Effects of the ligands of beef tryptophanyl-tRNA synthetase on the elementary steps of the tRNA(Trp) aminoacylation.
Tryptophanyl-tRNA synthetase catalyzed formation of Trp-tRNA(Trp) has been studied by mixing tRNA(Trp) 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) X 10(8) M-1 S-1, a dissociation rate constant kd = 143 +/- 73 S-1, and a dissociation constant Kd = 1.34 +/- 0.80 microM for tRNA(Trp). The maximum rate constant of tryptophan transfer to tRNA(Trp) 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(tryptophanyl 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 tRNA(Trp). Analysis of Trp-tRNA(Trp) formation from both complexes according to simple reaction schemes shows that the dissociation of tRNA(Trp) 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-tRNA(Trp) is 4.9 S-1 in the absence of free tryptophan, which is much slower than its rate of formation (33 S-1).(ABSTRACT TRUNCATED AT 250 WORDS)
Merle
M
M
Institut de Biochimie Cellulaire et Neurochimie--CNRS, Université de Bordeaux II, France.
Trezeguet
V
V
Gandar
J C
JC
Labouesse
B
B
D000215:Acylation; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000378:metabolism; D008433:Mathematics; D008962:Models, Theoretical; D010179:Pancreas / Q000201:enzymology; D012345:RNA, Transfer, Amino Acid-Specific / Q000378:metabolism*; D012364:RNA, Transfer, Trp / Q000378:metabolism*; D014369:Tryptophan-tRNA Ligase / Q000378:metabolism*
D008024:Ligands; D012345:RNA, Transfer, Amino Acid-Specific; D012364:RNA, Transfer, Trp; D000604:Amino Acyl-tRNA Synthetases; D014369:Tryptophan-tRNA Ligase