Cyclopropane-1,1-dicarboxylate is a slow-, tight-binding inhibitor of rice ketol-acid reductoisomerase

Ketol-acid reductoisomerase (EC 1.1.1.86) catalyses the second reaction in the biosynthesis of the branched-chain amino acids. The reaction catalyzed consists of two stages, the first of which is an alkyl migration from one carbon atom to its neighbour. The likely transition state is therefore a cyc...

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Bibliographic Details
Published in:Plant science (Limerick) 2005-04, Vol.168 (4), p.1035-1040
Main Authors: Lee, Yu-Ting, Ta, Hang Thu, Duggleby, Ronald G.
Format: Article
Language:English
Subjects:
alcohol oxidoreductases
Analytical, structural and metabolic biochemistry
Binding and carrier proteins
Biological and medical sciences
Branched-chain amino acids
chemical reactions
complementary DNA
Cyclopropane-1,1-dicarboxylate
dicarboxylic acids
Enzyme inhibition
enzyme inhibitors
enzyme kinetics
Escherichia coli
Fundamental and applied biological sciences. Psychology
gene expression
Herbicides
keto-acid reductoisomerase
Ketol-acid reductoisomerase
molecular sequence data
nucleotide sequences
Oryza sativa
Proteins
recombinant fusion proteins
Rice
sequence analysis
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Summary:Ketol-acid reductoisomerase (EC 1.1.1.86) catalyses the second reaction in the biosynthesis of the branched-chain amino acids. The reaction catalyzed consists of two stages, the first of which is an alkyl migration from one carbon atom to its neighbour. The likely transition state is therefore a cyclopropane derivative, and cyclopropane-1,1-dicarboxylate (CPD) has been reported to inhibit the Escherichia coli enzyme. In addition, this compound causes the accumulation of the substrate of ketol-acid reductoisomerase in plants. Here, we investigate the inhibition of the purified rice enzyme. The cDNA was cloned, and the recombinant protein was expressed in E. coli, purified and characterized kinetically. The purified enzyme is strongly inhibited by cyclopropane-1,1-dicarboxylate, with an inhibition constant of 90 nM. The inhibition is time-dependent and this is due to the low rate constants for formation (2.63 × 10 5 M −1 min −1) and dissociation (2.37 × 10 −2 min −1) of the enzyme-inhibitor complex. Other cyclopropane derivatives are much weaker inhibitors while dimethylmalonate is moderately effective.
ISSN:0168-9452
1873-2259
DOI:10.1016/j.plantsci.2004.11.020