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.
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, Ki = 50 nM and Ile-NHSO2-AMP, Ki = 1 nM;). PS-A (J) inhibits IleRS via a slow-tight binding competitive mechanism where E.J (Kj = approximately 2 nM), undergoes an isomerization to form a stabilized E*.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] >> Km, thus raising K*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.
Pope
A J
AJ
Department, SmithKline Beecham, New Frontiers Science Park, Harlow, Essex, United Kingdom. Andrew_J_Pope@SBPHRD.COM
Moore
K J
KJ
McVey
M
M
Mensah
L
L
Benson
N
N
Osbourne
N
N
Broom
N
N
Brown
M J
MJ
O'Hanlon
P
P
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D007532:Isoleucine / Q000378:metabolism; D007533:Isoleucine-tRNA Ligase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D016712:Mupirocin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D013211:Staphylococcus aureus / Q000201:enzymology*; D013329:Structure-Activity Relationship
D000900:Anti-Bacterial Agents; D007532:Isoleucine; D016712:Mupirocin; D007533:Isoleucine-tRNA Ligase