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

The S-methylated derivatives of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha SCH3) have been prepared by the reaction of both diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) with methyl iodide. At physiological pH ATP alpha SCH3 was unstable, decomposing predominantly to adenosine 5'-O-(S-methyl thiophosphate) (AMPSCH3) and pyrophosphate. 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 hexokinase and acetate kinase, and both diastereomers were active with fructose-6-phosphate kinase. The products of these reactions were the appropriate sugar or acyl phosphate, AMPSCH3, and inorganic phosphate, the latter two 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 creatine and phosphoglycerate kinase. These results are interpreted as meaning that creatine and phosphoglycerate kinase require Mg2+ coordination to the alpha-phosphate group during the enzyme-catalyzed reaction whereas the other three 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.