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 nucleoside phosphorylase to catalyze the N-glycosidic 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 non-destructive 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]Gln-tRNA 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 aspartyl-tRNA 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.
Lloyd
A J
AJ
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
Thomann
H U
HU
Ibba
M
M
Söll
D
D
D000215:Acylation; D000255:Adenosine Triphosphate / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001226:Aspartate-tRNA Ligase / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D011756:Diphosphates / Q000032:analysis; D004228:Dithionitrobenzoic Acid; D004926:Escherichia coli / Q000201:enzymology; D043564:Inorganic Pyrophosphatase; D011687:Purines / Q000032:analysis; D011755:Pyrophosphatases / Q000378:metabolism; D012352:RNA, Transfer, Glu / Q000378:metabolism; D012364:RNA, Transfer, Trp / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D014369:Tryptophan-tRNA Ligase / Q000378:metabolism
D011756:Diphosphates; D011687:Purines; D012352:RNA, Transfer, Glu; D012364:RNA, Transfer, Trp; D001224:Aspartic Acid; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D000604:Amino Acyl-tRNA Synthetases; D001226:Aspartate-tRNA Ligase; C020659:glutaminyl-tRNA synthetase; D014369:Tryptophan-tRNA Ligase