Abstract
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 (hDRS delta 32) 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-hDRS delta 32 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 hDRS delta 32 were purified by affinity chromatography on tRNA-Sepharose. Both hDRS and hDRS delta 32 were present as a mixture of monomeric and dimeric forms. GST-hDRS formed high molecular weight aggregates while GST-hDRS delta 32 was a dimeric protein. Both hDRS and hDRS delta 32 bound to hydrophobic interaction gels such as aminohexyl-agarose. In the absence of propylene glycol, hDRS bound to amino-hexyl-agarose weaker than hDRS delta 32, but, in the presence of 50% propylene glycol, hDRS bound tighter than hDRS delta 32. Both hDRS and hDRS delta 32 were fully active in aspartylation of mammalian tRNA and ATP-PPi exchange. In comparison to the N-terminal truncated form, the full-length enzyme showed greater thermal stability and ATP-PPi exchange activity but lower aminoacylation activity. The catalytic constant of hDRS delta 32 for aminoacylation of tRNA was 2-fold higher than that of hDRS. The Michaelis-Menten constants for aspartic acid and tRNAAsp were 302 microM and 13 nM for hDRS, and 29 microM and 130 nM for hDRS delta 32, 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.
Highlights
Aftercleavage of GST from the fusion proteins by synthetase complexes have provided an excellent model for thrombin, hDRSand hDRSA32 were purifiedby affin- the elucidation of the structural andfunctional basis of proity chromatography on tRNA-Sepharose
SimilatGro ST-hDRSG, STamplification of the cDNA for hDRS haDndRSA32 by polym- hDRSA32 is fully active in aspartylationof calf liver tRNA
It is intriguing that the small dispensable peptide has multiple effects on the enzyme properties and is a newly evolved sequence inaspartyl-tRNAsynthetase coinciding with the occurrence of the multienzyme complex of aminoacyl-tRNA synthetases inhigh eukaryotes
Summary
Full-length complementary DNA coding humanaspartyl-tRNA synthetase was described previously (Jacobo-Molina et al, 1989). At various time intervals at 37 "C, aliquots of 10-20 pl of the reaction mixture were assayed for transformants for eachrecombinant were isolated and examined by agarose gel electrophoresis, and sequences in the acid-precipitable radioactivity for the formation of ["Claspartyl- constructs were confirmed by sequence analyses. The concentration of IPTG for a maximal yield of expression of Michaelis-Mentenconstants for aspartic acid,ATP,andtRNAA"p were determined from the initial velocity of the aspartylation of calf liver tRNA under the standard conditions at saturating concentra-. Measurements of ATP-PPi Exchange-The standard reaction mixture contained 50 mM Tris-HC1, pH 8.0,8 mMMgC12, 2 mM ATP, 5 multiple protein bands with lower molecular weights as analyzed by SDS-gel electrophoresissuggesting partial degradamM aspartic acid, 2 mM pyrophosphate, and a limiting amount of tion of the fusion protein (data notshown).
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