METAL-ION DEPENDENCE OF PHOSPHOROTHIOATE ATP ANALOGS IN THE BACILLUS-STEAROTHERMOPHILUS TYROSYL-TRANSFER RNA-SYNTHETASE REACTION

Pre-steady-state kinetic analyses on the formation of tyrosyl adenylate from tyrosine and each of the four diastereomers of α- and β-phosphorothioate adenosine triphosphates [ATPαS and ATPβS; Eckstein, F., & Goody, R. (1976) Biochemistry 15, 1685-1691; Yee, D., Armstrong, V. W., & Eckstein, F. (1979) Biochemistry 18, 4116-4123] were performed in the presence of Mg2+, Co2+, and Cd2+ as the divalent metal ion cofactor. A modest preference of 5.5-fold in k3/KA′ (where is the rate constant for tyrosyl adenylate formation and KA′ is the dissociation constant for ATP, or phosphorothioate ATP, from the E•Tyr•metal•ATP complex) for the SP ATPαS diastereomer and the absence of an inversion of preference when the metal ion is changed suggest that there is a stereospecific enzyme-α-phosphate interaction and that there is no direct metal ion interaction with the α-phosphate. The extent of reaction of the ATPαS diastereomers (30-50%) implies that these analogues are more susceptible to the hydrolytic site reaction previously reported for this enzyme [Wells, T. N. C., & Fersht, A. R. (1986) Biochemistry 25, 1881-1886]. The strong preference in k3/KA′ for the Rp ATPβS diastereomer (16-fold for Mg2+ and 50-fold for Co2+) is indicative of a stereospecific interaction with the pro SPβ oxygen of ATP. The SP ATPβS diastereomer exhibits an unusually low extent of reaction (~10% versus 70-100% for the Rp diastereomer) that does not appear to be due to the hydrolytic side reaction. This low extent of reaction appears to mask the inversion of preference in k3 and k3/KA′ when the metal ion is changed. The observed change in preference in KA′ (Sp/Rp for Mg2+ = 1.8 and for Cd2+ = 0.36) is consistent with metal ion binding to the β-phosphate. A model of the E•Tyr•Mg•ATP complex is proposed that involves enzyme binding to the pro-Rpα oxygen and Mg2+ chelating to the pro-Spβ oxygen of ATP. © 1990, American Chemical Society. All rights reserved.