Use of phosphorus-31 nuclear magnetic resonance to distinguish bridge and nonbridge oxygens of oxygen-17-enriched nucleoside triphosphates. Stereochemistry of acetate activation by acetyl coenzyme A synthetase

Abstract

Adenosine 5''-(thiophosphate) (AMPS) contains a prochiral P center. Differentiation of the 2 diastereotopic O 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 AMPS was converted to the isomer A of adenosine 5''-(1-thiotriphosphate) (ATP.alpha.S), which is known to have S configuration at P.alpha., the pro-R O is incorporated into the bridge position, whereas the pro-S O is located at the nonbridge position. The 31P NMR spectra of the 17O-enriched compounds were used to distinguish between the bridge and nonbridge O based on the decrease in the peak intensity of 31P NMR signals caused by the directly bound 17O 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.alpha.S and that the nucleophilic displacement proceeds with net inversion of configuration of P.alpha. of ATP.alpha.S (B), supporting the in-line mechanism.