Probing the mechanism of the bifunctional enzyme ketol-acid reductoisomerase by site-directed mutagenesis of the active site.
Ketol-acid reductoisomerase (EC 1.1.1.86) is involved in the biosynthesis of the branched-chain amino acids. It is a bifunctional enzyme that catalyzes two quite different reactions at a common active site; an isomerization consisting of an alkyl migration, followed by an NADPH-dependent reduction of a 2-ketoacid. The 2-ketoacid formed by the alkyl migration is not released. Using the pure recombinant Escherichia coli enzyme, we show that the isomerization reaction has a highly unfavourable equilibrium constant. The reductase activity is shown to be relatively nonspecific and is capable of utilizing a variety of 2-ketoacids. The active site of the enzyme contains eight conserved polar amino acids and we have mutated each of these in order to dissect their contributions to the isomerase and reductase activities. Several mutations result in loss of the isomerase activity with retention of reductase activity. However, none of the 17 mutants examined have the isomerase activity only. We suggest a reason for this, involving direct reduction of a transition state formed during the isomerization, which is necessitated by the unfavourable equilibrium position of the isomerization. Our mechanism explains why the two activities must occur in a single active site without release of a 2-ketoacid and provides a rationale for the requirement for NADPH by the isomerase.
Tyagi
Rajiv
R
Department of Biochemistry and Molecular Biology, The University of Queensland, Brisbane, Qld 4072, Australia.
Lee
Yu-Ting
YT
Guddat
Luke W
LW
Duggleby
Ronald G
RG
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D050540:Ketol-Acid Reductoisomerase; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism
D009249:NADP; D000429:Alcohol Oxidoreductases; D050540:Ketol-Acid Reductoisomerase; D008274:Magnesium