Ligand Binding and Enzymic Catalysis Coupled through Subunits in Tyros yl - t RNA

The interaction of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus with its substrates in the aminoacyl adenylation reaction has been studied by stopped-flow fluorescence. The observed changes have been assigned to their chemical and physical processes by comparison with equilibrium dialysis, pyrophosphate exchange kinetics and rapid quenching and sampling techniques to give the rate constants for ligand binding, the formation of tyrosyl adenylate, and the reverse reaction. The stoichiometry of tyrosine and ATP binding in the catalytic process has been determined directly by equilibrium dialysis and equi I n the two preceding papers (Fersht, 1975; Fersht et al., 1975) it has been established that the tyrosyl-tRNA synthetase from Bacillus stearothermophilus rapidly forms one aminoacyl adenylate per dimer but the second site is only weakly active if at all. A general mechanism was postulated that could account for this type of behavior (Fersht, 1975). In this context we present a kinetic analysis of the formation of the aminoacyl adenylate using stopped-flow fluorescence, quenched flow, and conventional techniques. Holler and Calvin (1 972) used the fluorescence of an external probe to study the pre-steady-state kinetics of the isoleucyl activating enzyme from Escherichia coli. Blanquet et al. (1 972) have shown that the tryptophanyl fluorescence of the methionyl-enzyme may be used to monitor. ligand binding. In the present study the fluorescent changes that occur on mixing the tyrosyl-enzyme with ATP and tyrosine and the aminoacyl adenylate complex with pyrophosphate are directly correlated with ligand binding and chemical processes by comparison with more direct methods. It is also shown how the stoichiometry of ligand binding during the catalytic process may be determined by equilibrium dialysis or equilibrium gel filtration under the conditions of the pyrophosphate exchange reaction, i.e., where there is no net turnover of substrate, but the system is at equilibrium (or steady state) and the equilibrium position favors starting materials. This is an analogous procedure to that used to study the binding of a substrate to chymotrypsin by Xray diffraction (the “equilibrium” method, Fersht and Renard, 1974). Experimental Section Materials and apparatus have been described by Fersht et a / . (1 975) and Fersht (1 975). The quenched-flow apparatus will be described elsewhere. Methods. All experiments were performed under standard conditions of 25 f 0 . lo , Tris-C1 (144 mM, p = 0.1) (pH 7.78) and 10 mM MgCl2 unless otherwise stated.