EXPRESSION OF RAT ASPARTYL-TRANSFER-RNA SYNTHETASE IN SACCHAROMYCES-CEREVISIAE - ROLE OF THE NH2-TERMINAL POLYPEPTIDE EXTENSION ON ENZYME-ACTIVITY AND STABILITY

Cytoplasmic aspartyl-tRNA synthetase from mammals is one of the compon ents of a multienzyme complex comprising nine synthetase activities. T he presence of an amino-terminal extension composed of about 40 residu es is a characteristic of the eukaryotic enzyme. We report here the ex pression in the yeast Saccharomyces cerevisiae of a native form of rat aspartyl-tRNA synthetase and of two truncated derivatives lacking 20 or 36 amino acid residues from their amino-terminal polypeptide extens ion. The three recombinant enzyme species were purified to homogeneity . They behave as alpha(2) dimers and display catalytic parameters in t he tRNA aminoacylation reaction identical to those determined for the native, complex-associated form of aspartyl-tRNA synthetase isolated f rom rat liver. Because the dimer dissociation constant of rat AspRS is much higher than that of its bacterial and yeast counterparts, we cou ld establish a direct correlation between dissociation of the dimer an d inactivation of the enzyme. Our results clearly show that the monome r is devoid of amino acid activation and tRNA aminoacylation activitie s, indicating that dimerization is essential to confer an active confo rmation on the catalytic site. The two NH2-terminal truncated derivati ves were fully active, but proved to be more unstable than the recombi nant native enzyme, suggesting that the polypeptide extension fulfills structural rather than catalytic requirements.