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

被引:55
作者
Pope, AJ
Moore, KJ
McVey, M
Mensah, L
Benson, N
Osbourne, N
Broom, N
Brown, MJB
O'Hanlon, P
机构
[1] SmithKline Beecham Pharmaceut, Mol Interact & New Assay Technol, Dept Mol Recognit, Harlow CM19 5AW, Essex, England
[2] SmithKline Beecham Pharmaceut, Dept Med Chem, Harlow CM19 5AW, Essex, England
关键词
D O I
10.1074/jbc.273.48.31691
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 = similar to 2 nM), undergoes an isomerization to form a stabilized E.*J complex (K-j* = 50 pM). To overcome tight-binding artifacts when K-j* much less than [E], K-j* values were estimated from PPi/ATP exchange where [S] much greater than K-m, thus raising K-J,K-app* well above [E]. Using [H-3]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.
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页码:31691 / 31701
页数:11
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