7-Enriched Nucleoside Triphosphates. Stereochemistry of Acetate Activation by Acetyl Coenzyme A Synthetase?

Adenosine 5’-( thiophosphate) (AMPS) contains a prochiral phosphorus center. Differentiation of the two diastereotopic oxygens would allow elucidation of the stereochemical course of biological adenylyl transfer reactions. A general method was developed to distinguish between the “pro-R’ and “pro-S’ oxygens. When we converted the AMPS to the isomer A of adenosine S-( 1-thiotriphosphate) (ATP&), which is known to have S configuration a t P,, the pro-R oxygen is incorporated into the bridge position, whereas the p r o 4 oxygen is located a t the nonbridge position. The 31P A d e n y 1 y 1 transfer reactions involve nucleophilic displacement of the pyrophosphoryl group of ATP by the second substrate (Scheme I). In most cases the functional group which undergoes nucleophilic displacement contains oxygen, e.g., carboxyl, hydroxyl, and imidol [HN=C(OH)NH-] groups, or sulfate. This type of reaction is involved in many classes of important biological processes (Stadtman, 1973), e.g., carboxyl group activation (fatty acyl-CoA synthetases, aminoacyl-tRNA synthetases, lipoate activating enzymes, biotin activating enzymes, etc.), biosynthesis of phosphodiester derivatives of adenosine (RNA polymerases, adenylation of amino glycoside antibiotics, etc.), synthesis of adenosine diphosphate derivatives (NAD, FAD, ADPglucose, etc.), sulfate activation, synthesis of imidol adenylate derivatives, and adenylylation of functional groups of proteins. In many cases a third substrate displaces the A M P moiety, resulting in the incorporation of an oxygen from the second substrate into the phosphoryl group of AMP. Among the numerous enzymes catalyzing reactions involving chiral, prochiral, or proprochiral phosphorus centers, only a few have been analyzed unequivocally for their steric courses, and only two of them, e.g., DNA-dependent RNA polymerase (Eckstein et al., 1976) and tRNA nucleotidyltransferase (Eckstein et al., 1977), are adenylyl transfer reactions. Both enzymes specifically incorporate the isomer A of ATPaS.’ In both cases, the configuration of the thiophosphate diester bond From the Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907. Received November 28, 1978. This work was supported by a grant from the Purdue Cancer Research Committee and in part by National Institutes of Health Research Grant GM 18852. 0006-2960/79/04181468$01 .OO/O N M R spectra of the ”0-enriched compounds were used to distinguish between the bridge and nonbridge oxygens based on the decrease in the peak intensity of 31P N M R signals caused by the directly bound ”0 isotope. The method was used to elucidate the stereochemical course of acetate activation catalyzed by yeast acetyl coenzyme A (CoA) synthetase. The results indicate that yeast acetyl-coA synthetase is specific for the isomer B of ATP& and that the nucleophilic displacement proceeds with net inversion of configuration at P, of ATP& (B), supporting the ‘‘in-line” mechanism.