Characterization of isoleucyl-tRNA synthetase from Staphylococcus aureus. I: Kinetic mechanism of the substrate activation reaction studied by transient and steady-state techniques.
The kinetic mechanism for the amino acid activation reaction of Staphylococcus aureus isoleucyl-tRNA synthetase (IleRS; E) has been determined from stopped-flow measurements of the tryptophan fluorescence associated with the formation of the enzyme-bound aminoacyl adenylate (E.Ile-AMP; Scheme 1). Isoleucine (Ile) binds to the E.ATP complex (K4 = 1.7 +/- 0.9 microM) approximately 35-fold more tightly than to E (K1 = 50-100 microM), primarily due to a reduction in the Ile dissociation rate constant (k-1 approximately 100-150 s-1, cf. k-4 = 3 +/- 1.5 s-1). Similarly, ATP binds more tightly to E.Ile (K3 = approximately 70 microM) than to E (K2 = approximately 2.5 mM). The formation of the E.isoleucyl adenylate intermediate, E.Ile-AMP, resulted in a further increase in fluorescence allowing the catalytic step to be monitored (k+5 = approximately 60 s-1) and the reverse rate constant (k-5 = approximately 150-200 s-1) to be determined from pyrophosphorolysis of a pre-formed E.Ile-AMP complex (K6 = approximately 0.25 mM). Scheme 1 was able to globally predict all of the observed transient kinetic and steady-state PPi/ATP exchange properties of IleRS by simulation. A modification of Scheme 1 could also provide an adequate description of the kinetics of tRNA aminoacylation (kcat,tr = approximately 0.35 s-1) thus providing a framework for understanding the kinetic mechanism of aminoacylation in the presence of tRNA and of inhibitor binding to IleRS.
Pope
A J
AJ
Department of Molecular Recognition, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park North, Harlow, Essex, United Kingdom. Andrew_J_Pope@SBPHRD.COM
Lapointe
J
J
Mensah
L
L
Benson
N
N
Brown
M J
MJ
Moore
K J
KJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D007532:Isoleucine / Q000378:metabolism; D007533:Isoleucine-tRNA Ligase / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D013050:Spectrometry, Fluorescence; D013211:Staphylococcus aureus / Q000201:enzymology*
D007532:Isoleucine; D000255:Adenosine Triphosphate; D007533:Isoleucine-tRNA Ligase