Evolutionary aspects of accuracy of phenylalanyl-tRNA synthetase. A comparative study with enzymes from Escherichia coli, Saccharomyces cerevisiae, Neurospora crassa, and turkey liver using phenylalanine analogs

Abstract

The phenylalanyl-tRNA synthetases from Escherichia coli, Saccharomyces cerevisiae, Neurospora crassa, and turkey liver activate a number of phenylalanine analogues (tyrosine, leucine, methionine, p-fluorophenylalanine, beta-phenylserine, beta-thien-2-ylalanine, 2-amino-4-methylhex-4-enoic acid, mimosine, N-benzyl-L- or N-benzyl-D-phenylalanine, and ochratoxin A), as demonstrated by Km and kcat of the ATP/PPi pyrophosphate exchange. Upon complexation with tRNA, the enzyme-tRNAPhe complexes show a significantly increased initial discrimination of these amino acid analogues expressed in higher Km and lower kcat values, as determined by amino-acylation of tRNAPhe-C-C-A(3'NH2). The overall accuracy is further enhanced by a second discrimination, a proofreading step. The strategies employed by the enzymes with respect to accuracy differ. Better initial discrimination in the aminoacylation and less elaborated proofreading for the E. coli enzyme can be compared to a more efficient proofreading by other synthetases. In this way the comparatively poor initial amino acid recognition in the case of the S. cerevisiae and N. crassa enzymes is balanced. The extent of initial discrimination is therefore inversely coupled to the hydrolytic capacity of the proofreading. A striking difference can be noted for the proofreading mechanisms. Whereas the enzymes from E. coli, S. cerevisiae, and N. crassa follow the pathway of posttransfer proofreading, namely, enzymatic hydrolysis of the misaminoacylated tRNA, the turkey liver enzyme uses tRNA-dependent pretransfer proofreading in the case of natural amino acids. In spite of the same subunit structure and similar molecular weight, the phenylalanyl-tRNA synthetases from a prokaryotic and lower and higher eukaryotic organisms show obvious mechanistic differences in their strategy to achieve the necessary fidelity.

The proofreading of hydroxy analogues of leucine and isoleucine by leucyl-tRNA synthetases fromE. coliand yeast

“…These findings support earlier reports of a lower initial specificity of yeast aminoacyl-tRNA synthetases in comparison to their E. coli counterparts (16,31,33).…”

Real-time assay for testing components of protein synthesis

“…The apparent K m for Phe, estimated from the concentration dependence of either S 1 or A 1 , is ∼10–15 µM. This value, which represents the first reported estimation of K m for an amino acid in a CFPS system, is considerably higher than the value for Phe-RS (2 µM) determined in a highly purified system (42), perhaps reflecting competing binding to Phe-RS in the CFPS -Phe kit by near cognate amino acids and/or added Phe-RS inhibitor.…”

Editing of errors in selection of amino acids for protein synthesis.

“…Although this is true for charging of noncognate amino acids to tRNA (66, 147), direct measurements with microbial ArgRS, IleRS, MetRS, PheRS, TyrRS, and ValRS indicate that in the charging of a cognate amino acid to tRNA, the ATP/ aminoacyl-tRNA stoichiometry is one with an upper limit of experimental error of 0.05 (99). Similar ATP/aminoacyl-tRNA stoichiometries were also obtained in reactions catalyzed by plant SerRS (73) and avian PheRS (54).…”