1idd

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spinqualitylabelsall models 1idd, resolution 2.5Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

ISOCITRATE DEHYDROGENASE Y160F MUTANT APO ENZYME

Overview

Two site-directed mutants of isocitrate dehydrogenase (IDH) of Escherichia, coli have been studied by site-directed mutagenesis kinetic and structural, studies. Substitution of phenylalanine for tyrosine at position 160, (Y160F) showed 0.4% of the kcat of wild-type with isocitrate as substrate, while the Km for isocitrate remained unchanged. When the postulated, intermediate, oxalosuccinate, was enzymatically decarboxylated, Y160F, showed a higher kcat and a similar Km to the wild type values. The rate of, reduction of oxalosuccinate to isocitrate by the Y160F mutant was greatly, decreased relative to the wild-type. Substitution of methionine for lysine, at position 230 decreased kcat to 1.1% of that of the wild-type and Km, increased by a factor of 500-600. The decarboxylation of oxalosuccinate, was undetectable for the K230M mutant. The structure of the site-directed, mutants of IDH with and without a bound complex of isocitrate and Mg2+ was, solved at 2.5 A resolution and compared by difference mapping against, previously determined enzyme structures. The structural studies show that, (i) the overall protein-folding side chain conformations and active sites, of both mutants are isomorphous with wild-type enzyme, (ii) isocitrate and, magnesium bind to both enzyme mutants with the same relative conformation, and binding interactions as wild-type enzyme, and (iii) the mutated side, chains (Phe 160 and Met 230) are positioned for catalysis in a similar, conformation as that observed for the wild-type enzyme. Hence, the, alteration of the side chain functional groups is directly related to the, loss of enzyme activity. Possible roles of the active site tyrosine and, lysine are discussed.

About this Structure

1IDD is a Single protein structure of sequence from Escherichia coli. Active as Isocitrate dehydrogenase (NADP(+)), with EC number 1.1.1.42 Full crystallographic information is available from OCA.

Reference

Mutational analysis of the catalytic residues lysine 230 and tyrosine 160 in the NADP(+)-dependent isocitrate dehydrogenase from Escherichia coli., Lee ME, Dyer DH, Klein OD, Bolduc JM, Stoddard BL, Koshland DE Jr, Biochemistry. 1995 Jan 10;34(1):378-84. PMID:7819221

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