Studies of the action of adenylosuccinase with 6-thio analogues of adenylosuccinic acid.

Recent work has shown that certain growth inhibitory purine analogues undergo anabolism to ribonucleoside 5’-phosphates (1, 2). Ribonucleoside 5’-phosphate derivatives of purine analogues that effect temporary remission of some human leukemias have been synthesized (3-6), and effects of these nucleotides on enzymatic interconversions of naturally occurring purine nucleotides have been studied (7-9). A preliminary communication (7) reported that 6-mercapto-9-P-n-ribofuranosylpurine 5’-phosphate (B-thioinosinic acid) inhibited conversion of adenylosuccinic acid (Fig. 3, III) to adenosine phosphate by adenylosuccinase. This conversion, described by Carter and Cohen (lo), involves reversible cleavage of adenylosuccinic acid to adenosine phosphate and fumaric acid. A possible mechanism of inhibition could be addition of the 6-mercapto group of the nucleotide to the double bond of fumaric acid to form an inhibitory analogue (Fig. 3, IV) of adenylosuccinic acid in which the nitrogen at position 6 is replaced by sulfur. Synthesis of this analogue was desired to investigate this possibility and, in addition, to examine the potentialities of the analogue as a relatively selective inhibitor of adenylosuccinase. The present communication details the synthesis and purification of the 6-thio analogue of adenylosuccinic acid, adenylosuccinic acid itself, and the n-succino enantiomorphs of each of these nucleotides. An unexpected finding was that the 6-thio analogue is cleaved by the enzyme preparation in a manner analogous to the cleavage of adenylosuccinic acid. From studies of the analogues of adenylosuccinic acid as substrates and inhibitors of adenylosuccinase, and from studies of chemical properties of purine derivatives serving as models, hypotheses are suggested concerning the mechanism of enzymatic cleavage of adenylosuccinate and some of the characteristics of the active site of adenylosuccinase.