Demonstration of two active sites on a monomeric aminoacyl-tRNA synthetase. Possible roles of negative cooperativity and half-of-the-sites reactivity in oligomeric enzymes.
The dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus which binds (tightly) only one tyrosyl adenylate or tyrosine per dimer is shown from kinetic, equilibrium dialysis, and gel filtration methods to have a second active site. ATP and tyrosine bind strongly and synergistically to the tyrosyl-tRNA synthetase tyrosyl adenylate complex, [E with Tyr similar to AMP], to give the complex [E with Tyr similar to AMP,ATP,Tyr]. This complex probably slowly forms an [E with (Tyr similar to AMP)2] complex which hydrolyses rapidly and does not accumulate. Similarly, the monomeric valyl-enzyme is shown to have two active sites. An [E with Val similar AMP,ATP,Val] complex is formed which probably slowly gives an unstable [E with Val similar AMP)2] complex. In view of this and the recent demonstrations that several aminoacyl-tRNA synthetases are composed of repeating sequences it is suggested that all of these enzymes have at least two active sites. The second site is difficult to detect by normal steady-state kinetic measurements and binding assays as these enzymes exhibit negative cooperativity of substrate binding hand half-of-the sites reactivity. A mechanism based on interacting sites is proposed that could account for these observations: changes in binding energy at one site may be coupled with catalysis at the other to give large rate enhancements. Howeever, this cannot account for the high specificity in the acylation of tRNA, A "VERIFICATION" PROCEDURE SEEMS ESSENTIAL. The proposed mechanism is quite general for catalysis and could be a reason why so many nonregulatory enzymes have subunits.
D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology; D001665:Binding Sites; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D010761:Phosphorus Radioisotopes; D011485:Protein Binding; D014443:Tyrosine; D014633:Valine
D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D000249:Adenosine Monophosphate; D014443:Tyrosine; D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases; D014633:Valine