4. Binds to enzymes better (recognition tag). The 1. Reaction of Glycolysis Enzyme Class: Transferase Specifically, phosphotransferase or "kinase" CHO H HO H H OH H OH OH Enzyme CH2O H Electrophile (P) Nucleophile (-OH) H+ O -O P O - Glucose (Glc) ATP N O O P O O - NH2 N N O H OH H OH N O P O O - p. 24,26 CHO NH2 H HO H H OH H O OH OH CH2O-O P O - N O O P O O - N N O N O P O OH OH H OH CHO NH2 H HO H H OH H OOH OH CH2O OP OO O P OO O P O OH OH Glc-6-P ADP N N O N N H OH Energy coupling... p. 26 G Calculations on 1. Reaction in Glycolysis (Phosphorylation of Glucose) Summary of Chalk Board Calculations Go' or G (kJmol-1) Glc + Pi 5 mM 1 mM Glc-6-P + 0.083 mM H2O +13.9 +21.1 Keq 3.7x10-3 2.8x10-4 (intracellular concentrations) Go' = RTlnKeq Keq = e^(-Go'/RT) = e^(-13,900 Jmol-1/8.315 Jmol-1K-1 x 298 K) = 0.0037 G = Go' + RTln[P]/[S] G = +13.9 kJmol-1 + (8.315 Jmol-1K-1 x 310 K) x ln[83 x10-6]/[5 x 10-3][1 x 10-3] G = +13.9 kJmol-1 + (2.578 kJmol-1) x 2.81 G = +13.9 + 7.2 = +21.1 kJmol-1 (intracellular conditions are even more unfavorable than standard conditions for the reaction to proceed as desired) Q: How to drive glucose phosphorylation forward despite large positive G? A: Couple to much more favorable reaction (larger negative G) such as to the hydrolysis of ATP!! Go' or G (kJmol-1) ATP + H2O ADP + Pi -30.5 -46.5 Keq 2.2x105 6.8x107 Intracellular [ATP]/[ADP][Pi] = 500 or higher ("phosphorylation potential" ) G = Go' + RTln[P]/[S] G = - 30.5 + RTln 1/500 G = - 30.5 + (- 15.4) = - 46.5 kJmol-1 Combination (coupling) of both reactions via an enzyme (hexokinase): Go' or G (kJmol-1) Glc + Pi Glc-6-P Intracellular conditions ATP + H2O ADP Intracellular conditions + H2O +13.9 +21.1 -30.5 -46.5 ADP -16.6 -25.4 Keq 3.7x10-3 2.8x10-4 2.2x105 6.8x107 8.1x102 1.9x104 + Pi Glc + ATP Glc-6-P Intracellular conditions + Note: Coupling of a reaction to ATP hydrolysis can shift its Keq up to 108 fold !! (2.8x10-4 1.9x104) Hexokinase (induced fit) Isoenzymes: catalyze the same reaction but differ in properties (Liver) Aerobic Glycolysis (Overview) Fischer projection-open chain CHO H HO H H OH H HO CHO OH CH2OH O 1 CH2OPO322 O 1 CH2OPO322 O DHAP 1 H OH OH CH2OH 2 H OH OH CH2OPO32HO H H H OH OH CH2OPO32- 3 HO 3 4 5 6 4 H OH OH CH2OPO32H 3 CH2OH + 4 HC 5 O ATP ADP H H ATP ADP H H 5 OH G-3-P Haworth projection Ring form GLC GLC-6-P F-6-P F-1,6-bisP 6 CH OPO 22 3 CH2OH H OH OH H OH O H OH H CH2OPO32O OH H OH OH O3POH2C H O OH CH2OH O3POH2C H O OH CH2OPO32- HPO42OH 1 ATP OH H 2 H OH H OH 3 ATP ADP H OH H 6 NAD+ NADH + H+ ADP 2 Molecules -O - C O O C O -O -O OPO32C O C O 10 O OPO32CH2 9 H OPO32- 8 H OH CH2OPO32- 7 H 3,4 2,5 1,6 C O OH CH3 ATP ADP H2O CH2OH ATP ADP CH2OPO32- PYR PEP 2-PGA 3-PGA 