Substrate specificity of 5′-methylthioadenosine phosphorylase from human prostate

Abstract

5′-Methylthioadenosine phosphorylase was purified approx. 340-fold from human prostate by using affinity chromatography by Hg-coupled Sepharose. The enzyme, responsible for the breakdown of 5′-methylthioadenosine into adenine and methylthioribose 1-phosphate, was partially characterized. The apparent Km for 5′-methylthioadenosine is 25 microM. It is activated by thiols and shows an absolute requirement for phosphate ions. New analogues of 5′-methylthioadenosine were prepared and their activity as substrates or inhibitors of the reaction was investigated. The replacement of the 6-amino group of the adenine moiety by a hydroxy group, as well as the replacement of N-7 by a methinic radical, resulted in an almost complete loss of activity. Otherwise the replacement of sulphur by selenium, as well as that of the methyl group by an ethyl one, is compatible with the activity as substrate. The positively charged sulphonium group also prevents catalytic interaction with the enzyme. The inhibitory effect of 5′-methylthiotubercidin (competitive) and 5′-dimethylthioadenosine sulphonium salt (non-competitive) was also demonstrated. The reported results suggest three binding sites between the substrate and the enzyme.