Section 6 4. Gluconeogenesis Pyruvate oxidation

1. Section 64. GluconeogenesisPyruvate oxidation 10/25/05

2. Other roles of glycolysis & its intermediates • source of other glc & frc derivatives e.g., disaccharides, amino sugars, glycolipids • catabolism of other monosaccharides converted to glc 6-P & frc 6-P • DHAP → glycerol 3-P (glycerol part of triglycerides: FATS) • 1,3-BPG in RBCs, source of 2,3-BPG (↓O2 affinity of Hb) Comparison:anaerobicaerobic glycolysis advantages• ATP made fast •ATP yield high •1 compartment •acid build-up low •no mitochondria or O2 needed drawbacks•ATP yield low • slow •acid builds up • requires O2, mitochondria 1

3. Gluconeogenesis • synthesis of glucose units from noncarbohydrate precursors (e.g., pyruvate, glycerol, amino acids) • from pyruvate/lactate, the reverse of glycolysisexcept for the 3 irreversible steps (#1, 3 & 10) • bypasses of the 2 phosphorylation steps (glycolysis steps #1 & #3): • step 1'glc 6-P phosphataseglucose 6-P + H2O → glucose + Pi • step 3' 1,6 FBP phosphatasefructose 1,6 bisP + H2O → fructose 6-P + Pi glc¯ 1glc6P¯frc6P¯ 31,6FBP¯PEP¯ 10pyr 2

4. Bypass of glycolysis step 10 (PEP ® pyruvate) • 1 bypass enzyme uses the coenzyme, biotin, a carrier of carboxyl units (“activated CO2”) • carboxybiotin: activated precursor in carboxylations • functional form of biotin covalently linked to lys sidechain of enzyme • biotin-lys: long, flexible chain allows moving of COO–group from 1 site to another site reactive site 3

5. Gluconeogenesis: step10 bypass pyr ATP + CO2 ADP + Pi • first reaction: carboxylation of pyruvate* • mitochondrial matrixpyruvate + H+ transported across inner membrane by pyr carrier protein (S5L3sl7) • enzyme: pyruvate carboxylase (biotin-E) • second reaction: phosphorylation-coupled decarboxylation of oxaloacetate • cytosol • enzyme:PEP carboxykinase • summary of step 10 bypass (step 10') ATP + GTP + pyruvate → PEP + ADP + Pi + GDP 10'a oxalo-acetate 10'b GTP GDP + CO2 10'b PEP 4 * same reaction used to replenish Krebs cycle intermediates

6. Gluconeogenesis: summary & control • energetic cost for 2 lactate → glc: 6 ATP equivalents • 4 equivalents used to bypass step 10 • 2 to reverse step 7 • this pathway also used to synthesize glc from amino acids, glycerol, others • especially important during glc scarcity(fasting, low-carb diet) • control step enzyme inhibitor activator 10'a pyruvate carboxylase ADP acetyl CoA 3' FBP phosphatase AMP citrate* * provides coordination with Krebs (citric acid) cycle 5

7. Complete catabolism of glucose Stage I aerobic glycolysis glc → pyruvate (pyr) II pyr catabolism pyr → acetyl CoA III the Krebs cycle acetyl CoA → CO2 Pyruvate: a major intermediate glucose oxaloacetate pyruvate lactate manyamino acids acetyl CoA 6

8. Pyruvate catabolism: overall reaction • occurs in the mitochondrial matrix pyruvate + H+ enters via pyruvate carrier protein (S5L3sl7) • oxidative decarboxylation by pyruvate DHase • an organized enzyme assembly (60 polypeptide chains) • 3 enzyme types: E1, E2, E3 • these 3 enzymes use 5 different coenzymes 2 of these have stoichiometric (substrate/product) role • pyruvate + NAD+ + CoA →acetyl CoA + CO2 + NADH • conversion of 3-C compound to 2-C product & CO2 • irreversible, with no bypass significance: • relatively plentiful fatty acids not convertible to carbs • relatively scarce carbs depleted if rate not controlled 7

9. Pyr DHasecoenzymes NAD, FAD and TPP 8

10. Pyr DHasemechanism pyruvate oxidizedlipoyllysine (lp arm) lp arm the fate of pyruvate is traced in pink (boxes) acetyl-reducedlipoyllysine oxidizedlipoyllysine reducedlipoyllysine acetyl CoA Lehninger et al., Fig. 15-6(cf. Stryer Fig. 17.9) 9

11. Steps 1-5 in the oxidative decarboxylation of pyruvate to acetyl-CoA by the pyr DHase complex • E1-thiaminePP reacts with pyruvate; this enables decarboxylation • the hydroxyethyl group is oxidized to an acetyl group at the same time it is transferred to now-reduced lp arm • a transesterification in which the -SH group of CoA replaces the -SH group of lp arm-E2 to yield acetylCoA and the reduced form of the lp arm • dihydrolipoyl dehydrogenase (E3) catalyzes transfer of two hydrogen atoms (2 e–s + 2 H+) from the reduced lp arm of E2 to the FAD prosthetic group of E3, restoring the oxidized form of the lp arm of E2 • the reduced FADH2 group of E3 transfers a hydride ion (2 e–s + 1 H+) to NAD+, forming NADH. The enzyme complex is now ready for another catalytic cycle 10

12. Stoichiometries oxidative decarboxylation of pyruvate:ATP yield 2pyr + 2NAD+ + 2CoA → 2acetylCoA + 2CO2 + 2NADH ox phos:2NADH + 2H+ + O2→ 2NAD+ + 2H2O 5 stage II: 2H+ + 2pyr + O2 + 2CoA → 2acetylCoA + 2CO2 + 2H2O 5 stage I (aerobic glycolysis): glucose + O2→ 2pyr + 2H2O + 2H+ 5-7 stage I + II: glucose + 2O2 + 2CoA → 2acetylCoA + 2CO2 + 4H2O 10-12 11

13. Regulation of pyruvate DHase complex • irreversible with no bypass • relatively scarce carbohydrates would be depleted if rate not controlled • regulatory strategy: pyruvate → acetyl CoA only when ATP or its sources low • inhibition ATP, NADH, acetyl CoA, phosphorylation of E1 • activation AMP, dephosphorylation of E1 Model of pyr DHase complex E2 E1 lparmof E2 E3 12

14. Next:5. Pentose phosphate pathwayKrebs cycle