On the Mechanism of Ribonuclease A

The triethylammonium salt of uridine 3′‐O‐thiophosphate methyl ester crystallized in space group P21 with cell dimensions a= 684.5 pm, b= 1480.4 pm, c= 2254.2 pm, β= 92.8° and two molecules per asymmetric unit. Solution of the structure was accomplished by direct methods; the nucleotide molecules have been located unambiguously but the cations are disordered and appear only as highly noise background. The least‐squares refinement converged at a conventional R‐factor of 19.1%. The base in both molecules of the asymmetric unit is anti with respect to the sugar, the conformation about the C(4′)‐C(5′) bond is gauche, gauche, the sugar pucker is C(2′)‐endo. The conformation about the two P–O ester bonds is not as observed in double helical RNA's, suggesting that the sulphur atom directs the orientation of the ester bonds. Uridine 3′‐O‐thiophosphate methyl ester was obtained by pancreatic ribonuclease A catalyzed methanolysis of the endo‐isomer of uridine 2′: 3′‐cyclothiophosphate. As the thiophosphate diester moiety is in the R configuration with respect to the methyl group, the enzyme must have attacked the cyclothiophosphate via an in‐line mechanism.