Mutational analysis of the catalytic residues lysine 230 and tyrosine 160 in the NADP(+)-dependent isocitrate dehydrogenase from Escherichia coli.

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.