EXPRESSION OF HUMAN ASPARTYL-TRANSFER RNA-SYNTHETASE IN ESCHERICHIA-COLI - FUNCTIONAL-ANALYSIS OF THE N-TERMINAL PUTATIVE AMPHIPHILIC HELIX

Mammalian aspartyl-tRNA synthetase occurs in the multienzyme complex of aminoacyl-tRNA synthetases, while bacterial and yeast aspartyl-tRNA synthetases exist as free soluble enzymes. Cloning and sequencing of mammalian aspartyl-tRNA synthetase revealed a newly evolved N-terminal 32-amino-acid sequence, which contains a putative amphiphilic helix (Jacobo-Molina, A., Peterson, R., and Yang, D. C. H. (1989) J. Biol. Chem. 264, 16608-16612). Human aspartyl-tRNA synthetase (hDRS) and an N-terminal 32-residue truncated form of human aspartyl-tRNA synthetase (hDRSDELTA32) were expressed in Escherichia coli under the control of the inducible tac promoter as glutathione-S-transferase (GST) fusion proteins linked through a thrombin cleavage site. The GST-hDRS fusion protein and the GST-hDRSDELTA32 were purified by affinity chromatography on glutathione-agarose and were fully active in aspartylation of mammalian tRNA. After cleavage of GST from the fusion proteins by thrombin, hDRS and hDRSDELTA32 were purified by affinity chromatography on tRNA-Sepharose. Both hDRS and hDRSDELTA32 were present as a mixture of monomeric and dimeric forms. GST-hDRS formed high molecular weight aggregates while GST-hDRSDELTA32 was a dimeric protein. Both hDRS and hDRSDELTA32 bound to hydrophobic interaction gels such as aminohexyl-agarose. In the absence of propylene glycol, hDRS bound to aminohexyl-agarose weaker than hDRSDELTA32, but, in the presence of 50% propylene glycol, hDRS bound tighter than hDRSDELTA32. Both hDRS and hDRSDELTA32 were fully active in aspartylation of mammalian tRNA and ATP-PP(i) exchange. In comparison to the N-terminal truncated form, the full-length enzyme showed greater thermal stability and ATP-PP(i) exchange activity but lower aminoacylation activity. The catalytic constant of hDRSDELTA32 for aminoacylation of tRNA was 2-fold higher than that of hDRS. The Michaelis-Menten constants for aspartic acid and tRNA(Asp) were 302 muM and 13 nm for hDRS, and 29 muM and 130 nM for hDRSDELTA32, respectively. These results suggest that the newly evolved N-terminal peptide in hDRS may modulate the enzymatic activity, the stability, and the chromatographic behavior of hDRS. The structure and function of the N-terminal peptide in aspartyl-tRNA synthetase and in the synthetase complex will be discussed.