Glycolysis

Anaerobic degradation of glucose to yield lactate

or ethanol and CO2

Learning Objectives

• Sequence of Reactions

– Metabolites

– Enzymes

• Enzyme Mechanisms

• Energetics

• Regulation

Overview of Glycolysis

Glucose (C6) —> 2 Pyruvate (C3)

2 ADP + 2 Pi —> 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

Substrate Level Phosphorylation

—> Serine, Cysteine and Glycine

—> Aromatic Amino Acids

—> Alanine

Summary of Stage II

2 GA3P + 2 NAD+ + 4 ADP + 2 Pi

2 Pyruvate + 2 NADH + 2 H+ + 4 ATP

Summary of Glycolysis

Glucose + 2 NAD+ + 2 ADP + 2 Pi

2 Pyruvate + 2 NADH + 2 H+ + 2 ATP

NOTE: NAD+ must be regenerated!

Reactions of Glycolysis

Stage I

Hexokinase(First Use of ATP)

O

CH2OH

OH

OH

OHHO

O

CH2OPO3

OH

OH

OHHO

-D-glucose–6–P(G6P)

-D-glucose(Glc)

ATP ADP

Mg2+

2–

NOTE: Lack of Specificity

Go’ (kJ/mol) G (kJ/mol) Glucose + Pi G-6-P + H2O 13.8 20.5ATP + H2O ADP + Pi -30.5 -54.4

Glucose + ATP G-6-P + ADP -16.7 -33.9

Page 489

Role of Mg2+

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 CH2OH

• Proximity effect

Regulation of Hexokinase

Inhibition by glucose-6-P

Impermeability

Hexokinase versus Glucokinase

• Hexokinase (all tissues)– Non-specific

– KM = ~100 µM

– Inhibited by glucose-6-P

• Glucokinase (primarily in liver)– Specific

– KM = ~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

Glucose-6-P Fructose-6-P Glycolysis

Glycogen

Pentose-P Pathway (NADPH)

Regulation!

Phosphoglucose Isomerase

Go’ (kJ/mol) G (kJ/mol) Glucose-6-phosphate Fructose-6-phosphate 2.2 -1.4

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

Go’ (kJ/mol) G (kJ/mol) F-6-P + Pi F-1,6-bisP + H2O 16.3 36.0ATP + H2O ADP + Pi -30.5 -54.4

F-6-P + ATP F-1,6-bisP + ADP -14.2 -18.8

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

4

5

6

1

2

3

Carbon #from glucose

Go’ (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

CHO

CHOH

CH2OP

Glyceraldehyde-3-P(GA3P)

CH2OH

C

CH2OP

O

Dihydroxyacetone-P(DHAP)

Go’ (kJ/mol) G (kJ/mol) DHAP GAP 7.5 ~0

Part 494

Enzymatic Mechanism ofTriose 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

GAPDHCHO

CHOH

CH2OP

Glyceraldehyde-3-P(GA3P)

+ NAD+ Pi+

COOP

CHOH

CH2OP

+ NADH H++

1,3-Bisphosphoglycerate(BPG)

3,4

2,5

1,6

Go’ (kJ/mol) G (kJ/mol) GAP + NAD+ H2O 3-PG + NADH + H+ -43.1 36.03PG + Pi 1,3-BPG + H2O 49.4 -54.4

GAP + NAD+ + Pi 1,3-BPG + NADH + H+ 6.3 -18.8

Acylphosphate

C

CHOH

CH2OP

1,3-Bisphosphoglycerate(BPG)

O OP

R C OP

O

Acylphosphate("high energy")

Enzymatic Mechanism ofGlyceraldehyde-3-P

Dehydrogenase

Figure 15-9 part 1

Enzymatic Mechanism ofGlyceraldehyde-3-P

Dehydrogenase(Substrate Binding)

Figure 15-9 part 2

Enzymatic Mechanism ofGlyceraldehyde-3-P

Dehydrogenase(Thiol Addition)

Figure 15-9 part 3

Enzymatic Mechanism ofGlyceraldehyde-3-P

Dehydrogenase(Dehydrogenation)

Figure 15-9 part 4

Enzymatic Mechanism ofGlyceraldehyde-3-P

Dehydrogenase(Phosphate Binding)

Figure 15-9 part 5

Enzymatic Mechanism ofGlyceraldehyde-3-P

Dehydrogenase(Product Release)

2,3-bisphosphoglycerate

Rxn #8

Rxn #7

Rxn #6

Rxns #1-5 Hemoglobinregulation

Pyruvate kinase

Pyruvate

Rxn #9

Rxn #10

Glycolysis deficiencies affect oxygen delivery

Phosphoglycerate Kinase

Formation of first ATPs

Substrate-level Phosphorylation

Figure 15-10

Yeast Phosphoglycerate Kinase

Coupled Reactions

GA3P + NAD+ + H2O 3PGA + NADH + H+

3PGA + Pi 1,3BPG + H2O

GA3P + NAD+ + Pi

² Go' = –43.1 kJ / mol

² Go' = +49.4 kJ / mol

² Go' = +6.3 kJ / mol1,3BPG + NADH + H+

1,3BPG + ADP 3PGA + ATP ² Go' = –18.8 kJ / mol

GA3P + NAD+ + ADP + Pi3PGA + ATP + NADH + H+ ² Go' = –12.5 kJ /mol

G = ~0

Substrate Channeling

Phosphoglycerate Mutase

Go’ (kJ/mol) G (kJ/mol) 3-PGA 2-PGA 4.4 ~0

Page 500

Phosphohistidine Residue inPhosphoglycerate Mutase

Enzymatic Mechanism ofPhosphoglycerate Mutase

Figure 15-12 part 1

Enzymatic Mechanism ofPhosphoglycerate Mutase

(Substrate Binding)

