Glycolysis Anaerobic degradation of glucose to yield lactate or ethanol and CO 2

Learning Objectives Sequence of Reactions –Metabolites –Enzymes Enzyme Mechanisms Energetics Regulation

Overview of Glycolysis Glucose (C 6 ) —> 2 Pyruvate (C 3 ) 2 ADP + 2 P i —> 2 ATP

Figure 15-1 Glycolysis

Stage I of Glycolysis (Energy Investment) 2X

Summary of Stage I Glucose + 2 ATP ——> 2 GA3P + 2 ADP + 2 H +

Stage II of Glycolysis (Energy Recovery) Substrate Level Phosphorylation —> Serine, Cysteine and Glycine —> Aromatic Amino Acids —> Alanine

Summary of Stage II 2 GA3P + 2 NAD ADP + 2 P i 2 Pyruvate + 2 NADH + 2 H ATP

Summary of Glycolysis Glucose + 2 NAD ADP + 2 P i 2 Pyruvate + 2 NADH + 2 H ATP NOTE: NAD + must be regenerated!

Reactions of Glycolysis Stage I

Hexokinase (First Use of ATP) NOTE: Lack of Specificity  G o’ (kJ/mol)  G (kJ/mol) Glucose + P i  G-6-P + H 2 O ATP + H 2 O  ADP + P i Glucose + ATP  G-6-P + ADP

Page 489 Role of Mg 2+

Figure 15-2 Substrate-induced Conformational Changes in Yeast Hexokinase

Results of Conformational Change Formation of ATP binding site Exclusion of water Increased nucleophilicity of CH 2 OH Proximity effect

Regulation of Hexokinase Inhibition by glucose-6-P Impermeability

Hexokinase versus Glucokinase Hexokinase (all tissues) –Non-specific –K M = ~100 µM –Inhibited by glucose-6-P Glucokinase (primarily in liver) –Specific –K M = ~10 mM –Not inhibited by glucose-6-P

Functional Rationale Most tissues: metabolize blood glucose which enters cells –Glc-6-P impermeable to cell membrane –Product inhibition Liver: maintain blood glucose –High blood glucose: glycogen –Low blood glucose: glycolysis

Figure 22-4 Hexokinase versus Glucokinase

Metabolism of Glucose-6-P Regulation!

Phosphoglucose Isomerase  G o’ (kJ/mol)  G (kJ/mol) Glucose-6-phosphate  Fructose-6-phosphate

Reaction Mechanism of Phosphoglucose Isomerase

Figure 15-3 part 1 Reaction Mechanism of Phosphoglucose Isomerase (Substrate Binding)

Figure 15-3 part 2 Reaction Mechanism of Phosphoglucose Isomerase (Acid-Catalyzed Ring Opening)

Figure 15-3 part 3 Reaction Mechanism of Phosphoglucose Isomerase (Formation of cis-enediolate Intermediate)

Figure 15-3 part 4 Reaction Mechanism of Phosphoglucose Isomerase (Proton Transfer)

Figure 15-3 part 5 Reaction Mechanism of Phosphoglucose Isomerase (Base-Catalyzed Ring Closure)

Figure 15-3 part 1 Reaction Mechanism of Phosphoglucose Isomerase (Product Release)

Phosphofructokinase (Second Use of ATP) NOTE: bisphosphate versus diphosphate  G o’ (kJ/mol)  G (kJ/mol) F-6-P + P i  F-1,6-bisP + H 2 O ATP + H 2 O  ADP + P i F-6-P + ATP  F-1,6-bisP + ADP

Characteristics of Reaction Catalyzed by PFK Rate-determining reaction Reversed by Fructose-1,6-bisphosphatase Mechanism similar to Hexokinase

Regulatory Properties of PFK Main control point in glycolysis Allosteric enzyme –Positive effectors AMP Fructose-2,6-bisphosphate –Negative effectors ATP Citrate

Page 558  - D -Fructose-2,6-Bisphosphate

Formation and Degradation of  - D -Fructose-2,6-bisP High glucose Low glucose

Aldolase Carbon # from glucose  G o’ (kJ/mol)  G (kJ/mol) F-1,6-bisP  GAP + DHAP 23.8 ~0

Figure 15-4 Mechanism of Base-Catalyzed Aldol Cleavage NOTE: requirement for C=O at C2 Rationale for Phosphoglucose Isomerase

Enzymatic Mechanism of Aldolase

Figure 15-5 part 1 Enzymatic Mechanism of Aldolase (Substrate Binding)

Figure 15-5 part 2 Enzymatic Mechanism of Aldolase (Schiff Base (imine) Formation)

Figure 15-5 part 3 Enzymatic Mechanism of Aldolase (Aldol Cleavage)

Figure 15-5 part 4 Enzymatic Mechanism of Aldolase (Tautomerization and Protonation)

Figure 15-5 part 5 Enzymatic Mechanism of Aldolase (Schiff Base Hydrolysis and Product Release)

Triose Phosphate Isomerase  G o’ (kJ/mol)  G (kJ/mol) DHAP  GAP 7.5 ~0

Part 494 Enzymatic Mechanism of Triose Phosphate Isomerase

Part 494 Transition State Analog Inhibitors of Triose Phosphate Isomerase

Figure 15-7 Schematic Diagram of the First Stage of Glycolysis

Summary of Stage I Glucose + 2 ATP ——> 2 GA3P + 2 ADP + 2 H +

Reactions of Glycolysis Stage II

Glyceraldehyde-3-P Dehydrogenase GAPDH 3,4 2,5 1,6  G o’ (kJ/mol)  G (kJ/mol) GAP + NAD+ H 2 O  3-PG + NADH + H PG + P i  1,3-BPG + H 2 O GAP + NAD+ + P i  1,3-BPG + NADH + H

