Chapt. 22 GlycolysisGlycolysis overviewAnaerobic glycolysisGlycolysis phasesGlycolysis step 1Glycolysis phase IGlycolysis phase II**Alternatie fates of pyruvateAerobic: Glycerol 3-P shuttle carries NADHAnaerobic:Fate of lactateII. Other functions of glycolysisIII. Glycolysis is regulatedLevels of ATP, ADP, AMPRegulation of PFK-1Regulation of glycolysis enzymesLactic AcidemiaKey conceptsReview questionGlyceraldehyde 3-P dehydrogenaseChapt. 22 GlycolysisCh. 22 GlycolysisStudent 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 causesGlycolysis overviewFig. 1*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 ATPAnaerobic glycolysisFig. 2In absence of oxygen, anaerobic glycolysis:•Recycles NADH to permit glycolysis continue•Reduces pyruvate to lactate•Only 2 ATP per glucose•May cause lactic acidemiaGlycolysis phasesFig. 3Glycolysis phases:•Preparation: •Glucose phosphorylated•Cleaved to 2 triose phosphates•Costs 2 ATP•ATP-generating phase:•Triose phosphates oxidized more•Produces 2 NADH•Produces 4 ATPGlycolysis step 1Fig. 41. 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 hexokinasesGlycolysis phase IFig. 5 top2 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 stepGlycolysis phase IIFig. 5 lowerOxidation, substrate level phosphorylation yield2 NADH, 4 ATP from 1 Glyceraldehyde 3-PKey enzymes:Glyceraldehyde 3-P dehydrogenase•High-energy bond Pyruvate kinase:•Regulated step**Alternatie fates of pyruvateFate 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*Aerobic: Glycerol 3-P shuttle carries NADHAerobic: 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 NADHFig. 7Bacteria not need shuttle since only 1 compartmentFig. 5 topAnaerobic: Anaerobic glycolysis: NADH reduces pyruvate to lactate, regenerates NAD+ to continue glycolysis1 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 glucoseFig. 9Fate of lactateFig. 10Fate 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)II. Other functions of glycolysisGlycolysis generates precursors for other paths:•5-C sugars for NTPs•Amino acids•Fatty acids, glycerol•Liver is major site of biosynthesisFig. 11III. Glycolysis is regulatedGlycolysis 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. 12Levels of ATP, ADP, AMPFig. 13Levels of AMP in cytosol good indicator of rate ATP utilization2 ADP <-> AMP + ATP reaction of adenylate kinase•Hydrolysis ATP -> ADP increases ADP, AMP•ATP present highest conc:•Small dec ATP -> large AMPRegulation of PFK-1Regulation 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. 14Regulation of glycolysis enzymesRegulation 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 PO4 to inactivate•Rate of ATP utilization•NADH/NAD+ ratioFig. 12Lactic AcidemiaFig. 15Lactic acidosis:•Excess lactic acid in blood > 5mM•pH < 7.2•From increasedNADH/NAD+Many causes ->•Excess alcohol•Hypoxia•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 CReview questionWhich of the following statements correctly describes an aspect of glycolysis?a.ATP is formed by oxidative phosphorylationb.Two molecules of ATP are used in the beginning of the pathwayc.Pyruvate kinase is the rate-limiting enzymed.One molecule of pyruvate and 3 olecules of CO2 are formed from the oxidation of 1 glucosee.The reactions take place in the matrix of the mitochondriaGlyceraldehyde 3-P dehydrogenaseFig. 17Glyceraldehyde 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