Reginald Garrett and Charles Grisham Chapter 19 Glycolysis to accompany Biochemistry, 2/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

Overview of Glycolysis The 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

First Phase of Glycolysis 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 Be SURE you can interconvert Keq and standard state free energy change Be SURE you can use Eq. 3.12 to generate far right column of Table 19.1

Hexokinase 1st step in glycolysis; G large, negative Hexokinase (and glucokinase) act to phosphorylate glucose and keep it in the cell Km for glucose is 0.1 mM; cell has 4 mm glucose So hexokinase is normally active! Glucokinase (Kmglucose = 10 mM) only turns on when cell is rich in glucose Hexokinase is regulated - allosterically inhibited by (product) glucose-6-P - but is not the most important site of regulation of glycolysis - Why?

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 Be able to write a mechanism!

Rx 3: Phosphofructokinase PFK is the committed step in glycolysis! The second priming reaction of glycolysis Committed step and large, neg delta G - means PFK is highly regulated ATP inhibits, AMP reverses inhibition Citrate is also an allosteric inhibitor Fructose-2,6-bisphosphate is allosteric activator PFK increases activity when energy status is low PFK decreases activity when energy status is high

C6 cleaves to 2 C3s (DHAP, Gly-3-P) 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 Understand the evidence for Schiff base intermediate (box on page 622)

Rx 5: Triose Phosphate Isomerase DHAP converted to Gly-3-P An ene-diol mechanism (know it!) Active site Glu acts as general base Triose phosphate isomerase is a near-perfect enzyme - see Table 14.5

Glycolysis - Second Phase 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 is one we saw in Chapter 16 (see Figure 16.10!) Mechanism involves covalent catalysis and a nicotinamide coenzyme - know it

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

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

PEP to Pyruvate makes ATP 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 G - regulation! Allosterically activated by AMP, F-1,6-bisP Allosterically inhibited by ATP and acetyl-CoA Understand the keto-enol equilibrium of Py

The Fate of NADH and Pyruvate Aerobic or anaerobic?? NADH is energy - two possible fates: If O2 is available, NADH is re-oxidized in the electron transport pathway, making ATP in oxidative phosphorylation In anaerobic conditions, NADH is re-oxidized by lactate dehydrogenase (LDH), providing additional NAD+ for more glycolysis

The Fate of NADH and Py Aerobic or anaerobic?? Pyruvate is also energy - two possible fates: aerobic: citric acid cycle anaerobic: LDH makes lactate

Energetics of Glycolysis The elegant evidence of regulation! See Figure 16.31 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 Glycolysis Fructose, mannose and galactose Fructose and mannose are routed into glycolysis by fairly conventional means. See Figure 18.32 Galactose is more interesting - the Leloir pathway "converts" galactose to glucose - sort of.... See Figure 16.33