Acylphosphate

Enzymatic Mechanism of Glyceraldehyde-3-P Dehydrogenase

Figure 15-9 part 1 Enzymatic Mechanism of Glyceraldehyde-3-P Dehydrogenase (Substrate Binding)

Figure 15-9 part 2 Enzymatic Mechanism of Glyceraldehyde-3-P Dehydrogenase (Thiol Addition)

Figure 15-9 part 3 Enzymatic Mechanism of Glyceraldehyde-3-P Dehydrogenase (Dehydrogenation)

Figure 15-9 part 4 Enzymatic Mechanism of Glyceraldehyde-3-P Dehydrogenase (Phosphate Binding)

Figure 15-9 part 5 Enzymatic Mechanism of Glyceraldehyde-3-P Dehydrogenase (Product Release)

2,3-bisphosphoglycerate Rxn #8 Rxn #7 Rxn #6 Rxns #1-5 Hemoglobin regulation Pyruvate kinase Pyruvate Rxn #9 Rxn #10

Glycolysis deficiencies affect oxygen delivery

Phosphoglycerate Kinase Formation of first ATPs Substrate-level Phosphorylation

Figure Yeast Phosphoglycerate Kinase

Coupled Reactions       G = ~0

Substrate Channeling

Phosphoglycerate Mutase  G o’ (kJ/mol)  G (kJ/mol) 3-PGA  2-PGA 4.4 ~0

Page 500 Phosphohistidine Residue in Phosphoglycerate Mutase

Enzymatic Mechanism of Phosphoglycerate Mutase

Figure part 1 Enzymatic Mechanism of Phosphoglycerate Mutase (Substrate Binding)

Figure part 2 Enzymatic Mechanism of Phosphoglycerate Mutase (Phosphorylation of Substrate)

Figure part 3 Enzymatic Mechanism of Phosphoglycerate Mutase (Phosphorylation of Enzyme)

Figure part 4 Enzymatic Mechanism of Phosphoglycerate Mutase (Product Release)

Enolase Formation of “high energy” intermediate Inhibition by F –  G o’ (kJ/mol)  G (kJ/mol) 2-PGA  PEP

Pyruvate Kinase Formation of second ATPs Substrate-level Phosphorylation  G o’ (kJ/mol)  G (kJ/mol) PEP + H 2 O  Pyruvate + P i ADP + P i  ATP + H 2 O 30.5 PEP + ADP  Pyruvate + ATP

Figure Enzymatic Mechanism of Pyruvate Kinase

Figure Hydrolysis of PEP

Regulatory Properties of Pyruvate Kinase Secondary control point in glycolysis Allosteric enzyme –Positive effectors ADP Fructose-1,6-bisphosphate –Negative effectors ATP (energy charge) Acetyl-Coenzyme A

Figure Summary of Second Stage of Glycolysis

Summary of Stage II 2 GA3P + 2 NAD ADP + 2 P i 2 Pyruvate + 2 NADH + 2 H ATP

Summary of Glycolysis Glucose + 2 NAD ADP + 2 P i 2 Pyruvate + 2 NADH + 2 H ATP NOTE: NAD + must be regenerated!

Figure Metabolic Fates of Pyruvate

Recycling of NADH Anaerobic Fate of Pyruvate

Role of Anaerobic Glycolysis in Skeletal Muscle

Homolactate Fermentation

Page 505 Lactate Dehydrogenase

Mechanism of Lactate Dehydrogenase

Summary of Anaerobic Glycolysis Glucose + 2 ADP + 2 P i 2 Lactate + 2 ATP + 2 H 2 O + 2 H +

Energetics of Fermentation Glucose ——> 2 Lactate Glucose + 6 O 2 ——> 6 CO H 2 O ∆G o’ = -200 kJ/mol ∆G o’ = kJ/mol Most of the energy of glucose is still available following glycolysis!

Alcoholic Fermentation

Figure Alcoholic Fermentation

Figure part 1 Pyruvate Decarboxylase

Page 507 Thiamin Pyrophosphate Thiamine = Vitamin B 1

Figure Mechanism of Pyruvate Decarboxylase

Figure part 1 Mechanism of Pyruvate Decarboxylase (Nucleophilic Attack)

Figure part 2 Mechanism of Pyruvate Decarboxylase (CO 2 Elimination)

Figure part 3 Mechanism of Pyruvate Decarboxylase (Protonation of Carbanion)

Figure part 4 Mechanism of Pyruvate Decarboxylase (Product Release)

Figure part 2 Alcohol Dehydrogenase

Page 509 Mechanism of Alcohol Dehydrogenase

Regulation of Glycolysis and Gluconeogenesis

Table 15-1 Free Energy Changes of Glycolytic Reactions

Figure Diagram of Free Energy Changes in Glycolysis

Regulatory Properties of Hexokinase Inhibition by glucose-6-P

Metabolism of Glucose-6-P Regulation!

Regulatory Properties of Phosphofructokinase Main control point in glycolysis

Figure Regulation of Phosphofructokinase

Regulatory Properties of Pyruvate Kinase Secondary control point in glycolysis Allosteric enzyme –Positive effectors ADP Fructose-1,6-bisphosphate –Negative effectors ATP (energy charge) Acetyl-Coenzyme A

Gluconeogenesis

Necessity of Glucose-6-P and Glucose

Glycolysis and Gluconeogenesis

Figure Glycolysis and Gluconeogenesis

Figure Glycolysis and Gluconeogenesis

Coordinated Control of Glycolysis and Gluconeogenesis