Expression, purification, and characterization of human tyrosyl-tRNA synthetase.

Human tyrosyl-tRNA synthetase is a homodimeric enzyme and each subunit is near 58 KD. It catalyzes the aminoacylation of tRNA(Tyr) by L-tyrosine. The His(6)-tagged human TyrS gene was obtained by RT-PCR from total RNA of human lung giant-cell cancer strain 95 D. It was confirmed by sequencing and cloned into the expression vector pET-24 a (+) to yield pET-24 a (+)-HTyrRS, which was transfected into Escherichia coli BL21-CodonPlus-RIL. The induced-expression level of His(6)-tagged human TyrRS was about 24% of total cell proteins under IPTG inducing. The recombinant protein was conveniently purified in a single step by metal (Ni(2+)) chelate affinity chromatography. About 22.3mg purified enzyme could be obtained from 1L cell culture. The k(cat) value of His(6)-tagged human TyrRS in the second step of tRNA(Tyr) aminoacylation was 1.49 s(-1). The K(m) values of tyrosine and tRNA(Tyr) were 0.3 and 0.9 microM. Six His residues at the C terminus of human TyrRS have little effect on the activities of the enzyme compared with other eukaryotic TyrRSs.

Stabilization of the transition state for the transfer of tyrosine to tRNA(Tyr) by tyrosyl-tRNA synthetase.

Aminoacylation of tRNA(Tyr) involves two steps: (1) tyrosine activation to form the tyrosyl-adenylate intermediate; and (2) transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA(Tyr). In Bacillus stearothermophilus tyrosyl-tRNA synthetase, Asp78, Tyr169, and Gln173 have been shown to form hydrogen bonds with the alpha-ammonium group of the tyrosine substrate during the first step of the aminoacylation reaction. Asp194 and Gln195 stabilize the transition state complex for the first step of the reaction by hydrogen bonding with the 2'-hydroxyl group of AMP and the carboxylate oxygen atom of tyrosine, respectively. Here, the roles that Asp78, Tyr169, Gln173, Asp194, and Gln195 play in catalysis of the second step of the reaction are investigated. Pre-steady-state kinetic analyses of alanine variants at each of these positions shows that while the replacement of Gln173 by alanine does not affect the initial binding of the tRNA(Tyr) substrate, it destabilizes the transition state complex for the second step of the reaction by 2.3 kcal/mol. None of the other alanine substitutions affects either the initial binding of the tRNA(Tyr) substrate or the stability of the transition state for the second step of the aminoacylation reaction. Taken together, the results presented here and the accompanying paper are consistent with a concerted reaction mechanism for the transfer of tyrosine to tRNA(Tyr), and suggest that catalysis of the second step of tRNA(Tyr) aminoacylation involves stabilization of a transition state in which the scissile acylphosphate bond of the tyrosyl-adenylate species is strained. Cleavage of the scissile bond on the breakdown of the transition state alleviates this strain.

Efficient synthesis of a 72-kDa mitochondrial polypeptide using the yeast Ty expression system.

Using the Ty system from yeast we report the efficient expression of a heterologous eukaryotic gene encoding a 72 kDa mitochondrial polypeptide. The pFM2IIBgIII expression vector was initially modified for this purpose by inserting the factor X(a) protease cleavage site. The TyA gene, which encodes the structural component of the yeast virus-like particles (VLPs), and the eukaryotic yst1 gene, encoding a 72 kDa mitochondrial tyrosyl-tRNA synthetase from the filamentous fungus Podospora anserina, were subsequently fused to the factor X(a) cleavage site. The resulting chimeric gene, in which the two polypeptide coding sequences are separated by the factor X(a) cleavage site, was expressed in yeast. High yield expression of this foreign protein, which was isolated from yeast transformants as hybrid TyVLPs, was verified after factor X(a) treatment by SDS polyacrylamide gel electrophoresis and antibody detection. The strategy presented here should be useful for expressing a wide variety of eukaryotic genes.

[Isolation and characteristics of functionally active proteolytically modified forms of tyrosyl-tRNA synthetase from bovine liver]. / Vydelenie i kharkteristkia funktsional'no aktivnoi proteoliticheski modifitsirovannoi formy tirozil-tRNK-sintetazy iz pecheni byka.

Functionally active proteolytic modified form of tyrosyl-tRNA-synthetase has been isolated in a homogeneous form from the bovine liver under incomplete blocking of endogenous proteolysis. The isolation scheme is described. From the data of gel electrophoresis under denaturing conditions the molecular weight of this form is 39 +/- 1.5 kDa and from the data of gel filtration under native conditions -84 kDa. Thus, this form as well as the native enzyme is a dimer of the alpha 2-type. As compared to the native enzyme (Mm 2 x 59 kDa) a proteolytically modified form has a fragment of the polypeptide chain about 20 kDa long split out (this fragment is not essential for catalytic activity). The values of catalytic characteristics of the modified form in tRNA(Tyr) aminoacylation reaction (Km = 1.19 microM and kcat = 2.99 min-1) are close to those obtained for the main form of the enzyme (0.69 microM and 2.97 min-1, respectively). Amino acid composition of the low-molecular form of tyrosyl-tRNA-synthetase has been determined. It was found that the fragment split out in limited proteolysis was characterized by very high content of positively charged lysine residues (46 residues). A proteolytically modified form of tyrosyl-tRNA-synthetase possesses, like the main form, the affinity to high-molecular rRNA but it is eluted from the column filled with rRNA-sepharose at lower salt concentration (50 mM KCl) as compared to the main form of the enzyme (100 mM KCl).

The amino acid sequence of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus.

The primary structure of the tyrosyl-tRNA synthetase (TyrTS) of Bacillus stearothermophilus has been deduced from the nucleotide sequence of the cloned gene and from the amino acid sequence of peptides isolated from the purified enzyme. TyrTS (B. stearothermophilus) has a molecular weight of 47316 and the sequence is 56% homologous with that of TyrTS (Escherichia coli). The binding domain for the substrate intermediate tyrosyl adenylate is located in the N-terminal portion of the polypeptide and is highly conserved in both enzymes. Several lysine residues, which are shielded from acetylation in the TyrTS-tRNATyr complex, are also located in a stretch of highly conserved sequence.

Purification and some properties of rat liver tyrosyl-tRNA synthetase.

Rat liver cytoplasmic tyrosine:tRNA ligase (tyrosine:tRNA ligase, EC 6.1.1.1) was purified by ultracentrifugation, DEAE-cellulose chromatography and repeated phosphocellulose chromatography by more than 1500-fold. The molecular weight of the enzyme was approx. 150 000 as determined by Sephadex G-200 gel filtration. On the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme consisted of two subunits, each of 68 000 daltons. We found the following Km values for the enzyme: 13 micrometer for tyrosine and 1.7 mM for ATP in the ATP:PPi exchange reaction and 13 micrometer for tyrosine, 210 micrometer for ATP and 0.14 micrometer for tRNATyr in the aminoacylation reaction. The rate of tyrosyl-tRNA synthesis was 50-fold lower than that of ATP:PPi exchange. Addition of a saturating amount of tRNA did not affect the rate of ATP:PPi exchange.

Purification and properties of tyrosyl tRNA synthetase of rat liver.

Purification of rat liver tyrosine tRNA synthetase yields two protein fractions A and B and both fractions are required for charging of tyrosine to tRNAtyr. Fraction B catalyzes the activation of tyrosine. Fractions A and B have been purified to near homogeneity and they are composed of single polypeptide chains of 62,000 daltons each. Gel filtration studies suggest a molecular weight of 120,000 for the synthetase.