New aspects on the kinetics of activation of ribosomal peptidyltransferase-catalyzed peptide bond formation by monovalent ions and spermine.
The effect of NH4+ and K+ ions on the activity of ribosomal peptidyltransferase was investigated in a model system derived from Escherichia coli, in which AcPhe-puromycin is produced by a pseudo-first-order reaction between the preformed AcPhe-tRNA-poly(U)-ribosome complex (complex C) and excess puromycin. Detailed kinetic analysis suggests that both NH4+ and K+ ions act as essential activators of peptidyltransferase by filling randomly, but not cooperatively, multiple sites on the ribosome. With respect to the NH4+ effect at 25 degrees C. the values of the molecular interaction coefficient (n), the dissociation constant (KA), and the apparent catalytic rate constant (kmax) of peptidyltransferase at saturating levels of NH4+ and puromycin are 1.99, 268.7 mM and 24.8 min(-1), respectively. The stimulation of peptidyltransferase by K+ ions at 25 degrees C (n = 4.38, KA = 95.5 mM, kmax = 9.6 min[-1]) is not as marked as that caused by NH4+ ions. Furthermore, it is evident that NH4+ at high concentration (200 mM) is effective in filling regulatory sites of complex C, which are responsible for the modulatory effect of spermine. The combination of NH4+ ions (200 mM) with spermine (300 microM) produces an additive increase in peptidyltransferase activity. Taken together, these findings suggest the involvement of two related pathways in the regulation of peptidyltransferase activity, one mediated by specific monovalent cations and the other mediated by spermine.
Michelinaki
M
M
Department of Biochemistry, School of Medicine, University of Patras, Greece.
Spanos
A
A
Coutsogeorgopoulos
C
C
Kalpaxis
D L
DL
D002384:Catalysis; D002414:Cations, Monovalent; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D010455:Peptides / Q000378:metabolism*; D010458:Peptidyl Transferases / Q000187:drug effects / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism* / Q000494:pharmacology; D011691:Puromycin / Q000096:biosynthesis; D000644:Quaternary Ammonium Compounds / Q000378:metabolism* / Q000494:pharmacology; D012346:RNA, Transfer, Amino Acyl / Q000187:drug effects / Q000378:metabolism; D012270:Ribosomes / Q000201:enzymology*; D013096:Spermine / Q000378:metabolism* / Q000494:pharmacology
D002414:Cations, Monovalent; D010455:Peptides; D000644:Quaternary Ammonium Compounds; D012346:RNA, Transfer, Amino Acyl; C066516:tRNA, N-acetylphenylalanyl-phenylalanine-; D013096:Spermine; D011691:Puromycin; D010458:Peptidyl Transferases; D011188:Potassium