Kinetic isotope effect analysis of the ribosomal peptidyl transferase reaction.
The ribosome is the macromolecular machine responsible for protein synthesis in all cells. Here, we establish a kinetic framework for the 50S modified fragment reaction that makes it possible to measure the kinetic effects that result from isotopic substitution in either the A or P site of the ribosome. This simplified peptidyl transferase assay follows a rapid equilibrium random mechanism in which the reverse reaction is nonexistent and the forward commitment is negligible. A normal effect (1.009) is observed for (15)N substitution of the incoming nucleophile at both low and high pH. This suggests that the first irreversible step is the formation of the tetrahedral intermediate. The observation of a normal isotope effect that does not change as a function of pH suggests that the ribosome promotes peptide bond formation by a mechanism that differs in its details from an uncatalyzed aminolysis reaction in solution. This implies that the ribosome contributes chemically to catalysis of peptide bond formation.
Seila
Amy C
AC
Department of Molecular Biophysics, Yale University, New Haven, Connecticut 06520-8114, USA.
Okuda
Kensuke
K
Núñez
Sara
S
Seila
Andrew F
AF
Strobel
Scott A
SA
D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D041961:Deuterium Exchange Measurement / Q000379:methods; D015226:Dinucleoside Phosphates / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D009587:Nitrogen Isotopes / Q000378:metabolism*; D010458:Peptidyl Transferases / Q000737:chemistry* / Q000378:metabolism*; D021122:Protein Subunits / Q000096:biosynthesis / Q000378:metabolism; D011522:Protons; D011691:Puromycin / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012269:Ribosomal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D015226:Dinucleoside Phosphates; D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; D021122:Protein Subunits; D011522:Protons; D012346:RNA, Transfer, Amino Acyl; D012269:Ribosomal Proteins; C090494:biotinyl-6-aminocaproyl-6-aminocaproyl-phenylalanyl-leucyl-phenylalanyl-diphenylphosphonate; C019037:tRNA, peptidyl-; D011691:Puromycin; C017849:cytidylyl-3'-5'-(2'(3')-O-L-phenylalanyl)-L-adenosine; D001710:Biotin; D010458:Peptidyl Transferases