Studies on the catalytic rate constant of ribosomal peptidyltransferase.
A detailed kinetic analysis of a model reaction for the ribosomal peptidyltransferase is described, using fMet-tRNA or Ac-Phe-tRNA as the peptidyl donor and puromycin as the acceptor. The initiation complex (fMet-tRNA X AUG X 70 S ribosome) or (Ac-Phe-tRNA X poly(U) X 70 S ribosome) (complex C) is isolated and then reacted with excess puromycin (S) to give fMet-puromycin or Ac-Phe-puromycin. This reaction (puromycin reaction) is first order at all concentrations of S tested. An important asset of this kinetic analysis is the fact that the relationship between the first order rate constant kobs and [S] shows hyperbolic saturation and that the value of kobs at saturating [S] is a measure of the catalytic rate constant (k cat) of peptidyltransferase in the puromycin reaction. With fMet-tRNA as the donor, this kcat of peptidyltransferase is 8.3 min-1 when the 0.5 M NH4Cl ribosomal wash is present, compared to 3.8 min-1 in its absence. The kcat of peptidyltransferase is 2.0 min-1 when Ac-Phe-tRNA replaces fMet-tRNA in the presence of the ribosomal wash and decreases to 0.8 min-1 in its absence. This kinetic procedure is the best method available for evaluating changes in the activity of peptidyltransferase in vitro. The results suggest that peptidyltransferase is subjected to activation by the binding of fMet-tRNA to the 70 S initiation complex.
D000217:Acyltransferases / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008954:Models, Biological; D010455:Peptides / Q000378:metabolism; D010458:Peptidyl Transferases / Q000378:metabolism*; D011691:Puromycin / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012358:RNA, Transfer, Met; D012270:Ribosomes / Q000201:enzymology*
D010455:Peptides; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C006383:tRNA, N-acetylphenylalanine-; C024988:tRNA, formylmethionine-; D011691:Puromycin; D000217:Acyltransferases; D010458:Peptidyl Transferases