Glycolysis Degradation of Glucose to yield pyruvate
After this Lecture you will be able to answer: For each step of glycolysis: How does it occur? Why does it occur? Is it Regulated? How? What are the anaerboic fates of pyruvate? What is the purpose of gluconeogenesis? How are glycolysis and gluconeogenesis and glycolysis differentially regulated?
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 GAP + 2 ADP
Stage II of Glycolysis (Energy Recovery) Substrate Level Phosphorylation Substrate Level Phosphorylation
Summary of Stage II 2 GAP + 2 NAD + + 4 ADP + 2 P i 2 Pyruvate + 2 NADH + 2H+ + 4 ATP
Summary of Glycolysis Glucose + 2 NAD + + 2 ADP + 2 P i 2 Pyruvate + 2 NADH + 2 H + + 2 ATP NOTE: NAD + must be regenerated!
Reactions of Glycolysis Stage I
Hexokinase (First Use of ATP) CH 2 OH O ATP Mg 2+ ADP CH 2 OPO 3 O 2 HO OH OH HO OH OH OH OH -D-glucose (Glc) -D-glucose 6 P (G6P) G o (kj/mol) G (kj/mol) Glucose + P i G-6-P + H 2 O 14 20 ATP + H 2 O ADP + P i -30.5-54.8 Glucose + ATP G-6-P + ADP -16.5-34.8 NOTE: Lack of Specificity
Page 489 Role of Mg 2+
Substrate-induced Conformational Changes in Yeast Hexokinase Figure 15-2
Results of Conformational Change Formation of ATP binding site Exclusion of water Proximity effect
Regulation of Hexokinase Inhibition by glucose-6-p Impermeability
Hexokinase versus Glucokinase Hexokinase (all tissues) Non-specific (multiple hexoses) K M = ~100 µm Inhibited by glucose-6-p Glucokinase (primarily in liver) Specific (only glucose) 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: storage as glycogen Lower blood glucose: glycolysis for energy
Figure 22-4 Hexokinase versus Glucokinase
Glucose-6-P is a branch point Glycogen Glucose-6-P Fructose-6-P Glycolysis Pentose-P Pathway (NADPH) Regulation!
Phosphoglucose Isomerase: Fructose: C1 easier to phosphorylate Cleaved into 2 3-Carbon Molecules G o (kj/mol) G (kj/mol) Glucose-6-phosphate Fructose-6-phosphate 2-2
Reaction Mechanism of Phosphoglucose Isomerase
Reaction Mechanism of Phosphoglucose Isomerase (Substrate Binding) Figure 15-3 part 1
Reaction Mechanism of Phosphoglucose Isomerase (Acid-Catalyzed Ring Opening) Figure 15-3 part 2
Reaction Mechanism of Phosphoglucose Isomerase (Formation of cis-enediolate Intermediate) Figure 15-3 part 3
Reaction Mechanism of Phosphoglucose Isomerase (Proton Transfer) Figure 15-3 part 4
Reaction Mechanism of Phosphoglucose Isomerase (Base-Catalyzed Ring Closure) Figure 15-3 part 5
Reaction Mechanism of Phosphoglucose Isomerase (Product Release) Figure 15-3 part 1
Phosphofructokinase (Second Use of ATP) G o (kj/mol) G (kj/mol) F-6-P + P i F-1,6-bisP + H 2 O 16 36 ATP + H 2 O ADP + P i -30.5-54.8 F-6-P + ATP F-1,6-bisP + ADP -14.5-18.8 NOTE: bisphosphate versus diphosphate
Characteristics of Reaction Catalyzed by PFK Highly Thermodynamically favorable Irreversible First Commited step Therefore Regulated! -Rate determining step Reversed by Fructose-1,6-bisphosphatase Mechanism similar to Hexokinase
Regulatory Properties of PFK Main control point in glycolysis Allosteric enzyme Positive effectors AMP/ADP 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 Glycolysis Intermediate Low glucose Not Glycolysis Intermediate
Aldolase Carbon # from glucose 1 2 3 4 5 6 G o (kj/mol) G (kj/mol) F-1,6-bisP GAP + DHAP 24 ~0
Mechanism of Base-Catalyzed Aldol Cleavage NOTE: requirement for C=O at C2 Rationale for Phosphoglucose Isomerase Figure 15-4
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 CH 2 OH C O CH 2 OP CHO CHOH CH 2 OP Dihydroxyacetone-P (DHAP) Glyceraldehyde-3-P (GA3P) G o (kj/mol) G (kj/mol) DHAP GAP 7.5 ~0
The First Stage of Glycolysis Figure 15-7
Summary of Stage I Glucose + 2 ATP > 2 GAP + 2 ADP
Phosphofructokinase is allosterically by high concentrations of. I. activated; ATP II. inhibited; ATP III. inhibited; fructose-2,6-bisphosphate IV. activated; fructose -2,6-bisphosphate A) I, III B) II, III C) II, IV D) I, IV E) none of the above
Which one of the following does NOT occur in the reactions of glycolysis between glucose and fructose-1,6-bisphosphate? A. Product inhibition of an enzyme. B. An isomerization. C. Formation of a high energy intermediate. D. Phosphoryl transfer.
