Chapt. 22 Glycolysis

1. Chapt. 22 Glycolysis • Ch. 22 Glycolysis • Student Learning Outcomes: • Explain how glucose is universal fuel, oxidized in every tissue to form ATP • Describe the major steps of glycolysis • Explain decision point for pyruvate utilization depending on oxygen • Describe major enzymes regulated • Explain lactic acidemia and causes

2. Glycolysis overview • Overview of Glycolysis, TCA cycle, electron transport chain: • Starts 1 glucose phosphorylated • 2 ATP to start process • Oxidation to 2 pyruvates • yields 2 NADH, 4 ATP • Aerobic conditions: pyruvate to TCA cycle, complete oxidation • NADH from cytoplasm into mitochondria • to ETC (waste some) • Complete oxidation total 30-32 ATP Fig. 1*

3. Anaerobic glycolysis • In absence of oxygen, anaerobic glycolysis: • Recycles NADH to permit glycolysis continue • Reduces pyruvate to lactate • Only 2 ATP per glucose • May cause lactic acidemia Fig. 2

4. Glycolysis phases • Glycolysis phases: • Preparation: • Glucose phosphorylated • Cleaved to 2 triose phosphates • Costs 2 ATP • ATP-generating phase: • Triose phosphates oxidized more • Produces 2 NADH • Produces 4 ATP Fig. 3

5. Glycolysis step 1 • 1. Glucose is phosphorylated by Hexokinase with ATP: • Commitment step • G6-P not cross plasma membrane • Irreversible • Many pathway choices • Glycogen synthesis needs G1-P • Many tissue-specific isozymes of hexokinases Fig. 4

6. Glycolysis phase I • 2 ATP convert Glucose to Fructose 1,6 bis-P; • Fructose 1,6-bis-P split to 2 trioses • Glyceraldehyde 3-P (and DHAP isomerized) • Key enzymes: • Hexokinase • PFK-1 • Commits to glycolysis • Regulated step Fig. 5 top

7. Glycolysis phase II • Oxidation, substrate level phosphorylation yield • 2 NADH, 4 ATP from 1 Glyceraldehyde 3-P • Key enzymes: • Glyceraldehyde 3-P dehydrogenase • High-energy bond • Pyruvate kinase: • Regulated step Fig. 5 lower

8. **Alternatie fates of pyruvate • Fate of pyruvate depends on availability of oxygen: • Much more ATP from complete oxidation of glucose • Aerobic: shuttles carry NADH into mitochondria; pyruvate can be oxidized to Acetyl CoA and enter TCA • Anaerobic: pyruvate reduced by NADH to lactate, NAD+, H+ Fig. 6*

9. Aerobic: Glycerol 3-P shuttle carries NADH • Aerobic: Glycerol 3-P shuttle carries e- from NADH into mitochondrion; regenerates cytosol NAD+ • Glycerol 3-P diffuses across • outer memberane, donates e- • to inner membrane FAD enzyme • Loses some energy • FAD(2H) not NADH Bacteria not need shuttle since only 1 compartment Fig. 5 top Fig. 7

10. Anaerobic: • Anaerobic glycolysis: NADH reduces pyruvate to lactate, regenerates NAD+ to continue glycolysis • 1 glucose + 2 ADP + 2 Pi -> 2 lactate + 2 ATP + 2 H2O + 2 H+ • Lactate and H+ transported to blood; can have lactic acidosis • Red blood cell, muscle, eye, other tissues • To maintain cell: • Run faster • More enzymes • Use lot glucose Fig. 9

11. Fate of lactate • Fate of lactate: • Used to make glucose (liver) – Cori cycle • Reoxidized to pyruvate (liver, heart, skeletal muscle) • lactate + NAD+ -> pyruvate + NADH • Lactate dehydrogenase (LDH) favors lactate, but if NADH used in ETC (or gluconeogenesis), then other direction • Heart can use lactate -> pyruvate for energy • Isoforms of LDH: M4 muscle; H4 heart; mixed others) Fig. 10

12. II. Other functions of glycolysis • Glycolysis generates precursors for other paths: • 5-C sugars for NTPs • Amino acids • Fatty acids, glycerol • Liver is major site of • biosynthesis Fig. 11

13. III. Glycolysis is regulated • Glycolysis is regulated by need for ATP: • Hexokinase • Tissue specific isoforms • Inhibited by G-6-P • Except for liver • PFK-1 • Pyruvate kinase • Pyruvate dehydrogenase • (PDH or PDC) Fig. 12

14. Levels of ATP, ADP, AMP • Levels of AMP in cytosol good indicator of rate ATP utilization • 2 ADP <-> AMP + ATP • reaction of adenylate kinase • Hydrolysis ATP -> ADP • increases ADP, AMP • ATP present highest conc: • Small dec ATP -> large AMP Fig. 13

15. Regulation of PFK-1 • Regulation of PFK-1: • Rate-limiting step, tetramer • 6 binding sites: • 2 substrates: ATP, Fructose 6-P • 4 allosteric: inhibit ATP • Activate by AMP • Activate by fructose 2,6 bis-P (product when excess glucose in blood) Fig. 14

16. Regulation of glycolysis enzymes • Regulation of pyruvate kinase: • R form (RBC), L (liver); M1/M2 muscle, others • Liver enz allosteric inhibition by compound in fasting; • also inhibited by PO4 from Protein Kinase A • Regulation of PDH (PDC): • By PO4to inactivate • Rate of ATP utilization • NADH/NAD+ ratio Fig. 12

17. Lactic Acidemia Fig. 15 • Lactic acidosis: • Excess lactic acid in blood > 5mM • pH < 7.2 • From increased • NADH/NAD+ • Many causes -> • Excess alcohol • Hypoxia

18. Key concepts • Glycolysis is universal pathway by which glucose is oxidized and cleaved to pyruvate • Enzymes are in cytosol • Generates 2 molecules of ATP (substrate-level phosphorylation) and 2 NADH • Pyruvate can enter mitochondria for complete oxidation to CO2 in TCA + electron transport chain • Anaerobic glycolysis reduces pyruvate to lactate, and recycles (wastes) NADH -> NAD+ • Key enzymes of glycolysis are regulated: hexokinase, PFK-1, pyruvate kinase, PDH C

19. Review question • Which of the following statements correctly describes an aspect of glycolysis? • ATP is formed by oxidative phosphorylation • Two molecules of ATP are used in the beginning of the pathway • Pyruvatekinase is the rate-limiting enzyme • One molecule of pyruvate and 3 olecules of CO2 are formed from the oxidation of 1 glucose • The reactions take place in the matrix of the mitochondria

20. Glyceraldehyde 3-P dehydrogenase • Glyceraldehyde 3-P dehydrogenase uses covalent linkage of substrate to S of cys to form ~P: • Covalent link to S of Cys; NAD+ nearby • Oxidation forms NADH + H+; ~S bond • NADH leaves, new NAD+ • Pi attacks thioester • Enzyme reforemd Fig. 17