THE ACTIVE-SITE OF YEAST ASPARTYL-TRANSFER-RNA SYNTHETASE - STRUCTURAL AND FUNCTIONAL-ASPECTS OF THE AMINOACYLATION REACTION

The crystal structures of the various complexes formed by yeast aspart yl-tRNA synthetase (AspRS) and its substrates provide snapshots of the active site corresponding to different steps of the aminoacylation re action. Native crystals of the binary complex tRNA-AspRS were soaked i n solutions containing the two other substrates, ATP (or its analog AM PPcP) and aspartic acid. When all substrates are present in the crysta l, this leads to the formation of the aspartyl-adenylate and/or the as partyl-tRNA. A class II-specific pathway for the aminoacylation reacti on is proposed which explains the known functional differences between the two classes while preserving a common framework. Extended signatu re sequences characteristic of class II aaRS (motifs 2 and 3) constitu te the basic functional unit. The ATP molecule adopts a bent conformat ion, stabilized by the invariant Arg531 of motif 3 and a magnesium ion coordinated to the pyrophosphate group and to two class-invariant aci dic residues. The aspartic acid substrate is positioned by a class II invariant acidic residue, Asp342, interacting with the amino group and by amino acids conserved in the aspartyl synthetase family. The amino acids in contact with the substrates have been probed by site-directe d mutagenesis for their functional implication.