A broadly applicable continuous spectrophotometric assay for measuring aminoacyl-tRNA synthetase activity

We describe a convenient, simple and novel continuous spectrophotometric method for the determination of aminoacyl-tRNA synthetase activity. The assay relies upon the measurement of inorganic pyrophosphate generated in the first step of the aminoacylation of a tRNA. Pyrophosphate release is coupled to inorganic pyrophosphatase, to generate phosphate, which in turn is used as the substrate of purine nucleoslde phosphorylase to catalyze the N-glycosldic cleavage of 2-amino 6-mercapto 7-methylpurine ribonucleoside. Of the reaction products, ribose 1-phosphate and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorbance at 360 nm relative to the nucleoside and hence provides a spectrophotometric signal that can be continuously followed. The nondestructive nature of the spectrophotometric assay allowed the re-use of the tRNAs in question in successive experiments. The usefulness of this method was demonstrated for glutaminyl-tRNA synthetase (GlnRS) and tryptophanyl-tRNA synthetase. Initial velocities measured using this assay correlate closely with those assayed by quantitation of [3H] pHJGIntRNA or [14C] Trp-tRNA formation respectively. In both cases amino acid transfer from the aminoacyl adenylate to the tRNA represents the rate determining step. In addition, aminoacyl adenylate formation by aspartyltRNA synthetase was followed and provided a more sensitive means of active site titration than existing techniques. Finally, this novel method was used to provide direct evidence for the cooperativity of tRNA and ATP binding to GlnRS.