1. Lehninger Ch. 14 BIO 322 Recitation 1 / Spring 2013 GLYCOLYSIS, GLUCONEOGENESIS, AND THE PENTOSE PHOSPHATE PATHWAY

2. OUTLINE 2 Glycolysis  Fates of Pyruvate  Regulation of Glycolysis (Chapter 15) Gluconeogenesis Pentose Phosphate Pathway

3. Fates of Glucose 3

4. Glycolysis 4 D-Glucose (6C) 2 molecules of Pyruvate (3C) ATP, NADH conserve some released energy In some mammalian tissues/cell types sole source of energy Ten steps: 5 in preparatory phase & 5 in payoff phase

5. First 5 steps – Prepatory Phase 2 ATP invested, raising the free energy content of intermediates Metobolized hexoses converted to common product G3P. One molecule of glucose yields to two molecules of G3P. Last 5 steps – Payoff Phase 4 ATP produced, Net yield 2 ATP Energy Conserved via 2 NADH per glucose molecule. Three types of chemical transformation: 1)Degradation of glucose carbon skeleton to yield pyruvate. 2)ADP into ATP 3)NAD+ - NADH 5 ‘Lysis’ step Triose Phosphates Phosphorylation without ATP

6. Under STD conditions, glycolysis is irreversible, completed by a large net decrease in free energy At cellular conditions, energy recovered as ATP with an efficiency of more than %60 Most of the energy is still in pyruvate and can be extracted by oxidative reactions in Citric Acid Cycle (Ch. 16) Oxidative Phosphorylation (Ch. 19) Fates of Pyruvate 6

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8. STEP 1: Phosphorylation of Glucose Glucose Phosphorylation at C-6 Irreversible step Hexokinase Requires Mg (True Substrate Mg+ATP) Mg shields negative charges of phosphoryl groups of ATP Mg makes terminal phosphorus easier target for nucleophilic attack Soluble, cytosolic Protein Hepatocytes – an extra hexokinase called hexokinase IV or glucokinase – different from other hexokinase in kinetic and regulatory properties 8

9. STEP 3: Phosphorylation of Fructose 6- Phosphate to Fructose 1,6-Bisphosphate Irreversible under celluar conditions Commited Step, since G6P and F6P has other fates, but F16BP is targeted for glycolysis. Major regulatory point in glycolysis. PFK-1 activity increased, when ATP is low or ADP and AMP are in excess PFK-1 inhibited by ATP, activated by F26BP (product of PFK-2) – Next week in regulation 9

10. STEP 10: Transfer of the Phosphoryl Group from Phosphoenolpyruvate to ADP PEP + ADP –> Pyruvate + ATP Pyruvate kinase Mg, K, Mn Substrate Level Phosphorylation At pH 7, keto form dominates (non-enzymatic process) Due to keto form, large negative standard free energy change (-31,4) – large driving force pushing reaction forward to ATP synthesis PEP hydrolysis (-61.9), ATP formation (-30,5) Irreversible and important site of regulation 10

11. Glycogen phosphorylase (alpha 1-4) at non-reducing end until alpha 1-6 branch point. (debranching enzyme) Starch by alpha-amylase (mouth), by pancreatic alpha amylase – maltose (1,4), dextrin (1,6) 11

12. Lactic Acid Fermentation Hypoxic conditions; NADH generated by glycolysis cannot be reoxidixed by oxygen Glycolysis would stop due to lack NAD+  no electron acceptor for the oxidation of G3P. Regeneration required in an other way. NAD+ regenerated from NADH by reduction of pyruvate to lactate via lactate dehydrogenase. (Exergonic) No net change in NAD+ or NADH (erythrocytes- no mitochondria- produce lactate even under aerobic conditions) Lactate from muscle or erythrocytes to blood, targeted to liver, converted back to glucose – back to muscle (Cori cycle) Fermentation 12

13. Ethanol Fermentation 1.Pyruvate carboxylase (Mg, Thiamine pyrophosphate (coenzyme)) Irreversible Present in brewers`and baker`s yeast Carbon dioxide Absent in vertebrate tissues and in organisms that carry lactic acid fermentation. 2. Alcohol dehydrogenase (Zn) -is present in many organisms that metabolize ethanol, including humans. -human liver, oxidation of ethanol with reduction in NAD+/NADH ratio TPP : Derived from vitamin B1 Deficiency  beriberi 13 Cleavage of bonds adjacent to carbonyl group.

14. Brain – 120 g of glucose, more than half of the glucose stored as glycogen in muscle and liver. Source: Lactate, pyruvate, glycerol, certain AA (3C) In mammals - takes place in the liver and renal cortex 7/10 identical rxns to glycolysis Hexokinase, PFK-1 and Pyruvate Kinase  All have large negative free energy change  Irreversible Whereas others have free energy change near to zero. In gluconeogenesis 3 separate set of enyzmes catalyze exergonic reactions.  Both irreversible processes. Gluconeogenesis 14

15. 1.Conversion of Pyruvate to PEP Pyruvate Carboxylase:  Pyruvate to oxaloacetate by pyruvate carboxylase (mitoch enzyme, coenzyme biotin -carries HCO3-)  First regulatory enzyme, requires Acetyl-CoA as positive regulator (accumulation is a sign of FA availability as fuel) No mitoch transporter for oxaloacetate, malate dehydrogenase reduces it to malate for export to cytosol. (malate leaves mitoch via specific transporter, back to oxaloacetate in cytosol) PEP Carboxykinase: Oxaloacetate to PEP by PEP carboxykinase (Mg dependent, GTP as the phophoryl donor)  Pyruvate  cytosol to mitochondria for conversion  Or can be generated from alanine within mitochondria by transamination  Overall actual free energy change -25 kj/mol due to high PEP consumption in other rxns  The reaction must be effectively irreversible. Bypass of Irreversible Steps 15

16. Produced and consumed NADH in balance Lactate as glucogenic precursor  Lactate to pyruvate in cytosol by lactate dehydrogenase  Pyruvate to oxaloacetate by pyruvate carboxylase  Mitochondrial isozyme of PEP carboxykinase  PEP transported out. 16

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18. G6P into pentose phosphates Oxidative pathway NADP elecctron acceptor  NADPH Rapidly dividing cells such as bone marrow, skin, intestinal mucosa use pentoses to make RNA, DNA, ATP, NADH, FADH2, CoA Other tissues – product is just NADPH Needed for reductive biosynthesis To prevent oxidative damage by maintaining high NADPH/NADP+ Glucose 6-Phosphate dehydrogenrase oxidizes glucose 6-phosphate 6-phosphogluconate oxidized and decarboxylated NADP+ electron acceptor Pentose Phosphate Pathway of Glucose Oxidation 18

19. Nonoxidative Pathway In tissues require primarly NADPH, the pentose phophates produced in oxidative phase are recyled into G6P. 19

20. Glucose 6-phosphate Glycolysis PPP High [NADP+]  PPP Low [NADP+]  Glycolysis 20