Handout Enzyme Kinetics and Control Reactions ANSC 689 PHYSIOLOGICAL CHEMISTRY OF LIVESTOCK SPECIDS Enzyme Kinetics and Control Reactions I. Kinetics A. Reaction rates 1. First order (reaction rate is proportional to the amount of substrate). Mixed order (reaction rate is not quite proportional to the amount of substrate) 3. Zero order (reaction rate no longer responds to the amount of substrate) B. The Michaelis-Menton (hyperbolic) curve 1. Michaelis-Menton equation: v = V max x [S] K,m + [S]. Significance of K m 3. V max a. Concentration of substrate at which the velocity is half-maximal b. Typically near the concentration of substrate in the cell a. Maximal velocity of the enzyme b. Dependent on number of apparent substrate binding sites 1 8 6 6 8 1
II. Sigmoidal kinetics A. Sigmoidal kinetics -- K s 1. Indicates cooperativity a. Indicates at least two subunits of an enzyme b. The first substrate molecule binds to a subunit with difficulty (low affinity). c. Subsequent substrate molecules bind to the remaining subunits more easily (high affinity). d. This allows substrate concentrations to be higher in the concentrations in the cell (for competing reactions).. Hyperbolic curves can be changed to sigmoidal curves by allosteric effectors, ph, and salts. B. Allosteric effectors 1. Inhibitors increase the K m (decrease substrate affinity) and make the curve more sigmoidal.. Activators decrease the K m (increase substrate affinity) and make the curve more hyperbolic. 3. Increasing sigmoidicity allows decision making between competing pathways. 1 8 6 5 1 15
III. Regulation of cellular processes A. Change in amount of enzyme 1. Adaptive vs constuitive a. Adaptive: responds to external stimulus, e.g., change in nutrient intake b. Constuitive: rate of synthesis of enzyme does not respond to external stimulus, e.g., change in nutrient intake. Time required is slow relative to effects of allosteric effectors or enzyme phosphorylation. 3. Doubling the amount of enzyme doubles the number of substrate binding sites. 1 8 6 6 8 B. Phosphorylation of enzymes 1. Glycogen metabolism (glycogen synthase and glycogen phosphorylase). Lipid metabolism (acetyl-coa carboxylase, hormone sensitive lipase) 3. Time required to change enzyme activity is fast relative to changes in total enzyme amount.. Phosphorylation events that decrease activity decrease the number of apparent substrate binding sites. 3
IV. Identification of control reactions in a pathway A. Where is the regulatory reaction? 1. At unidirectional reactions (a, c or e).. Early in the sequence or pathway (a). 3. At branch points (c and d).. At steps where the reverse reaction is catalyzed by a different enzyme (f). B. Kinetic considerations 1. Maximal reaction rate (i.e., number of binding sites), V max. Affinity of the enzyme for its substrate, K m (or K s.5 ) 3. Equilibrium constants and mass action ratios. Allosteric activators and inhibitors C. Possible control reactions and respective allosteric effectors 1. Hexokinase G-6-P strongly inhibits. Glycogen synthase G-6-P activates 3. Glycogen phosphorylase AMP and Ca ++ activate; ATP inhibits. 6-Phosphofructokinase ATP + citrate inhibit; F-6-P, AMP, P i, 6-PG, F-,6-P activate 5. Glyceraldehyde-3-P-DH NAD/NADH ratio 6. Pyruvate kinase F-1,6-P, 6-PG, F-,6- P (+), ATP ( ) D. Equilibriium constants (K eq ) and Mass Action Ratio (MAR) 1. K eq is measured under set conditions of concentration, temperature, and pressure.. MAR is calculated from actual intracellular concentrations of reactants and products. e.g., F-6-P + ATP à F-1,6-P + ADP
K eq = [F-1,6-P ] x [ADP] [F-6-P] x [ATP] under set conditions and MAR = F-1,6-P ] x [ADP] actual cellular conditions [F-6-P] x [ATP] Enzyme Activities K eq MAR Hexokinase 1.5,.8 Phosphoglucoisomerase 176.. 6-Phosphofructokinase 56 1,.3 Aldolase 78.1.1 Triosephosphate isomerase,65.. Glyceraldehyde-3-phosphate dehydrogenase /169 1, 9 plus phosphoglycerate kinase Phosphoglycerate mutase 1.1.1 Enolase 158 3.5 1. Pyruvate kinase 387 -, 5