Glycolysis

1. Glycolysis • Glucose → pyruvate (+ ATP, NADH) • Preparatory phase + Payoff phase • Enzymes • Highly regulated (eg. PFK-1 inhibited by ATP) • Form multi-enzyme complexes • Pass products/substrates along: efficiency

2. Overall balance sheet Glucose + 2NAD+ + 2ADP + 2Pi→ 2 pyruvate + 2NADH + 2H+ + 2ATP + 2H2O

3. Fermentation pathwaysAlternate fate of pyruvate • “Fermentation”: carbohydrate metabolism that generates ATP but doesn’t change oxidation state (no O2 used, no net change in NAD+/NADH) • Fermentation of pyruvate to lactate • Cells with no mitochondria (erythrocytes) • anerobic conditions • Regeneration of 2 NAD+ to sustain operation of glycolysis • No net change in oxidation state (glucose vs lactate) • Lactate is recycled to glucose (post-exercise)

4. Fermentation pathways • Fermentation of pyruvate to EtOH • Yeast and microorganisms • No net oxidation (glucose to ethanol) • EtOH and CO2 generated

5. Aerobic respiration of glucose (etc) • Glycolysis: • Start with glucose (6 carbon) • Generate some ATP, some NADH, pyruvate (2 x 3 carbon) • TCA cycle • Start with pyruvate • Generate acetate • Generate CO2 and reduced NADH and FADH2 • Electron transport • Start with NADH/FADH2 • Generate electrochemical H+ gradient • Oxidative phosphorylation • Start with H+ gradient and O2 (and ADP + Pi) • Generate ATP and H2O

6. Aerobic respiration • Stage 1: • Acetyl CoA production • Some ATP and reduced electron carriers (NADH) • Glycolysis (for glucose), pre-TCA • Stage 2: • Acetyl CoA oxidation • Some ATP, lots of reduced e- carriers (NADH/FADH2) • TCA cycle/Krebs cycle/Citric acid cycle • Stage 3: • Electron transfer and oxidative phosphorylation • Generate and use H+ electrochemical gradient • Use of reduced e- to generate ATP

7. Fate of pyruvate under aerobic conditions: TCA cycle (Ch. 16) • Oxidation of pyruvate in ‘pre-TCA cycle’ • Generation of acetyl CoA (2 carbons) • CO2 • NADH • Acetyl CoA → TCA cycle • Generation of ATP, NADH

8. Pre-TCA cycle • Pyruvate acetyl CoA • Via ‘pyruvate dehydrogenase complex’ • 3 enzymes • 5 coenzymes • ~irreversible • 3 steps • Decarboxylation • Oxidation • Transfer of acetyl groups to CoA • Mitochondria • Transport of pyruvate

9. Pre-TCA cycle • Coenzymes involved (vitamins) • Catalytic role • Thiamin pyrophosphate (TPP) • Thiamin • decarboxylation • Lipoic acid • 2 thiols disulfide formation • E- carrier and acyl carrier • FAD • Riboflavin • e- carrier • Stoichiometric role • CoA • Pantothenic acid • Thioester formation acyl carrier • NAD+ • Niacin • e- carrier

10. Pre-TCA cycle • Enzymes involved pyruvate dehydrogenase complex • multiprotein complex • Pyruvate dehydrogenase (24) (E1) • Bound TPP • Dihydrolipoyl transacetylase (60) (E2) • Bound lipoic acid • Dihydrolipoyl dehydrogenase (12) (E3) • Bound FAD • 2 regulatory proteins • Kinase and phosphatase

11. Pre-TCA cycle • Step 1: • Catalyzed by pyruvate dehydrogenase • Decarboxylation using TPP • C1 is released • C2, C3 attached to TPP as hydroxyethyl

12. Pre-TCA • Step 2 • Hydroxyethyl TPP is oxidized to form acetyl linked-lipoamide • Lipoamide (S-S) is reduced in process • Catalyzed by pyruvate dehydrogenase (E1) • Step 3 • Acetyl group is transferred to CoA • Oxidation energy (step 2) drives formation of thioester (acetyl CoA) • Catalyzed by dihydrolipoyl transacetylase (E2) • Step 4 • Dihydrolipoamide is oxidized/regenerated to lipoamide • 2 e- transfer to FAD, then to NAD+ • Catalyzed by dihydrolipoyl dehydrogenase (E3)

13. Overall…. • Pyruvate acetyl CoA • Via ‘pyruvate dehydrogenase complex’ • 4 step process • Decarboxylation of pyruvate and link to TPP • Oxidation of hydroxyethyl TPP and reduction/acetylation of lipoamide • Transfer of acetyl group to CoA • Oxidation of lipoamide via FAD (and e- transfer to NAD+)

14. Overall…. • Pyruvate acetyl CoA • Via ‘pyruvate dehydrogenase complex’ • 4 step process • Decarboxylation of pyruvate and link to TPP • Oxidation of hydroxyethyl TPP and reduction/acetylation of lipoamide • Transfer of acetyl group to CoA • Oxidation of lipoamide via FAD (and e- transfer to NAD+)

15. Pre-TCA • Substrate channeling • Multi enzyme complex •  rxn rate • Facilitated by E2 • ‘swinging’ lipoamide • accept e- and acetyl from E1 and transfer to E3 • Pathology: mutations in complex/thiamin deficiency

16. Regulation of pre-TCA • PDH complex • Inhibited by • Acetyl CoA, ATP, NADH, fatty acids • Activated by • CoA, AMP, NAD+ • Phosphorylation • Serine in E1 phosphorylated by kinase • Inactive E1 • Kinase activated by ATP, NADH, acetyl CoA… • Regulatory phosphatase hydrolyzes the phosphoryl • Activates E1 • Ca2+ and insulin stimulate

17. TCA cycle • Aerobic process • “Generates” energy • Occurs in mitochondria • 8 step process • 4 are oxidations • Energy ‘conserved’ in formation of NADH and FADH2 • Regenerated via oxidative phosphorylation • Acetyl group → 2 CO2 • Not the C from the acetyl group • Oxaloacetate required in ‘catalytic’ amounts • Some intermediates • Other biological purposes

18. Step 1: condensation of oxaloacetate with acetyl CoA citrate • Via citrate synthase • Conformational change upon binding • Oxaloacetate binds 1st • Conf change to create acetyl CoA site • Citrate synthase • Conformational changes upon binding of oxaloacetate TCA cycle unbound bound

19. TCA cycle • Mechanism of citrate synthase • 2 His and 1 Asp • 2 reactions • 1st rxn (condensation) • 2 steps • Highly unfavorable because of low oxaloacetate • 2nd rxn (hydrolysis) • Highly favorable because of thioester cleavage • Drives 1st rxn forward • CoA is recycled back to the pre TCA cycle