Glycolysis PDF

Chapter 19 Glycolysis based on...

Chapter 19 Glycolysis based on Biochemistry, 4/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 Outline  19.1 Overview of Glycolysis  19.2 Coupled Reactions in Glycolysis  19.3 First Phase of Glycolysis  19.4 Second Phase of Glycolysis  19.5 Metabolic Fates of NADH and Pyruvate  19.6 Anaerobic Pathways for Pyruvate  19.7 Energetic Elegance of Glycolysis  19.8 Other Substrates in Glycolysis GLYCOLYSIS  Stepwise degradation of glucose  Occurs in the cytosol  Basically anaerobic process  Principal steps occur with no requirement for oxygen  First two billion years of biological evolution on earth Overview of Glycolysis (Embden-Meyerhof (Warburg) Pathway)  Essentially all cells carry out glycolysis  Ten reactions - same in all cells - but rates differ  Two phases:  First phase converts glucose to two G-3-P  Second phase produces two pyruvates  Products are pyruvate, ATP and NADH  Three possible fates for pyruvate The Fate of NADH and Pyruvate Aerobic or anaerobic??  Pyruvate has two possible fates:  aerobic: citric acid cycle  anaerobic: LDH makes lactate The Fate of NADH and Pyruvate Aerobic or anaerobic??  NADH is energy - two possible fates:  IfO2 is available, NADH is re-oxidized in the electron transport pathway, making ATP in oxidative phosphorylation  Inanaerobic conditions, NADH is re-oxidized by lactate dehydrogenase (LDH), providing additional NAD+ for more glycolysis Phosphorylation  To synthesize ATP using the metabolic free energy contained in the glucose molecule would be to convert glucose into one (or more) of the high-energy phosphates that have standard-state free energies of hydrolysis more negative than that of ATP.  synthesized easily from glucose § Phosphoenolpyruvate § 1,3-bisphosphoglycerate § acetyl phosphate.  The first reaction - phosphorylation of glucose  Hexokinase or glucokinase  This is a priming reaction - ATP is consumed here in order to get more later  ATP makes the phosphorylation of glucose spontaneous Hexokinase 1st step in glycolysis ΔG large, negative (-33kJ/mol) under cellular conditions  Hexokinase (and glucokinase) act to phosphorylate glucose and keep it in the cell  Km for glucose is 0.1 mM; efficient at cell concentration of 4 mM glucose  So hexokinase is normally active!  Hexokinase is regulated - allosterically inhibited by (product) glucose-6-P - but is not the most important site of regulation of glycolysis - Why? Glucokinase (in the liver) 1st step in glycolysis ΔG large, negative (-33kJ/mol) under cellular conditions  Glucokinase (Kmglucose = 10 mM) only turns on when the cell is rich in glucose  Highly specific for glucose  Hexokinase coverts to glucokinase at high glucose concentration.  Inducible enzyme – the amount present is controlled by insulin Rx 2: Phosphoglucoisomerase Glucose-6-P to Fructose-6-P  Why does this reaction occur??  next step (phosphorylation at C-1) would be tough for hemiacetal -OH, but easy for primary -OH  isomerization activates C-3 for cleavage in aldolase reaction  Ene-diol intermediate in this reaction Rx 3: Phosphofructokinase (PFK) PFK is the committed step in glycolysis!  The second priming reaction of glycolysis  Committed step and large, neg ΔG (-18.8 kJ/mol in erythrocytes) means PFK is highly regulated  Most important site of regulation Rx 3: Phosphofructokinase (PFK)  PFK increases activity when energy status is low  PFK decreases activity when energy status is high Phosphofructokinase with ADP shown in white and fructose-6-P in red.  ATP inhibits, AMP reverses inhibition  Citrate is also an allosteric inhibitor  Fructose-2,6-bisphosphate is allosteric activator Rx 4: Aldolase C6 cleaves to 2 C3s (DHAP, Gly-3-P)  Animal aldolases are Class I aldolases  Class I aldolases form covalent Schiff base intermediate between substrate and active site lysine Rx 5: Triose Phosphate Isomerase DHAP converted to Gly-3-P  An ene-diol mechanism  Active site Glu acts as general base  Triose phosphate isomerase is a near-perfect enzyme  Catalytic perfection – turnover number near the diffusion limit Metabolic energy produces 4 ATP  Net ATP yield for glycolysis is two ATP  Second phase involves two very high energy phosphate intermediates 1,3 - BPG Phosphoenolpyruvate Rx 6: Gly-3-Dehydrogenase Gly-3P is oxidized to 1,3-BPG  Energy yield from converting an aldehyde to a carboxylic acid is used to make 1,3-BPG and NADH  Mechanism involves covalent catalysis and a nicotinamide coenzyme Rx 7: Phosphoglycerate Kinase ATP synthesis from a high-energy phosphate  This is referred to as "substrate-level phosphorylation"  2,3-BPG (for hemoglobin) is made by circumventing the PGK reaction  FIGURE 19.21 Formation and decomposition of 2,3-bisphosphoglycerate. Rx 8: Phosphoglycerate Mutase Phosphoryl group from C-3 to C-2  Rationale for this enzyme - repositions the phosphate to make PEP  Note the phospho-histidine intermediates!  Zelda Rose showed that a bit of 2,3-BPG is required to phosphorylate His Rx 9: Enolase 2-P-Gly to PEP  Overall G is 1.8 kJ/mol  How can such a reaction create a PEP?  "Energy content" of 2-PG and PEP are similar  Enolase just rearranges to a form from which more energy can be released in hydrolysis Rx 10: Pyruvate Kinase PEP to Pyruvate makes ATP  These two ATP (from one glucose) can be viewed as the "payoff" of glycolysis  Large, negative ΔG - regulation!  Allosterically activated by AMP, F-1,6-bisP  Allosterically inhibited by ATP and acetyl-CoA  Understand the keto-enol equilibrium of Pyruvate FIGURE 19.28 The conversion of phosphoenolpyruvate (PEP) to pyruvate may be viewed as involving two steps: phosphoryl transfer followed by an enol-keto tautomerization. The tautomerization is spontaneous (ΔG° = 35–40 kJ/mol) and accounts for much of the free energy change for PEP hydrolysis. Energetics of Glycolysis The elegant evidence of regulation!  Standard state G values are scattered: + and - · G in cells is revealing:  Most values near zero  3 of 10 Rxns have large, negative  G  Large negative G rxns are sites of regulation! Other Substrates for Fructose, mannose and galactose Fructose and mannose are routed into Glycolysis glycolysis by fairly conventional means. Galactose is more interesting - the Leloir pathway "converts" galactose to glucose - sort of.... Compartmentalization of glycolysis, the citric acid cycle, and oxidative phosphorylation.