How much carbon dioxide is produced from the complete aerobic catabolism of F-1,6-BP via catabolic pathways? A. 0 CO 2 B. 3 CO 2 C. 4 CO 2 D. 5 CO 2 E. 6 CO 2
The reaction catalyzed by the enzyme aldolase has a ΔG ' +23 kj/mol. In muscle cells, the reaction proceeds in this same, forward direction. How can this occur? A) This ΔG ' means it is thermodynamically favored. B) The enzyme changes the G of the reaction in cells to something favorable. C) In cells the concentration of reactant(s) must be significantly greater than at equilibrium. D) In cells the concentration of product(s) must be significantly greater than at equilibrium. E) none of the above
Reactions of Glycolysis Stage II
Glyceraldehyde-3-P Dehydrogenase GAPDH 3,4 CHO COOP 2,5 CHOH + NAD + + P i CHOH + NADH H + + 1,6 CH 2 OP CH 2 OP Glyceraldehyde-3-P (GA3P) 1,3-Bisphosphoglycerate (BPG) G o (kj/mol) GAP + NAD+ H 2 O 3-PG + NADH + H+ -43.1 3PG + P i 1,3-BPG + H 2 O 49.4 G (kj/mol) GAP + NAD+ + P i 1,3-BPG + NADH + H+ 6.3 ~0
Acylphosphate O R C OP O OP C CHOH CH 2 OP Acylphosphate ("high energy") 1,3-Bisphosphoglycerate (BPG)
Enzymatic Mechanism of Glyceraldehyde-3-P Dehydrogenase
Enzymatic Mechanism of Glyceraldehyde-3-P Dehydrogenase (Substrate Binding) Figure 15-9 part 1
Enzymatic Mechanism of Glyceraldehyde-3-P Dehydrogenase (Thiol Addition) Figure 15-9 part 2
Enzymatic Mechanism of Glyceraldehyde-3-P Dehydrogenase (Dehydrogenation) Figure 15-9 part 3
Enzymatic Mechanism of Glyceraldehyde-3-P Dehydrogenase (Phosphate Binding) Figure 15-9 part 4
Enzymatic Mechanism of Glyceraldehyde-3-P Dehydrogenase (Product Release) Figure 15-9 part 5
2,3-bisphosphoglycerate Rxns #1-5 Hemoglobin regulation Rxn #6 Rxn #7 Rxn #8 Rxn #9 Rxn #10 Pyruvate kinase Pyruvate
Glycolysis deficiencies affect oxygen delivery
Phosphoglycerate Kinase Formation of first ATPs Substrate-level Phosphorylation
Coupled Reactions GA3P + NAD + + H 2 O 3PGA + NADH + H + ² G o ' = 43.1 kj/mol 3PGA + P i GA3P + NAD + + P i 1,3BPG + H 2 O 1,3BPG + NADH + H + ² G o ' = +49.4 kj/mol ² G o ' = +6.3 kj/mol 1,3BPG + ADP 3PGA + ATP ² G o ' = 18.8 kj/mol GA3P + NAD + + ADP + P i 3PGA + ATP + NADH + H + ² G o ' = 12.5 kj/mol G = ~0
Substrate Channeling GAPDH GAPDH PGK PGK
Phosphoglycerate Mutase G o (kj/mol) G (kj/mol) 3-PGA 2-PGA 5 ~0
Phosphohistidine Residue in Phosphoglycerate Mutase Page 500
Enzymatic Mechanism of Phosphoglycerate Mutase
Enzymatic Mechanism of Phosphoglycerate Mutase (Substrate Binding) Figure 15-12 part 1
Enzymatic Mechanism of Phosphoglycerate Mutase (Phosphorylation of Substrate) Figure 15-12 part 2
Enzymatic Mechanism of Phosphoglycerate Mutase (Phosphorylation of Enzyme) Figure 15-12 part 3
Enzymatic Mechanism of Phosphoglycerate Mutase (Product Release) Figure 15-12 part 4
Enolase G o (kj/mol) G (kj/mol) 2-PGA PEP 2-2 Formation of high energy intermediate
Pyruvate Kinase G o (kj/mol) PEP + H 2 O Pyruvate + P i -61.9 ADP + P i ATP + H 2 O 30.5 Formation of second ATPs Substrate-level Phosphorylation G (kj/mol) PEP + ADP Pyruvate + ATP -31.4-16.7
Enzymatic Mechanism of Pyruvate Kinase Figure 15-13
Figure 15-14 Hydrolysis of PEP
Regulatory Properties of Pyruvate Kinase Secondary control point in glycolysis Allosteric enzyme Positive effectors AMP/ADP Fructose-1,6-bisphosphate Negative effectors ATP (energy charge) Acetyl-Coenzyme A NADH
Summary of Second Stage of Glycolysis Figure 15-15
Summary of Stage II 2 GAP + 2 NAD + + 4 ADP + 2 P i 2 Pyruvate + 2 NADH + 2H+ + 4 ATP
Summary of Glycolysis Glucose + 2 NAD + + 2 ADP + 2 P i 2 Pyruvate + 2 NADH + 2 H + + 2 ATP NOTE: NAD + must be regenerated!