Figure 15-12 part 2

Enzymatic Mechanism ofPhosphoglycerate Mutase(Phosphorylation of Substrate)

Figure 15-12 part 3

Enzymatic Mechanism ofPhosphoglycerate Mutase(Phosphorylation of Enzyme)

Figure 15-12 part 4

Enzymatic Mechanism ofPhosphoglycerate Mutase

(Product Release)

Enolase

Formation of “high energy” intermediate

Inhibition by F–

Go’ (kJ/mol) G (kJ/mol) 2-PGA PEP -3.2 -2.4

Pyruvate Kinase

Formation of second ATPs

Substrate-level Phosphorylation

Go’ (kJ/mol) G (kJ/mol) PEP + H2O Pyruvate + Pi -61.9 ADP + Pi ATP + H2O 30.5

PEP + ADP Pyruvate + ATP -31.4 -16.7

Figure 15-13

Enzymatic Mechanism of Pyruvate Kinase

Figure 15-14

Hydrolysis of PEP

Regulatory Properties ofPyruvate Kinase

• Secondary control point in glycolysis

• Allosteric enzyme– Positive effectors

•ADP•Fructose-1,6-bisphosphate

– Negative effectors•ATP (energy charge)•Acetyl-Coenzyme A

Figure 15-15

Summary of Second Stage

of Glycolysis

Summary of Stage II

2 GA3P + 2 NAD+ + 4 ADP + 2 Pi

2 Pyruvate + 2 NADH + 2 H+ + 4 ATP

Summary of Glycolysis

Glucose + 2 NAD+ + 2 ADP + 2 Pi

2 Pyruvate + 2 NADH + 2 H+ + 2 ATP

NOTE: NAD+ must be regenerated!

Figure 15-16

Metabolic Fates of Pyruvate

Recycling of NADH

Anaerobic Fate of Pyruvate

Role of Anaerobic Glycolysis in Skeletal

Muscle

Homolactate Fermentation

NADH + H+ NAD+

LactateLactate

Dehydrogenase

Pyruvate

Page 505

Lactate Dehydrogenase

Mechanismof

LactateDehydrogenase

Summary of Anaerobic Glycolysis

Glucose + 2 ADP + 2 Pi

2 Lactate + 2 ATP + 2 H2O + 2 H+

Energetics of Fermentation

Glucose ——> 2 Lactate

Glucose + 6 O2 ——> 6 CO2 + 6 H2O

∆Go’ = -200 kJ/mol

∆Go’ = -2866 kJ/mol

Most of the energy of glucose is still available following glycolysis!

Alcoholic Fermentation

CO2 NADH + H+ NAD+

Pyruvate EthanolAlcohol

Dehydrogenase

AcetaldehydePyruvate

Decarboxylase

Figure 15-18

Alcoholic Fermentation

Figure 15-18 part 1

Pyruvate Decarboxylase

Page 507

Thiamin Pyrophosphate

Thiamine = Vitamin B1

Figure 15-20

Mechanism ofPyruvate Decarboxylase

Figure 15-20 part 1

Mechanism ofPyruvate Decarboxylase

(Nucleophilic Attack)

Figure 15-20 part 2

Mechanism ofPyruvate Decarboxylase

(CO2 Elimination)

Figure 15-20 part 3

Mechanism ofPyruvate Decarboxylase(Protonation of Carbanion)

Figure 15-20 part 4

Mechanism ofPyruvate Decarboxylase

(Product Release)

Figure 15-18 part 2

Alcohol Dehydrogenase

Page 509

Mechanism ofAlcohol Dehydrogenase

Regulation of Glycolysisand Gluconeogenesis

Table 15-1

Free Energy Changes of Glycolytic Reactions

Figure 15-21

Diagram of Free Energy Changes in Glycolysis

Regulatory Properties of Hexokinase

Inhibition by glucose-6-P

Metabolism of Glucose-6-P

Glucose-6-P Fructose-6-P Glycolysis

Glycogen

Pentose-P Pathway (NADPH)

Regulation!

Regulatory Properties ofPhosphofructokinase

• Main control point in glycolysis

Figure 15-23

Regulation of Phosphofructokinase

Regulatory Properties ofPyruvate 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

Glucose-6-P Fructose-6-P Glycolysis

Glycogen

Pentose-P Pathway (NADPH)

Glucose

Glycolysisand

Gluconeogenesis

Glycolysis and Gluconeogenesis

Figure 16-21

Glycolysisand

Gluconeogenesis

Figure 16-21

Glycolysis and Gluconeogenesis

Coordinated Control of Glycolysis and Gluconeogenesis