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 + 2P i → 2 pyruvate + 2NADH + 2H + + 2ATP + 2H 2 O

3. Fermentation pathways Alternate fate of pyruvate “Fermentation”: carbohydrate metabolism that generates ATP but doesn’t change oxidation state (no O 2 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 CO 2 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 CO 2 and reduced NADH and FADH 2 Electron transport –Start with NADH/FADH 2 –Generate electrochemical H + gradient Oxidative phosphorylation –Start with H+ gradient and O 2 (and ADP + P i ) –Generate ATP and H 2 O

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/FADH 2 ) 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) –CO 2 –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) (E 1 ) Bound TPP –Dihydrolipoyl transacetylase (60) (E 2 ) Bound lipoic acid –Dihydrolipoyl dehydrogenase (12) (E 3 ) Bound FAD 2 regulatory proteins –Kinase and phosphatase

11. Step 1: –Catalyzed by pyruvate dehydrogenase Decarboxylation using TPP C 1 is released C 2, C 3 attached to TPP as hydroxyethyl Pre-TCA cycle

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 (E 1 ) Step 3 –Acetyl group is transferred to CoA –Oxidation energy (step 2) drives formation of thioester (acetyl CoA) –Catalyzed by dihydrolipoyl transacetylase (E 2 ) Step 4 –Dihydrolipoamide is oxidized/regenerated to lipoamide –2 e- transfer to FAD, then to NAD+ –Catalyzed by dihydrolipoyl dehydrogenase (E 3 )

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 E 2 –‘swinging’ lipoamide –accept e - and acetyl from E 1 and transfer to E 3 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 E 1 phosphorylated by kinase –Inactive E1 –Kinase activated by ATP, NADH, acetyl CoA… Regulatory phosphatase hydrolyzes the phosphoryl –Activates E1 –Ca 2+ 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 FADH 2 –Regenerated via oxidative phosphorylation –Acetyl group → 2 CO 2 Not the C from the acetyl group –Oxaloacetate required in ‘catalytic’ amounts –Some intermediates Other biological purposes

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

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