Complete kinetic mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae.
The kinetic mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae was determined using initial velocity studies in the absence and presence of product and dead end inhibitors in both reaction directions. Data suggest a steady state random kinetic mechanism. The dissociation constant of the Mg-homoisocitrate complex (MgHIc) was estimated to be 11 +/- 2 mM as measured using Mg2+ as a shift reagent. Initial velocity data indicate the MgHIc complex is the reactant in the direction of oxidative decarboxylation, while in the reverse reaction direction, the enzyme likely binds uncomplexed Mg2+ and alpha-ketoadipate. Curvature is observed in the double-reciprocal plots for product inhibition by NADH and the dead-end inhibition by 3-acetylpyridine adenine dinucleotide phosphate when MgHIc is the varied substrate. At low concentrations of MgHIc, the inhibition by both nucleotides is competitive, but as the MgHIc concentration increases, the inhibition changes to uncompetitive, consistent with a steady state random mechanism with preferred binding of MgHIc before NAD. Release of product is preferred and ordered with respect to CO2, alpha-ketoadipate, and NADH. Isocitrate is a slow substrate with a rate (V/E(t)) 216-fold slower than that measured with HIc. In contrast to HIc, the uncomplexed form of isocitrate and Mg2+ bind to the enzyme. The kinetic mechanism in the direction of oxidative decarboxylation of isocitrate, on the basis of initial velocity studies in the absence and presence of dead-end inhibitors, suggests random addition of NAD and isocitrate with Mg2+ binding before isocitrate in rapid equilibrium, and the mechanism approximates rapid equilibrium random. The Keq for the overall reaction measured directly using the change in NADH as a probe is 0.45 M.
Lin
Ying
Y
Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, Oklahoma 73019, USA.
Alguindigue
Susan S
SS
Volkman
Jerome
J
Nicholas
Kenneth M
KM
West
Ann H
AH
Cook
Paul F
PF
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007523:Isocitrates / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014233:Tricarboxylic Acids / Q000378:metabolism
D007523:Isocitrates; D014233:Tricarboxylic Acids; C517550:homoisocitric acid; C034219:isocitric acid; D000429:Alcohol Oxidoreductases; C021368:homoisocitrate dehydrogenase; D008274:Magnesium