title: The pH dependence of the kinetic parameters of ketol acid reductoisomerase indicates a proton shuttle mechanism for alkyl migration.
abstract: The enzyme ketol acid reductoisomerase catalyzes the second common reaction in the biosynthesis of the branched chain amino acids. The reaction is complex as an alkyl migration and a ketone reduction apparently occur as separate steps during the conversion of acetolactate to 2,3-dihydroxy-3-methylbutyrate. This paper reports on the pH dependence of the kinetic parameters of the enzyme. The pH variation of log(V/K) for acetolactate was fit to an equation describing a bell-shaped curve, indicating an acid and a base catalyst for the reaction. In the reverse direction, V/K for 2,3-dihydroxy-3-methylbutyrate is constant over the pH range 8 to 10 and decreases below pH 8 with the ionization of two catalytic groups. The pH dependence of the V/K values for reduction of the kinetically competent intermediate and analogs of this intermediate are also described by a bell-shaped curve. The pH dependence of the V/K for alkyl migration of this intermediate indicates a single base catalyst for this reaction. We observe no deuterium kinetic isotope effect on V or V/K for the reaction of acetolactate at any pH. We observe a pH-dependent kinetic isotope effect on V/K for the reduction of the intermediate, the magnitude of which is metal ion dependent. Larger KIE's are observed in the presence of Mn2+ as opposed to Mg2+. In the reverse reaction there is a pH-dependent kinetic isotope effect on V/K. Based on the pH dependence of the kinetic parameters and the kinetic isotope effects, we propose a base-catalyzed proton shuttle mechanism for the alkyl migration reaction followed by an acid-assisted ketone reduction by NADPH.
authors.affiliation: Department of Chemistry, University of Akron, Ohio 44325-3601.
authors.forename: G T
authors.initials: GT
authors.lastname: Mrachko
authors.forename: S K
authors.initials: SK
authors.lastname: Chunduru
authors.forename: K C
authors.initials: KC
authors.lastname: Calvo
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D002087:Butyrates / Q000138:chemical synthesis / Q000378:metabolism; D002413:Cations, Divalent; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling; D050540:Ketol-Acid Reductoisomerase; D007700:Kinetics; D015394:Molecular Structure; D013379:Substrate Specificity; D014316:Tritium
chemical_list: D002087:Butyrates; D002413:Cations, Divalent; D014316:Tritium; D000429:Alcohol Oxidoreductases; D050540:Ketol-Acid Reductoisomerase