Determination of eukaryotic peptidyltransferase activity by pseudo-first-order kinetic analysis.
We have developed an in vitro system for the determination of peptidyltransferase activity in rabbit reticulocyte ribosomes. Using this system, a detailed kinetic analysis of a model reaction for peptidyltransferase is described, with AcPhe-tRNA as the peptidyl donor and puromycin as the acceptor. The [AcPhe-tRNA-poly(U)-80S ribosome] complex (complex C) is isolated and then reacted with excess puromycin to give AcPhe-puromycin. This reaction (puromycin reaction) follows first-order kinetics at all concentrations of puromycin tested. At saturating concentrations of puromycin, the first-order rate (k3) constant is identical to the catalytic rate constant (kcat) of peptidyltransferase. This k3 of peptidyltransferase is equal to 2.9 min-1 at 37 degrees C. Moreover, the ratio k3/ Ks, which is an accurate measure of peptidyltransferase activity, was increased 80-fold when salt-washed ribosomes were replaced by unwashed ribosomes. Finally, the puromycin reaction was inhibited by several well-known antibiotics acting on the eukaryotic peptidyltransferase.
Ioannou
M
M
Department of Biochemistry, School of Medicine, University of Patras, Greece.
Coutsogeorgopoulos
C
C
Drainas
D
D
D000818:Animals; D000900:Anti-Bacterial Agents; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D010458:Peptidyl Transferases / Q000032:analysis* / Q000097:blood / Q000378:metabolism*; D011072:Poly U; D011500:Protein Synthesis Inhibitors; D011691:Puromycin; D012346:RNA, Transfer, Amino Acyl; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology; D012270:Ribosomes / Q000201:enzymology; D012996:Solutions
D000900:Anti-Bacterial Agents; D011500:Protein Synthesis Inhibitors; D012346:RNA, Transfer, Amino Acyl; D012996:Solutions; C006383:tRNA, N-acetylphenylalanine-; D011072:Poly U; D011691:Puromycin; D010458:Peptidyl Transferases