Isoleucyl transfer ribonucleic acid synthetase. Steady-state kinetic analysis

A steady-state kinetic analysis was conducted of the overall aminoacylation reaction catalyzed by isoleucyl-tRNA synthetase. The patterns of Lineweaver-Burk plots obtained indicated that tRNA adds to the enzyme only after isoleucyl adenylate formation and pyrophosphate release. These kinetic patterns were consistent with the bi-uni-uni-bi Ping Pong mechanism generally accepted for this aminoacyl-tRNA synthetase, but they could also be accommodated by a mechanism in which a second molecule of L-isoleucine added to the enzyme between isoleucyl adenylate formation and aminoacylation of tRNA [Fersht, A.R., & Kaethner, M.M. (1976) Biochemistry 15, 818]. The values of the kinetic parameters favor the latter mechanism. The results of this kinetic analysis indicated that the affinity of isoleucyl-tRNA synthetase for Mg.ATP was enhanced upon binding of L-isoleucine and vice versa. It also indicated that the affinity of the enzyme for L-isoleucine is decreased upon binding tRNA and vice versa. The values of dissociation constants calculated for each of the substrates by this study generally compared well with those determined by other authors using a variety of kinetic and equilibrium methods.