Mutational analysis of the active site of indoleglycerol phosphate synthase fromEscherichia coli

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Abstract
Indoleglycerol phosphate synthase catalyzes the ring closure of 1‐(2‐carboxyphenylamino)‐1‐deoxyribulose 5′‐phosphate to indoleglycerol phosphate, the fifth step in the pathway of tryptophan biosynthesis from chorismate. Because chemical synthesis of indole derivatives from arylamino ketones requires drastic solvent conditions, it is interesting by what mechanism the enzyme catalyzes the same condensation reaction. Seven invariant polar residues in the active site of the enzyme fromEscherichia colihave been mutated directly or randomly, to identify the catalytically essential ones. A strain ofE. colisuitable for selecting and classifying active mutants by functional complementation was constructed by precise deletion of thetrpC gene from the genome. Judged by growth rates of transformants on selective media, mutants with either S58 or S60 replaced by alanine were indistinguishable from the wild‐type, but R186 replaced by alanine was still partially active. Saturation random mutagenesis of individual codons showed that E53 was partially replaceable by aspartate and cysteine, whereas K114, E163, and N184 could not be replaced by any other residue. Partially active mutant proteins were purified and their steady‐state kinetic and inhibitor binding constants determined. Their relative catalytic efficiencies paralleled their relative complementation efficiencies. These results are compatible with the location of the essential residues in the active site of the enzyme and support a chemically plausible catalytic mechanism. It involves two enzyme‐bound intermediates and general acid‐base catalysis by K114 and E163 with the support of E53 and N184.

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