IDENTIFICATION OF AN ACTIVE-SITE CYSTEINE RESIDUE IN HUMAN TYPE-I INS(1,4,5)P-3 5-PHOSPHATASE BY CHEMICAL MODIFICATION AND SITE-DIRECTED MUTAGENESIS

Chemical modification using thiol-directed agents and site-directed mu tagenesis have been used to investigate the crucial role of an active site cysteine residue within the substrate-binding domain of human typ e I Ins(1,4,5)P-3 5-phosphatase. Irreversible inhibition of enzymic ac tivity is provoked by chemical modification of the enzyme by N-ethylma leimide (NEM), 5,5'-dithio-2-nitrobenzoic acid, iodoacetate and to a m uch smaller extent by iodoacetamide. The alkylation reaction by NEM is prevented in the presence of Ins(1,4,5)P-3. The results indicate that NEM binds at the active site of the enzyme with a stoichiometry of 0. 9 mol of NEM per mol of enzyme. A single [C-14]NEM-modified peptide wa s isolated after alpha-chymotrypsin proteolysis of the radiolabelled e nzyme and reverse-phase HPLC. Sequence analysis of the active site-lab elled peptide (i.e. MNTRCPAWCD) demonstrated that Cys(348) contained t he radiolabel. Furthermore two mutant enzymes were obtained by site-di rected mutagenesis of the cysteine residue to serine and alanine respe ctively. Both mutant enzymes had identical UV CD spectra. The two muta nts (i.e. Cys(348) --> Ser and Cys(348) --> Ala) show a marked loss of enzymic activity (more than 98% compared with the wild-type enzyme). Thus we have directly identified a reactive cysteine residue as part o f the active site, i.e. the substrate-binding domain, of Ins(1,4,5)P-3 5-phosphatase. This cysteine residue is part of a sequence 10 amino a cids long that is well conserved among the primary structures of inosi tol and phosphatidylinositol polyphosphate 5-phosphatases.