Isoleucyl-tRNA synthetase from bakers' yeast: variable discrimination between tRNAIle and tRNAVal and different pathways of cognate and noncognate aminoacylation under standard conditions, in the presence of pyrophosphatase, elongation factor Tu-GTP complex, and spermine.

Abstract

Error rates in discrimination between cognate tRNAIle and noncognate tRNAVal in the aminoacylation reaction with isoleucine catalyzed by isoleucyl-tRNA synthetase from yeast have been investigated in three sets of experiments under different assay conditions. The overall discrimination factor was first determined by isoleucylation of tRNAVal/tRNAIle mixtures. In the second set of experiments, the number of AMP molecules formed per Ile-tRNA in the cognate and noncognate reactions was measured. The higher AMP formation in the noncognate aminoacylation is assigned to a proofreading reaction step. The calculated proofreading factors and an estimated initial discrimination factor yield overall discriminations that are consistent with those obtained from the first set of experiments. In the third series of studies, the orders of substrate addition and product release of cognate and noncognate isoleucylation reactions were investigated by initial rate kinetic methods. From kcat and Km values, the overall discrimination factors were calculated and showed again a good coincidence with those observed in the preceding sets of experiments. Besides under standard assay conditions, aminoacylation reactions were studied in the presence of pyrophosphatase or elongation factor Tu-GTP complex, under addition of both these proteins, in presence of these two additional proteins and spermine at high and low magnesium concentrations, and under special conditions that favor misacylations. Furthermore, isoleucylation of tRNAIle was tested at increased and decreased pH in the standard enzyme assay. Variation of the assay conditions results in changing discrimination factors, which differ by a factor of about 10. Substitution of tRNAIle by tRNAVal in the isoleucylation reaction causes changes in substrate addition and product release orders and thus of the whole catalytic cycle. For aminoacylation of tRNAIle, four different orders of substrate addition and product release appear: the sequential ordered ter-ter, the rapid equilibrium sequential random ter-ter, the random bi-uni uni-bi ping-pong, and a bi-bi uni-uni ping-pong mechanism with a rapid equilibrium segment. tRNAVal is aminoacylated in rapid equilibrium random ter-ter order, in a bi-bi uni-uni ping-pong mechanism with a rapid equilibrium segment, and in two bi-uni uni-bi ping-pong mechanisms. It is assumed that the different assay conditions can be regarded as a stepwise approximation to physiological conditions and that considerable changes in error rates may be also possible in vivo up to 1 order of magnitude.