Characterization of isoleucyl-tRNA synthetase from Staphylococcus aureus -: I:: Kinetic mechanism of the substrate activation reaction studied by transient and steady-state techniques

The kinetic mechanism for the amino acid activation reaction of Staphylococcus aureus isoleucyl-tRNA synthetase (IleRS; E) has been determined from stopped-flow measurements of the tryptophan fluorescence associated with the formation of the enzyme-bound aminoacyl adenylate (E.Ile-AMP; Scheme 1). Isoleucine (Ile) binds to the E.ATP complex (K-4 = 1.7 +/- 0.9 mu M) similar to 35-fold more tightly than to E (K-1 = 50-100 mu M), primarily due to a reduction in the Be dissociation rate constant (k(-1) approximate to 100-150 s(-1), cf. k(-4) = 3 +/- 1.5 s(-1)). Similarly, ATP binds more tightly to E.Ile (K-3 = similar to 70 mu M) than to E (K-2 = similar to 2.5 mM). The formation of the E.isoleucyl adenylate intermediate, E.Ile-AMP, resulted in a further increase in fluorescence allowing the catalytic step to be monitored (k(+5) = similar to 60 s(-1)) and the reverse rate constant (k(-5) = similar to 150-200 s(-1)) to be determined from pyrophosphorolysis of a pre-formed E.Ile-AMP complex (K-6 = similar to 0.25 mM). Scheme 1 was able to globally predict all of the observed transient kinetic and steady-state PPi/ATP exchange properties of IleRS by simulation. A modification of Scheme 1 could also provide an adequate description of the kinetics of tRNA aminoacylation (k(cat,tr) = similar to 0.35 s(-1)) thus providing a framework for understanding the kinetic mechanism of aminoacylation in the presence of tRNA and of inhibitor binding to IleRS.