1,3 bisPGA p. 25 Fischer projection-open chain CHO H HO H H OH H HO CHO OH CH2OH O 1 H OH OH CH2OH 2 H OH OH CH2OPO32HO H H H OH OH CH2OPO32- ATP ADP H H Haworth projection Ring form GLC GLC-6-P Isomerization F-6-P Reaction 2: Phosphogluco isomerase or Glucose-6-P ketolisomerase (see p 24) Go' = 1.67 kJ/mol G = - 2.92 kJ/mol p. 25 CH2OH O 1 CH2OPO322 O 1 CH2OPO322 O DHAP 3 HO H H H OH OH CH2OPO32HO 3 4 5 6 4 H OH OH CH2OPO32H 3 CH2OH + 4 HC 5 O ATP ADP H H 5 OH G-3-P Phosphorylation F-6-P F-1,6-bisP 6 CH OPO 22 3 Reaction 3: Phosphofructokinase-1 (PFK-1) or ATP:Fructose-6-P 1-phosphotransferase Go' = -14.2 kJ/mol G erythrocyte = -18.8 kJ/mol p. 25 CH2OH O 1 CH2OPO322 O 1 CH2OPO322 O DHAP 3 HO H H H OH OH CH2OPO32HO 3 4 5 6 4 H OH OH CH2OPO32H 3 CH2OH + 4 HC 5 O ATP ADP H H Cleavage 5 OH G-3-P F-6-P F-1,6-bisP 6 CH OPO 22 3 Reaction 4: Aldolase or Fructose-1,6-BisP glyceraldehyde-3-P lyase p. 25 Go' = +23.9 kJ/mol G = -0.23 kJ/mol Aldol Cleavage in Glycolysis (Reaction No. 4) CH2OP C O H CH2OP C O H O H HO C H H HO H C C Rest C Rest O Aldol Cleavage in Glycolysis Requirements for cleavage: C-OH must be to carbonyl carbon R1 R1 H C R2 R5 O R3 O H R2 C C O R5 C C R4 + R3 C R4 O H R1 C R2 C H O R3 p. 27 Reverse Reaction (Aldol Condensation) Condensation Requirements for condensation: H on C that is to carbonyl carbon on substrate 1 (C-H acidic) and need for carbonyl group on substrate 2 Substrate 1 R1 C R2 C H O R3 R2 Resonance stabilized H R1 C C O R3 R2 R1 C C O R3 H R5 C R4 O R2 R5 R1 C C C R4 O R3 OH Substrate 2 p. 27 F-1,6-BP DHAP G3P How can we force this reaction to go forward?? Le Chatelier's Principle Aldolase Go'= + 23.9 kJ/mol Go'= - 14.2 kJ/mol G = - 18.8 kJ/mol PFK-1 F6P Common Intermediate F-1,6-BP DHAP G3P Aldolase Go'= + 23.9 kJ/mol Go'= - 14.2 kJ/mol G = - 18.8 kJ/mol Phosphogluco isomerase Go'= + 1.7 kJ/mol G = - 2.9 kJ/mol Go'= - 16.6 kJ/mol G = - 24.8 kJ/mol PFK-1 F6P Hexokinase F-1,6-BP DHAP G3P Aldolase Go'= + 23.9 kJ/mol Go'= - 14.2 kJ/mol G = - 18.8 kJ/mol Go'= - 16.6 kJ/mol G = - 24.8 kJ/mol PFK-1 F6P Hexokinase F-1,6-BP DHAP G3P "ripple effect" Pyruvate Kinase Aldolase Go'= + 23.9 kJ/mol G = - 0.23 kJ/mol Go'= - 31.7 kJ/mol G = - 23.0 kJ/mol CH2OH O 1 CH2OPO322 O 1 CH2OPO322 O DHAP 3 HO H H H OH OH CH2OPO32HO 3 4 5 6 4 H OH OH CH2OPO32H 3 CH2OH + 4 HC 5 O ATP ADP H H 5 OH G-3-P F-6-P F-1,6-bisP 6 CH OPO 22 3 Reaction 5: Triose phosphate isomerase (ketolisomerase) For first 5 rxns, the total Go' = +2.2 kJ/mol G = -53.4 kJ/mol p. 25 4 HC H O 5 OH G-3-P Reaction 6: The Big Deal! 6 CH OPO 22 3 23 6 NAD HPO42+ Glyceraldehyde-3-P dehydrogenase or Glyceraldehyde-3-P NAD+ oxidoreductase (phosphorylating) NADH + H+ OPO32- H 3,4 2,5 1,6 C O OH CH2OPO32- 1,3 bisPGA