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 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 5'-(1-thiotriphosphate) (ATPalphaS), which is known to have S configuration at Palpha, the pro-R oxygen is incorporated into the bridge position, whereas the pro-S oxygen is located at the nonbridge position. The 31P NMR spectra of the 17O-enriched compounds were used to distinguish between the bridge and nonbridge oxygens 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 ATPalphaS and that the nucleophilic displacement proceeds with net inversion of configuration at Palpha of ATPalphaS (B), supporting the "in-line" mechanism.

Oxygen Chiral Phosphate Esters

“…The second important observation that was made concerning the effect of oxygen isotope substitution on the 31P NMR spectral properties of phosphate esters was reported independently by Tsai (14) and Lowe (1 2). Tsai first documented the 31P NMR spectral properties of phosphates in which " 0 was directly bonded to phosphorus.…”

“…In phosphate esters and nucleotides, the only effect of I7O on directly bonded 31P nuclei that has been observed is extensive line broadening, and this phenomenon has been used quite profitably in the problem of configurational analyses of both phosphate esters and inorganic thiophosphate. Two types of applications have been described: the first in which the isotopic identity of the thiophosphoryl oxygensin a chiral sample of [1701]AMPS was established by examination of the "P NMR spectrum of the sample of the S , diastereomer of ATPuS obtained by the coupled action of adenylate kinase and pyruvate kinase (14), and the second in which the presence of I7O was used t o allow configurational analysis of 160,170,'80-labeled phosphate esters and nucleotides by measurements of l80 perturbations on the materials that were not isotopically substituted with 170. Tsai isolated a sample of ['701]AMPS which arose via displacement of pyrophosphate by the labeled acetate followed by acetylation of CoA by the acetyl adenylate in a process that is accompanied by C-0 bond cleavage.…”

“…In 1979, Tsai described the quadrupolar effect of I7O on directly bonded "P nuclei (extensive line broadening) and used this phenomenon in the configurational analysis of [a-"O]ATPaS (14).…”

The effect of 17O on the relaxation of an amide proton within a hydrogen bond

“…The triethylamine had been stirred with Call 2 and distilled. Isotopic enrichment of the [I70]GDP was estimated to be 50% by integration of the 31p signals of the tx-and ]3-phosphates (Tsai, 1979).…”

18O and 17O Effects on 31P NMR as Probes of Enzymatic Reactions of Phosphate Compounds

“…The simplest example is that of the adenyl transfers to form carboxyadenylates. In the reactions listed in Table 3 a diastereomer of ATPaS was allowed to react with acetate e70) (46) or amino acid esO) (33), and the chirality of the AMPS CSO) product formed after transfer of the acyl group was determined by incorporating the AMPS enzymatically with known stereochemistry in ATPaS CSO) or C70). The labeled 0 is either in the af3 bridge position or bonded to a-P in a nonbridge position, depending on the chirality of AMPS e80) or e70).…”