Substrate specificity of 5′-methylthioadenosine phosphorylase from human prostate

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Abstract
5''-Methylthioadenosine phosphorylase was purified approximately 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 .mu.M. 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 and the replacement of N-7 by a methinic radical, resulted in an almost complete loss of activity. Otherwise the replacement of S by Se and that of the methyl group by an ethyl one, is compatible with the activity as substrate. The positively charged sulfonium group alos prevents catalytic interaction with the enzyme. The inhibitory effect of 5''-methylthiotubercidin (competitive) and 5''-dimethylthioadenosine sulfonium salt (non-competitive) was also demonstrated. Three binding sites between the substrate and the enzyme are suggested.