Adenosine 5′-phosphorothioate, an analog of adenosine 5′-phosphate, has been prepared from adenosine and thiophosphoryl chloride in triethyl phosphate in 65% yield and isolated as the sodium salt. The adenosine 5′-phosphate content of this product (measured with adenylate kinase) was less than 0.5% and the content of adenosine derivatives other than the 5′- phosphorothioate (measured as unchanged material after treatment with adenylate deaminase or with 5′- nucleotidase) was 1-3%. Adenosine 5′-phosphorothioate (0.34 mM) was phosphorylated by adenosine triphosphate in the presence of muscle adenylate kinase at less than 0.3% of the rate with adenosine 5′-phosphate at the same concentration, but 0.25 mM adenosine 5′-phosphorothioate increased the adenosine 5′-phosphate concentration for half-maximal activity, [S]0.5, from 0.15 to 0.18 mM and decreased the maximal velocity (V, micromoles per minute per milligram of protein) from 120 to 91 without changing the slope (1.35) of the Hill plot. Adenosine 5′-phosphorothioate was deaminated by adenylate deaminase from rat skeletal muscle. The Michaelis constant, Km, was 1.6 mM and V was 125, 9% of the maximal velocity with adenosine 5′-phosphate (V = 1330; Km = 0.9 mM). With 5′-nucleotidase from Crotalus venom liberation of adenosine from adenosine 5′-phosphorothioate was much slower than that from adenosine 5′-phosphate ( V = 0.026 and 1.39, respectively) but Km for adenosine 5′-phosphorothioate (0.02 mM) was less than that for adenosine 5′-phosphate (0.03-0.035 mM). Adenosine 5′-phosphorothioate is a competitive inhibitor of dephosphorylation of adenosine 5′-phosphate, with inhibitor constant, K, of 0.02 mM. Adenosine 5′-phosphorothioate activates the allosteric enzyme yeast diphosphopyridine nucleotide- isocitrate dehydrogenase almost as effectively as the known activator adenosine 5′-phosphate. At 0.31 mM D-isocitrate, half-maximal activation was given by 0.23 mM adenosine 5′-phosphorothioate and 0.19 mM adenosine 5′-phosphate; 0.35 mM adenosine 5′-phosphorothioate decreased [S]0.5 for isocitrate from 1.64 to 0.29 mM with little effect on the slope of the Hill plot (3.84.0) or on the maximal velocity. Adenosine 5′-phosphorothioate was less effective than adenosine 5′-phosphate as an inhibitor of fructose 1,6-diphosphatase from rat liver. At 0.1 mM fructose 1,6-diphosphate with 10 mM Mg2+ 50% inhibition was given by 0.16 mM adenosine 5′-phosphate and 1.6 mM adenosine 5′-phosphorothioate. Adenosine 5′phosphorothioate was more effective than adenosine 5′-phosphate as an activator of phosphorylase b from rabbit skeletal muscle with half- maximal activation of phosphate liberation from 20 mM glucose 1-phosphate at 13 μu adenosine 5′-phosphorothioate and 40 μu adenosine 5′-phosphate. The maximal activation with adenosine 5′-phosphorothioate was 1.3 times that with adenosine 5′-phosphate. © 1968, American Chemical Society. All rights reserved.
