Effects of substrate and inhibitor binding on proteolysis of isoleucyl-tRNA synthetase from Staphylococcus aureus.
Binding of ligands to isoleucyl-tRNA synthetase (IleRS; E) from Staphylococcus aureus was investigated through effects on proteolytic digestion. Approximately 50-fold higher concentrations of protease (trypsin or chymotrypsin) were required to inactivate IleRS after incubation with substrates and formation of the E. Ile-AMP intermediate compared with free E. Binding of pseudomonic acid A (PS-A) or isoleucynol adenylate (Ile-ol-AMP) also induced resistance to proteolysis and altered the patterns of IleRS cleavage fragments in an inhibitor-class specific manner. The determinants for PS-A binding were investigated via proteolysis of E.[3H]PS-A. Limited proteolysis of E.[3H]PS-A (excising residues 186-407) could be achieved without significant loss of bound inhibitor, eliminating this region as contributing to inhibitor binding. Assays were developed which allowed IleRS proteolysis to be readily followed using fluorescence polarization. Inhibitor-protected IleRS was labeled with fluorescein isothiocyanate with only a small effect upon catalytic activity (Fl-IleRS). The (pseudo) kinetics of proteolytic cleavage of Fl-IleRS could be measured at low nanomolar Fl-IleRS concentrations in 96/384-well microtiter plates, allowing real-time monitoring of dose-dependent protection from proteolysis. Thus, inhibitor (and substrate) binding could be reproducibly assessed in the absence of measurements of catalytic acitvity. This could potentially form the basis of novel screening assays for ligands to other proteins.
Pope
A J
AJ
Department of Molecular Recognition, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park North, Harlow, Essex, CM195AW United Kingdom. Andrew_J_Pope@SBPHRD.COM
McVey
M
M
Fantom
K
K
Moore
K J
KJ
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D002918:Chymotrypsin / Q000378:metabolism*; D007532:Isoleucine / Q000378:metabolism*; D007533:Isoleucine-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D013050:Spectrometry, Fluorescence; D013211:Staphylococcus aureus / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism*
D007532:Isoleucine; D000255:Adenosine Triphosphate; D002918:Chymotrypsin; D014357:Trypsin; D007533:Isoleucine-tRNA Ligase