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

Abstract 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 FS1). Isoleucine (Ile) binds to the E·ATP complex (K 4 = 1.7 ± 0.9 μm) ∼35-fold more tightly than to E(K 1 = 50–100 μm), primarily due to a reduction in the Ile dissociation rate constant (k -1 ≈ 100–150 s−1,cf. k -4 = 3 ± 1.5 s−1). Similarly, ATP binds more tightly toE·Ile (K 3 = ∼70 μm) than to E (K 2 = ∼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 = ∼60 s−1) and the reverse rate constant (k -5 = ∼150–200 s−1) to be determined from pyrophosphorolysis of a pre-formed E·Ile-AMP complex (K 6= ∼0.25 mm). Scheme FS1 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 FS1could also provide an adequate description of the kinetics of tRNA aminoacylation (k cat,tr = ∼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.