Modification of a catalytically important residue of indoleglycerol-phosphate synthase from Escherichia coli

Abstract

The active-site residues of indoleglycerol-phosphate synthase from Escherichia coli were tentatively localized by comparing crystallographic data with the amino acid identities among the known indoleglycerol-phosphate synthase sequences. To test the validity of the resulting model of catalysis one of the residues in the presumptive active site, Lys 55, was changed to serine using oligonucleotide-directed mutagenesis. The specificity constant k cat/ K m of the mutant is 3 × 10 4-times lower than that of the wild-type enzyme, due to a 60-fold decrease in k cat and a 450-fold increase in K m. This finding shows that Lys 55 is important for both catalysis and substrate binding.