Mutagenesis and Laue structures of enzyme intermediates: isocitrate dehydrogenase.

Site-directed mutagenesis and Laue diffraction data to 2.5 A resolution were used to solve the structures of two sequential intermediates formed during the catalytic actions of isocitrate dehydrogenase. Both intermediates are distinct from the enzyme-substrate and enzyme-product complexes. Mutation of key catalytic residues changed the rate determining steps so that protein and substrate intermediates within the overall reaction pathway could be visualized.

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.

Purification, crystallization, and preliminary X-ray studies of 10-formyltetrahydrofolate synthetase from Clostridia acidici-urici.

The monofunctional enzyme 10-formyltetrahydrofolate synthetase (THFS), which is responsible for the recruitment of single carbon units from the formate pool into a variety of folate-dependent biosynthetic pathways, has been subcloned, purified, and crystallized. The crystals belong to space group P2(1), with unit cell dimensions a = 102.4 A, b = 116.5 A, c = 115.8 A, and beta = 103.5. The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme tetramer, and a specific volume of the unit cell of 2.7 A3/ Da. The crystals diffract to at least 2.3 A resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector.