Effects of the ligands of beef tryptophanyl-tRNA synthetase on the elementary steps of the tRNATrp aminoacylation

Publisher Website
Google Scholar
Abstract
Tryptophanyl-tRNA synthetase catalyzed formation of TRp-tRNATrp has been studied by mixing tRNATrp with a preformed bis(tryptophanyl adenylate)-enzyme complex in the 0-60-ms time range, on a quenched-flow apparatus. Analyzing the data gives an association rate constant ka = (1.22 .+-. 0.47) .times. 108 M-1 s-1, a dissociation rate constant kd = 143 .+-. 73 s-1, and a dissociation constant Kd = 1.34 .+-. 0.80 .mu.M for tRNATrp. The maximum rate constant of tryptophan transfer to tRNATrp is kt = 33 .+-. 3 s-1. When starting the aminoacylation reaction with a mono (tryptophanyl adenylate)-enzyme complex, one obtains different kinetic profiles than when using a bis(trytophanyl adenylate)-enzyme complex. Over a 0-400-ms time range, the monoadenylate-enzyme complex yields an apparent first-order reaction, while the bis-adenylate-enzyme complex yields a biphasic aminoacylation of tRNATrp. Analysis of Trp-tRNATrp formation from both complexes according to simple raction schemes shows that the dissociation of tRNATrp from an enzyme subunit carrying no adenylate is 6.9-fold slower than from an enzyme subunit carrying an adenylate. The apparent rate constant of dissociation of nascent tryptophanyl-tRNATrp is 4.9 s-1 in the absence of free tryptophan, which is mush slower than its rate of formation (33 s-1). Study of the concentration dependence of the enzyme-catalyzed hydrolysis of preformed Trp-tRNATrp yields of Kd of 296 .+-. 44 nM for this derivative, corresponding to a rate constant of dissociation of the order of 36 s-1, when it is assumed that its rate constant of association with the enzyme is the same as that of tRNATrp. These data are interpreted as showing that a rate-limiting conformation change of the enzyme-(nascent tryptophanyl-tRNATrp) complex follows the formation of Trp-tRNATrp and precedes its dissociation. Study of the ATP-PPi isotopic exchange catalyzed by the enzyme in the presence of tRNATrp shows that tRNATrp is a noncompetitive inhibitor of tryptophanyl adenylate formation. The maximum inhibition factor is either 33% when one considers that tRNA inhibits both subunits or 66% when one considers that it inhibits only the subunit on which it is bound. The data are discussed in light of the half-of-the-sites reactivity of the enzyme an d of the activatory effect of trypotophan on the overall aminoacylation of tRNATrp [Trezequet, V., Merle, M., Gandar, J. C., and Labouesse, B. (1986) Biochemistry 25, 7125-7136]. They lead to the conclusion that at saturating substrate concentration there are four rate-determining steps, tryptophan activation, tryptophan transfer to tRNATrp, a conformation change following tRNATrp aminoacylation, and Trp-tRNATrp dissociation, while at low tryptophan concentration the conformation change following Trp-tRNATrp aminoacylation becomes a major rate-limiting step.

This publication has 27 references indexed in Scilit: