Non-equivalence of the sites of yeast phenylalanyl-tRNA synthetase during catalysis.
Yeast phenylalanyl-tRNA synthetase, an enzyme with an alpha2beta2 structure, has two active sites for phenylalanine, tRNAphe, phenylalanyladenylate and phenylalanyl-tRNAphe. Determination of phenylalanine binding properties to the free enzyme by equilibrium dialysis shows that only one mole of amino acid binds per mole of enzyme, i.e. absolute negative cooperativity. Binding of the amino acid in the presence of tRNA or of ATP and PPi unmasks the second phenylalanine binding site. The difference between the affinities at the tight and loose binding sites under such conditions is about 10--15. Titration of phenylalanyladenylate sites by the burst of ATP consumption shows the formation of a (enzyme-phenylalanyladenylate)2 complex in the presence of pyrophosphatase; however, the two sites differ widely in their affinity as shown by dialysis experiments. Measurements of hydrolysis rates of enzyme-bound phenylalanyladenylate suggests that when only the high-affinity adenylate site is occupied, the other protomer can still bind phenylalanine and ATP (in the presence of phenylalanine). Two moles of Phe-tRNAphe bind to the enzyme with a very high affinity (Kd less than 48 nM). The presence of millimolar concentrations of ATP, phenylalanine and pyrophosphate triggers negative cooperativity and under these conditions only one mole of Phe-tRNAphe is bound per mole of enzyme with a Kd value of 0.15 muM. The present results give support to interprotomer catalytic cooperativity in the mechanism of action of yeast phenylalanyl-tRNA synthetase.
D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D001665:Binding Sites; D007700:Kinetics; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism; D011485:Protein Binding; D011755:Pyrophosphatases; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000255:Adenosine Triphosphate; D011755:Pyrophosphatases; D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase