Direct mass spectroscopic method for determination of oxygen isotope position in adenosine 5'-O-(1-thiotriphosphate). Determination of the stereochemical course of the yeast phenylalanyl-tRNA synthetase reaction.

Abstract

Listen

Translate article

Negative ion fast atom bombardment mass spectrometry has been used to distinguish between (Sp)-adenosine 5'-O-(1-thiotriphosphate) containing either an alpha- nonbridging or an alpha-beta-bridging 18O label. The method does not require any nucleotide derivatization and so avoids the excessive manipulations and purifications necessary to distinguish between the above two species using conventional mass spectroscopy. Furthermore, it is between 50 and 200 times more sensitive than other direct methods based on 31P nuclear magnetic resonance spectroscopy. Routinely, 100 nmol of nucleoside phosphorothioate is ample to establish the 18O isotope position by normal as well as linked scan mass spectrometry. In cases where normal mass spectrometry is considered adequate, 10 nmol of material suffices. This technique should be useful in determining the stereochemical course of enzymatic nucleotidyl transfer and nuclease-catalyzed hydrolysis reactions under conditions of limiting availability of enzyme or substrate. Yeast phenylalanyl-tRNA synthetase was used to prepare the 18O-labeled adenosine 5'-O-(1-thiotriphosphate) species, and this enzyme was concomitantly shown to catalyze adenylyl transfer with inversion of configuration at phosphorus.