THE ROLE OF O-ACETYLSERINE SULFHYDRYLASE IN THE CONVERSION OF SODIUM AZIDE INTO A MUTAGENIC METABOLITE

The role of the enzyme O-acetylserine sulfhydrylase in the conversion of azide to a mutagenic metabolite was investigated. Organisms which lack azide mutagenicity were assayed for O-acetylserine sulfhydrylase activity. Arabidopsis, Drosophila, and Neurospora not only contained significant levels of enzyme activity, but also produced a product in vitro which was mutagenic in Salmonella typhimurium TA1530.;The synthesis of azide metabolite was further investigated. The kinetic properties of barley and bacterial O-acetylserine sulfhydrylase and the physical properties of in vitro barley and in vivo bacterial metabolite were compared to determine if additional metabolism of the initial enzymatic product occurred. The barley and bacterial enzyme kinetic properties were similar. Furthermore, azide metabolite biosynthesis in barley and bacteria is similar and azide and sulfide use the same catalytic site on the enzyme. It appears that the product of the initial enzymatic reaction is (beta)-azidoalanine.;The physical properties of the in vitro barley metabolite and in vivo bacterial metabolite also indicated both metabolites may be (beta)-azidoalanine. Both had retention times on a Beckman 121MB amino acid analyzer strongly suggestive of a carboxyl group. Infra red spectroscopy for the in vitro barley metabolite showed an absorption in the carboxyl region and an absorption indicative of an azide moiety. Mass spectra for the in vivo bacterial and in vitro barley metabolite were similar and consistent with fragments which could be obtained for (beta)-azidoalanine. That both metabolites are similar strongly suggests that no further metabolism of the initial enzymatic product occurs.;Barley O-acetylserine sulfhydrylase was purified to homogeneity by gel filtration, anion exchange chromatography, isoelectric focusing and discontinuous nondenaturing polyacrylamide gel electrophoresis. The purified enzyme produced a product from azide and O-acetylserine which increased the frequency of histidine reversions in Salmonella typhimurium TA1530. This conclusively demonstrates the role of this enzyme in the initial synthesis of azide metabolite in barley.