Shown below is the structure of 1,3-BPG. Which of the phosphate groups (labeled 1 or 2) has a very large, negative G of hydrolysis and why? A. 1, because it is an acyl phosphate. B. 1, because it oxidizes the carbon atom of glyceraldehyde-3-phosphate. C. 2, because it is transferred to ADP in the next step of glycolysis. D. 2, because it is more highly stabilized by resonance than the hydrolysis products. 1 2
Experimental evidence indicates that glyceraldehyde-3-phosphate dehydrogenase contains a critical residue in its active site, as shown by its inactivation by iodoacetamide. A) alanine B) aspartate C) cysteine D) methionine E) lysine
Consider the outline of glycolysis shown below. Which reactions are considered energy capture steps? A. 1 and 3 B. 7 and 10 C. 6 and 9 D. 6, 7 and 10 E. 1, 3, 6 and 9
If glucose labeled at the C-1 position with 14 C passes through glycolysis, on which carbon of pyruvate will the radiolabel be found? A) 1 B) 2 C) 3 D) It will be released in CO 2 rather than present in pyruvate. E) Not enough information is given to predict.
Metabolic Fates of Pyruvate Recycling of NADH Aerobic Anaerobic Figure 15-16
Homolactate Fermentation Anaerobic Glycolysis in muscle cells Lactate Dehydrogenase NADH + H + NAD + Lactate Pyruvate Lactate Dehydrogenase
Summary of Anaerobic Glycolysis Glucose + 2 ADP + 2 P i 2 Lactate + 2 ATP + 2 H 2 O
Energetics of Fermentation Glucose > 2 Lactate Glucose + 6 O 2 > 6 CO 2 + 6 H 2 O G o = -200 kj/mol G o = -2866 kj/mol Most of the energy of glucose is still available following glycolysis!
Figure 15-18 Alcoholic Fermentation Anaerobic glycolysis in Yeast
Regulation of Glycolysis and Gluconeogenesis
Free Energy Changes of Glycolytic Reactions Table 15-1
Figure 15-21 All Pathways are Thermodynamically Favorable and Irreversible
Regulatory Properties of Hexokinase Inhibition by glucose-6-p Because it s a branch point Glycogen Glucose-6-P Fructose-6-P Glycolysis Pentose-P Pathway (NADPH)
Regulatory Properties of Phosphofructokinase Main control point in glycolysis
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 NADH
Glycolysis and Gluconeogenesis
Necessity of Glucose-6-P and Glucose Brain, nervous system, and red blood cells use only Glucose for ATP production. Prolonged fasting and vigorous exercise can deplete glycogen Gluconeogenesis regenerates glucose
Glycolysis and Gluconeogenesis
Figure 16-21 Glycolysis and Gluconeogenesis
Gluconeogenesis and Glycolysis G = -36.3 kj/mol G = -83.8 kj/mol Figure 16-21
Coordinated Control of Glycolysis and Gluconeogenesis Increased Hormone signalling Increased glycogen breakdown
When glucose is metabolized to lactate in skeletal muscle how is ATP synthesized? A. Substrate-level phosphorylation B. Oxidative phosphorylation C. Covalent modification D. Both A and B
MCAT: Glycolysis is regulated by allosteric enzyme inhibition. Which of the following would be expected to decrease the rate of glycolysis? A. High levels of ATP B. High levels of AMP C. Increased blood glucose D. A high-fructose meal
Which of the following statements is not true concerning glycolysis? A. It is activated by high [AMP]. B. It results in net synthesis of ATP. C. It is an endergonic process. D. It results in synthesis of NADH. E. Its rate is slowed by a high [ATP]/[ADP] ratio.
For each step of glycolysis: How does it occur? Why does it occur? Is it Regulated? How? What are the anaerboic fates of pyruvate? What is the purpose of gluconeogenesis? How are glycolysis and gluconeogenesis and glycolysis differentially regulated?