Differences in the Magnesium Dependences of the Class I and Class II Aminoacyl‐tRNA Synthetases from Escherichia coli

Abstract

The magnesium dependences of the ATPJPP, exchange and tRNA aminoacylation of reactions were measured for six aminoacyl-tRNA synthetases (isoleucyl-, tyrosyl-and arginyl-tRNA synthetases from class I, and histidyl-, lysyl-and phenylalanyl-tRNA synthetases from class 11). The measured values were subjected to best-fit analyses using sum square error calculations between the data and the calculated curves in order to find the mode of participation of the Mg" and to optimize the sets of the kinetic constants.The following four dependences were observed: the class I1 synthetases require three Mgz+ for the activation reaction (including the one in MgATP), but the class I synthetases require only one Mgz+ (in MgATP) ; in class I1 synthetases both MgPP, and Mg,PP, participate in the pyrophosphorolysis of the aminoacyl adenylate. Arginyl-tRNA synthetase from class I also shows a better fit if also Mg,PP, reacts, but in the isoleucyl-and tyrosyl-tRNA synthetases only MgPP, but not Mg,PP, is used in the pyrophosphorolysis. Different synthetases have different requirements for the tRNA-bound Mg2+ and spermidine, independent of the enzyme class. 1-4 Mg" or spermidines are required in the best fit models. At the end of the reaction in all the synthetases analysed the dissociation of Mg" from the product aminoacyltRNA essentially enhances the subsequent dissociation of the aminoacyl-tRNA from the enzyme. The binding of ATP to the E . aminoacyl-tRNA complex also speeds up the dissociation of the aminoacyltRNA from most of these enzymes.

The crystal structure of asparaginyl-tRNA synthetase from Thermus thermophilus and its complexes with ATP and asparaginyl-adenylate: the mechanism of discrimination between asparagine and aspartic acid

“…These results based on crystallographic analysis are consistent with an independent study of the magnesium dependence of ATP/PPi exchange and aminoacylation reactions of three class I and three class II E.coli aminoacyl-tRNA synthetases. This confirmed that the magnesium dependence of class 2954 I and class II synthetases is distinct, one magnesium being required for class I and three for the class II enzymes (Airas, 1996).…”

Histidyl-tRNA Synthetase

“…et al, 1974). Recent kinetic studies of class I and class II aaRS from E. coli are consistent with these results (Airas, 1996) ions since an Arg (Arg259 in HisRSec) substitutes for the third ion, as described above. Arg259, part of the highly conserved Histidine A motif (Figures 2, 4), is critical for HisRS activity; mutating it to glutamine, lysine (Rühlmann et al, 1997) or histidine reduces the activity by several orders of magnitude without affecting histidine or ATP binding.…”

Studying base pair open–close kinetics of tRNALeu by TROSY‐based proton exchange NMR spectroscopy

“…As LeuRS is a class I aaRS, Mg 2+ is not involved in aminoacylation of LeuRS directly [24]. Besides, only the free form Mg 2+ was considered in the aminoacylation assay buffer condition; effects of complex form Mg 2+ bound by ATP, AMP and pyrophosphate were excluded.…”

“…Aminoacylation of Ec tRNA Leu (CAG) was assayed as described in [7] except that [Mg 2+ ]/[tRNA] was increased from 1:1 to 15:1, which was calculated based on concentration of the free Mg 2+ [24].…”