Describe the major steps of glycolysis Chapt. 22 Glycolysis Ch. 22 Glycolysis Student 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 causes

Overview of Glycolysis, TCA cycle, electron transport chain: Glycolysis overview 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 ATP Fig. 1*

In absence of oxygen, anaerobic glycolysis: Recycles NADH to permit glycolysis continue Reduces pyruvate to lactate Only 2 ATP per glucose May cause lactic acidemia Fig. 2

Glucose phosphorylated Cleaved to 2 triose phosphates Costs 2 ATP Glycolysis phases Glycolysis phases: Preparation: Glucose phosphorylated Cleaved to 2 triose phosphates Costs 2 ATP ATP-generating phase: Triose phosphates oxidized more Produces 2 NADH Produces 4 ATP Fig. 3

1. Glucose is phosphorylated by Hexokinase with ATP: Commitment step Glycolysis step 1 1. 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 hexokinases Fig. 4

2 ATP convert Glucose to Fructose 1,6 bis-P; Glycolysis phase I 2 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 step Fig. 5 top

Oxidation, substrate level phosphorylation yield Glycolysis phase II Oxidation, substrate level phosphorylation yield 2 NADH, 4 ATP from 1 Glyceraldehyde 3-P Key enzymes: Glyceraldehyde 3-P dehydrogenase High-energy bond Pyruvate kinase: Regulated step Fig. 5 lower

**Alternatie fates of pyruvate Fate 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 NADH Aerobic: 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 NADH Bacteria not need shuttle since only 1 compartment Fig. 5 top Fig. 7

Anaerobic: Anaerobic glycolysis: NADH reduces pyruvate to lactate, regenerates NAD+ to continue glycolysis 1 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 glucose Fig. 9

Used to make glucose (liver) – Cori cycle Fate of lactate Fate 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) Fig. 10

II. Other functions of glycolysis Glycolysis generates precursors for other paths: 5-C sugars for NTPs Amino acids Fatty acids, glycerol Liver is major site of biosynthesis Fig. 11

III. Glycolysis is regulated Glycolysis 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. 12

Levels of AMP in cytosol good indicator of rate ATP utilization Levels of ATP, ADP, AMP Levels of AMP in cytosol good indicator of rate ATP utilization 2 ADP <-> AMP + ATP reaction of adenylate kinase Hydrolysis ATP -> ADP increases ADP, AMP ATP present highest conc: Small dec ATP -> large AMP Fig. 13

Rate-limiting step, tetramer 6 binding sites: Regulation of PFK-1 Regulation 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. 14

Regulation of glycolysis enzymes Regulation 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+ ratio Fig. 12

Lactic acidosis: Lactic Acidemia Fig. 15 Excess lactic acid in blood > 5mM pH < 7.2 From increased NADH/NAD+ Many causes -> Excess alcohol Hypoxia Fig. 15

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 C

ATP is formed by oxidative phosphorylation Review question Which of the following statements correctly describes an aspect of glycolysis? ATP is formed by oxidative phosphorylation Two molecules of ATP are used in the beginning of the pathway Pyruvate kinase is the rate-limiting enzyme One molecule of pyruvate and 3 olecules of CO2 are formed from the oxidation of 1 glucose The reactions take place in the matrix of the mitochondria

Glyceraldehyde 3-P dehydrogenase Glyceraldehyde 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 reforemd Fig. 17