S-Methylated nucleoside phosphorothioates as probes of enzyme metal.cntdot.nucleotide binding sites

Abstract

The S-methylated derivatives of adenosine 5''-O-(1-thiotriphosphate) (ATP.alpha.SCH3) were prepared by the reaction of both diastereomers of adenosine 5''-O-(1-thiotriphosphate) with methyl iodide. At physiological pH ATP.alpha.SCH3 was unstable, decomposing predominantly to adenosine 5''-O-(S-methyl thiophosphate) (AMPSCH3) and PPi. A minor degradation pathway also yielded ATP and methyl mercaptan. Greatly enhanced stability was observed at lower pH. The SP diastereomer of ATP.alpha.SCH3 was a substrate for [yeast] hexokinase and [Escherichia coli] acetate kinase, and both diastereomers were active with [rabbit muscle] fructose-6-phosphate kinase. The products of these reactions were the appropriate sugar or acyl phosphate, AMPSCH3 and Pi, the latter 2 species arising from the breakdown of the transient intermediate 5''-O-(S-methyl 1-thiodiphosphate (ADP.alpha.SCH3). No measurable substrate activities were observed with [rabbit muscle] creatine kinase and [yeast] phosphoglycerate kinase. Creatine and phosphoglycerate kinase probably require Mg2+ coordination to the .alpha.-phosphate group during the enzyme-catalyzed reaction whereas the other 3 enzymes do not. Attempts to prepare adenosine 5''-O-(S-methyl 2-thiotriphosphate) (ATP.beta.SCH3) and ADP.alpha.SCH3 by similar methods were unsuccessful with adenosine 5''-O-(S-methyl 2-thiodiphosphate) (ADP.beta.S) and AMPSCH3 being respectively isolated as the major products.