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Volume 271, Number 46, Issue of November 15, 1996 pp. 29295-29303
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Expression of Rat Aspartyl-tRNA Synthetase in Saccharomyces cerevisiae
ROLE OF THE NH₂-TERMINAL POLYPEPTIDE EXTENSION ON ENZYME ACTIVITY AND STABILITY

(Received for publication, March 12, 1996, and in revised form, August 13, 1996)

From the Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Gif sur Yvette, France

Cytoplasmic aspartyl-tRNA synthetase from mammals is one of the components of a multienzyme complex comprising nine synthetase activities. The presence of an amino-terminal extension composed of about 40 residues is a characteristic of the eukaryotic enzyme. We report here the expression 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 extension. The three recombinant enzyme species were purified to homogeneity. They behave as ₂ dimers and display catalytic parameters in the tRNA aminoacylation reaction identical to those determined for the native, complex-associated form of aspartyl-tRNA synthetase isolated from rat liver. Because the dimer dissociation constant of rat AspRS is much higher than that of its bacterial and yeast counterparts, we could establish a direct correlation between dissociation of the dimer and inactivation of the enzyme. Our results clearly show that the monomer is devoid of amino acid activation and tRNA aminoacylation activities, indicating that dimerization is essential to confer an active conformation on the catalytic site. The two NH₂-terminal truncated derivatives were fully active, but proved to be more unstable than the recombinant native enzyme, suggesting that the polypeptide extension fulfills structural rather than catalytic requirements.

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