tailieunhanh - Báo cáo khoa học: Probing the mechanism of the bifunctional enzyme ketol-acid reductoisomerase by site-directed mutagenesis of the active site

Ketol-acid reductoisomerase (EC ) is involved in the biosynthesis of the branched-chain amino acids. It is a bifunctional enzyme that cata-lyzes two quite different reactions at a common active site; an isomeriza-tion 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 colienzyme, we show that the isomerization reaction has a highly unfavourable equili-brium constant | ềFEBS Journal Probing the mechanism of the bifunctional enzyme ketol-acid reductoisomerase by site-directed mutagenesis of the active site Rajiv Tyagi Yu-Ting Lee Luke W. Guddat and Ronald G. Duggleby Department of Biochemistry and Molecular Biology The University of Queensland Brisbane Australia Keywords alkylmigration reaction branched-chain amino acids equilibrium constant ketoacid reductase transition state Correspondence R. G. Duggleby Department of Biochemistry and Molecular Biology The University of Queensland Brisbane Qld 4072 Australia Fax 617 3365 4699 Tel 617 3365 4615 E-mail Received 11 October 2004 revised 18 November 2004 accepted 29 November 2004 doi Ketol-acid reductoisomerase EC 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 .

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