Effects of Substrate and Inhibitor Binding on Proteolysis of Isoleucyl-tRNA Synthetase from Staphylococcus aureus

Abstract

Pseudomonic acid A (PS-A)1 is a potent antibiotic that acts through inhibition of bacterial isoleucyl-tRNA synthetase (IleRS, E) (1, 2). As part of an effort to understand the precise mechanism of the interaction of PS-A and its analogues with IleRS we have undertaken a detailed characterization of the kinetics and thermodynamics of substrate and inhibitor binding by this enzyme (3, 4). These experiments showed that PS-A analogues are reversible, slow tight-binding inhibitors that form an highly stabilized inhibitor complex with overall dissociation constants in the picomolar range. Comparison of PS-A analogues with inhibitors that were non-hydrolyzable analogues of the normal activated enzyme intermdiate, Ile-AMP, showed that both classes of inhibitor bound to IleRS in a manner that was mutually exclusive with each other and with the substrates, L-isoleucine (Ile) and Mg⅐ATP, but that very different enzyme conformations were induced upon binding (as shown by differences in IleRS tryptophan fluorescence) (4). In this report, we describe experiments in which we attempted to gain additional information on the binding of substrates and inhibitors to IleRS using different approaches to the more classical techniques which we had previously employed (e.g. state-state and transient kinetics (3, 4)).One technique for investigating changes in the conformation of proteins as a result of ligand binding is to test their effect upon proteolysis (e.g. Ref. 5), and this has previously been shown to yield useful information on several aminoacyl-tRNA synthetases (e.g. Refs. 6 -9). Here, we used this approach in two distinct ways. First, the effect of substrate and inhibitor binding on IleRS proteolysis patterns was investigated in an attempt to identify protein determinants for binding. Second, a homogeneous real-time assay capable of monitoring ligand protection from IleRS proteolysis using fluorescence polarization was developed which could be potentially used to screen for new inhibitors.

Characterization of Isoleucyl-tRNA Synthetase from Staphylococcus aureus

“…Of course, Scheme 4 does not define the order of tRNA binding and dissociation which was not the purpose of the current study. However, since tRNA can bind to free IleRS with an affinity comparable to K m,tRNA Ј in the tRNA aminoacylation reaction (13), the most simple mechanism appears to involve tRNA binding throughout the reaction cycle as shown in Scheme 4. Although it is possible that the rather striking similarity of the tRNA aminoacylation kinetics predicted from Scheme 4 and of those observed experimentally (Fig.…”

“…Binding of Ile-ol-AMP (k ϩi and k Ϫi )-See below 2 for explanation of the notation used throughout this and accompanying (12,13) papers. The binding of excess Ile-ol-AMP to IleRS was monitored directly by rapid mixing of the enzyme and inhibitor in a stopped-flow apparatus ( Fig.…”

“…In this paper, we describe the construction of a minimal reaction mechanism for amino acid activation by IleRS which adequately describes all of the steady-state and transient kinetic properties of this enzyme. Accompanying papers describe a detailed characterization of the kinetics and mechanism of inhibitor binding (12), and an analysis of the effects of ligand binding upon proteolysis of IleRS (13).…”

A Homogeneous Method to Measure Aminoacyl-tRNA Synthetase Aminoacylation Activity Using Scintillation Proximity Assay Technology

“…To confirm the validity of the SPA method for the determination of activation and inhibition constants for aaRS enzymes, we selected IleRS as a model system, since this enzyme has been studied in some detail in our laboratories (6,10,11). The effect of Ile and ATP on the substrate activation kinetics of S. aureus IleRS was determined using both SPA and filtration methods under equivalent assay conditions (Figs.…”

“…tRNA aminoacylation activity was either measured using modifications to standard methods (10,11,13) or the SPA method, the development of which is described in this paper. Recombinant aaRS stocks were diluted, just before use, in 50 mM Tris-HCl, pH 7.9, containing 2 mM DTT and 0.3 mg/ml BSA.…”