Phenylalanyl-tRNA synthetase of Escherichia coli K10. Effects of zinc(II) on partial reactions of diadenosine 5',5"'-P1,P4-tetraphosphate synthesis, conformation, and protein aggregation.
The synthesis of diadenosine 5',5"'-P1-,P4-tetraphosphate (Ap4A) catalyzed by phenylalanyl-tRNA synthetase in the presence of Zn2+ involves the same partial reactions (synthesis of phenylalanyladenylate and transfer of the adenylate moiety to ATP) as occur in the absence of this metal ion. However, transfer is strongly stimulated while adenylate synthesis is depressed. Also inhibited are pyrophosphorolysis of phenylalanyladenylate and transfer of phenylalanine from the adenylate to cognate tRNA, because overall tRNA phenylalanylation is depressed [Mayaux, J.-F., & Blanquet, S. (1981) Biochemistry 20, 4647-4654], whereas binding of tRNA to the synthetase is not. At moderate concentrations of Zn2+, and in the presence of 5 microM phenylalanine and 0.5 mM ATP, transfer of AMP is rate limiting, while at higher concentrations of Zn2+ synthesis of adenylate is rate determining. The Zn2+ concentration optimum for stimulation depends on the concentration of phenylalanine and ATP. The effects of Zn2+ are mediated through two classes of binding site(s) on the synthetase, the half-saturations of which are 1-4 and 20-30 microM Zn2+, respectively. Binding of Zn2+ to the second class of site(s) causes inhibition of the synthetase, whereas binding to the first class is responsible for activation and inhibition, which may be caused by a conformational change. Evidence for the latter is the observed decrease in protein intrinsic fluorescence intensity and the decrease in fluorescence intensity of 6-(p-toluidinyl)naphthalene-2-sulfonate, which is used as a reporter group. The kinetics of the binding reaction show a saturation dependence on Zn2+, also suggesting that a conformational change occurs.(ABSTRACT TRUNCATED AT 250 WORDS)
D000227:Adenine Nucleotides / Q000096:biosynthesis*; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D015226:Dinucleoside Phosphates; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D015032:Zinc / Q000494:pharmacology*
D000227:Adenine Nucleotides; D015226:Dinucleoside Phosphates; D046911:Macromolecular Substances; C020733:diadenosine tetraphosphate; D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase; D015032:Zinc