BINDING OF ZINC TO ESCHERICHIA-COLI PHENYLALANYL TRANSFER RIBONUCLEIC-ACID SYNTHETASE - COMPARISON WITH OTHER AMINOACYL TRANSFER RIBONUCLEIC-ACID SYNTHETASES

The Zn content and the effect of exogenous Zn on enzymatic activities were explored in a series of aminoacyl-tRNA synthetases representing the various structural classes in this family of enzymes. The following situations can be distinguished. Homogeneous E. coli tyrosyl-tRNA synthetase, an .alpha.2 enzyme of MW 2 .times. 45K [kilodaltons], does not contain Zn. Its tRNA aminoacylation activity is insensitive to the addition of metal complexing agents and to the addition of up to 60 .mu.M Zn2+. Homogeneous E. coli isoleucyl-tRNA synthetase, an .alpha. enzyme (102K), has nearly 1 Zn atom strongly bound/polypeptide chain. The tRNA aminoacylation activity of this enzyme is inhibited by 1,10-phenanthroline but remains unaffected by the addition of 60 .mu.M Zn2+ in the assay. Homogeneous E. coli and Bacillus stearothermophilus methionyl-tRNA synthetases, dimers of MW 2 .times. 85K, each contain 1 Zn atom strongly bound/enzyme subunit. In the case of the E. coli or the B. stearothermophilus enzyme, the bound Zn is retained upon proteolytic modification. The tRNA aminoacylation activity of the E. coli enzyme is inhibited by 1,10-phenanthroline but is insensitive to the addition of Zn2+. Homogeneous E. coli phenylalanyl-tRNA synthetase, an .alpha.2.beta.2 enzyme, has no Zn strongly bound. Its tRNA aminoacylation activity is impaired by the addition of Zn2+ to the assay. A similar effect of Zn2+ can be observed in the L-phenylalanine-dependent isotopic ATP-PPi exchange reaction. The inhibition is fully reversible by the addition of Zn complexing agents. The effect of Zn occurs through direct binding of the metal ion to the enzyme. A stoichiometry of 8 Zn atoms/.alpha.2.beta.2 enzyme was determined by equilibrium dialysis and protein fluorescence measurements. The corresponding apparent affinity constant is equal to 0.8 .times. 106 M-1 (pH 8.0, 4.degree. C) in 150 mM KCl plus 7 mM MgCl2. A parallel effect of Zn is observed on the tRNA aminoacylation activity of yeast phenylalanyl-tRNA synthetase. The aminoacylation activity of E. coli glycyl-tRNA synthetase, another .alpha.2.beta.2 enzyme, appears unaffected by the presence of up to 20 .mu.M ZnCl2. In the accompanying paper (Plateau, P., et al., 1981), it is shown that important synthesis of diadenosine 5'',5",P1,P4-tetraphosphate by E. coli and yeast phenylalanyl-tRNA synthetase can be triggered in the presence of Zn.