Characterization of Isoleucyl-tRNA Synthetase from Staphylococcus aureus II. MECHANISM OF INHIBITION BY REACTION INTERMEDIATE AND PSEUDOMONIC ACID ANALOGUES STUDIED USING TRANSIENT AND STEADY-STATE KINETICS

Abstract The interactions of isoleucyl-tRNA synthetase (IleRS, E) from Staphylococcus aureus with both intermediate analogues and pseudomonic acid (PS-A) have been investigated using transient and steady-state techniques. Non-hydrolyzable analogues of isoleucyl-AMP (I) were simple competitive inhibitors (Ile-ol-AMP, K i = 50 nm and Ile-NHSO2-AMP, K i = 1 nm;). PS-A (J) inhibits IleRS via a slow-tight binding competitive mechanism where E·J (K j = ∼2 nm), undergoes an isomerization to form a stabilizedE *·J complex (K * j = 50 pm). To overcome tight-binding artifacts when K * j ≪ [E], K * j values were estimated from PPi/ATP exchange where [S] ≫K m, thus raisingK * j ,app well above [E]. Using [3H]PS-A, it was confirmed that binding occurs with 1:1 stoichiometry and is reversible. Formation of inhibitor complexes was monitored directly through changes in enzyme tryptophan fluorescence. For Ile-ol-AMP and Ile-NHSO2-AMP, the fluorescence intensity of E·I was identical to that when E·Ile-AMP forms catalytically. Binding of PS-A induced only a small change in IleRS fluorescence that was characterized using transient kinetic competition. SB-205952, a PS-A analogue, produced a 37% quenching of IleRS fluorescence upon binding as a result of radiationless energy transfer. Inhibitor reversal rates were obtained by measuring relaxation between spectroscopically different complexes. Together, these data represent a comprehensive solution to the kinetics of inhibition by these compounds.