p. 25 The Mechanism of Reaction 6 Role of Thioesters in Energy Transduction Disclaimer: Do NOT memorize mechanism, understand what happens here! Esters versus Thioesters O R1 C Ester - O O R2 Resonance stabilization - R1 C O - R2 O R1 C - No resonance stabilization of thioesters (more "strained" molecules) S R2 Thioester Therefore, Go' of thioester hydrolysis is highly negative (about 30 kJ/mol). See Table 4. Mechanism of Glyceraldehyde-3-P Dehydrogenase CH2OPO3 2CHOH H C O- SH HC H O OH CH2OPO32B NAD+ Enzyme + BH S NAD+ + Glyceraldehyde-3-P CH2OPO32CHOH Hydride removal to NAD+ H C O- S + HB NAD+ p. 29 CH2OPO3 2CHOH C O CH2OPO32CHOH C O O -O P OH O- HB + S NAD+ + S HB NADH + NAD NADH Thioester-linked substrate 2O3PO C H SH O OH CH2OPO32- NAD+ + B Enzyme + H+ 1,3-Bisphosphoglycerate Redox reaction p. 29 NAD(P)H: Safe and Soluble Carrier of "Hydrogen" From vitamin B (niacin) H O C NH2 2H H+ H H O C NH2 (Nicotinamide) + N Hydride acceptor/donor .. N R O H O H H OH N Ribose NH2 N NADH or NADPH -O P O H OH O N O P OO H H OH OX O H H N Adenine PPi Ribose Dehydrogenases p. 28 NAD+ or NADP+ G/E Calculations on 6. Reaction in Glycolysis Summary of Chalk Board Calculations Glyceraldehyde-3-P + Pi + NAD+ 1,3-Bisphoshoglycerate + NADH + H+ Can be formally written as two reactions (coupled by enzyme): I. Glyceraldehyde-3-P + H2O + NAD+ 3-Phoshoglycerate + NADH + H+ II. 3-Phoshoglycerate + Pi 1,3-Bisphoshoglycerate + H2O Compare Go' of both reactions I. Glyceraldehyde-3-P + H2O + NAD+ 3-Phoshoglycerate + NADH + H+ Similar to oxidation of acetaldehyde to acetate (see Table 3, reactions 3 and 12) Acetaldehyde + H2O + NAD+ Go' = -nFEo Acetate + NADH + H+ Eo = EoOxidant EoReductant Eo = EoNAD+ EoAcetaldehyde Eo = - 0.32V (-0.58V) Eo = + 0.26V Go' = - 2 (electrons) x 96.5 kJmol-1V-1 x 0.26V Go' = - 50.2 kJmol-1 II. 3-Phoshoglycerate + Pi See Table 4 for Go' of 1,3-Bisphoshoglycerate + H2O Hydrolysis of 1,3-Bisphosphoglycerate (Go' = - 49.6 kJmol-1) or Formation of 1,3-Bisphosphoglycerate (Go' = + 49.6 kJmol-1) Conclusion: In a first approximation (because we are only looking at Go' values), the oxidation of glyceraldehyde-3-P to 3-phosphoglycerate yields about the same amount of energy (-50.2 kJmol-1) as is required to produce 1,3-bisphosphoglycerate from 3-phosphoglycerate and Pi (+49.6 kJmol-1). Again, these two reactions do not occur in isolation but are coupled or combined by the enzyme Glyceraldehyde-3-P Dehydrogenase. Therefore, the Go' (and in fact G) of the overall reaction is close to zero. ...
View Full Document

This note was uploaded on 09/06/2009 for the course BIS 103 taught by Professor Abel during the Spring '08 term at UC Davis.

Ask a homework question - tutors are online