Mechanism of ketol acid reductoisomerase--steady-state analysis and metal ion requirement.
Ketol acid reductoisomerase is an enzyme of the branched-chain amino acid biosynthetic pathway. It catalyzes two separate reactions: an acetoin rearrangement and a reduction. This paper reports on the purification of the enzyme from a recombinant Escherichia coli and on the steady-state kinetics of the enzyme. The kinetics of the reaction were determined for the forward and reverse reaction by using the appropriate chiral substrates. At saturating metal ion concentrations the mechanism follows an ordered pathway where NADPH binds before acetolactate. The product of the rearrangement of acetolactate, 3-hydroxy-3-methyl-2-oxobutyrate, is shown to be kinetically competent as an intermediate in the enzyme-catalyzed reaction. Starting with acetolactate, Mg2+ is the only divalent metal ion that will support enzyme catalysis. For the reduction of 3-hydroxy-3-methyl-2-oxobutyrate, Mn2+ is catalytically active. Product and dead-end inhibition studies indicate that the binding of metal ion and NADPH occurs randomly. In the forward reaction direction, the deuterium kinetic isotope effect on V/K is 1.07 when acetolactate is the substrate and 1.39 when 3-hydroxy-3-methyl-2-oxobutyrate is the substrate.
Chunduru
S K
SK
Department of Chemistry, University of Akron, Ohio 44325.
Mrachko
G T
GT
Calvo
K C
KC
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D002087:Butyrates / Q000138:chemical synthesis; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000201:enzymology; D007202:Indicators and Reagents; D050540:Ketol-Acid Reductoisomerase; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology; D013379:Substrate Specificity
D002087:Butyrates; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D008345:Manganese; D000429:Alcohol Oxidoreductases; D050540:Ketol-Acid Reductoisomerase; D008274:Magnesium