SINGLE TURNOVER KINETIC STUDIES OF GUANOSINE TRIPHOSPHATE HYDROLYSIS AND PEPTIDE FORMATION IN THE ELONGATION-FACTOR TU-DEPENDENT BINDING OF AMINOACYL-TRANSFER RNA TO ESCHERICHIA-COLI RIBOSOMES

Abstract

The rates of GTP hydrolysis and peptide formation during the reaction of Phe-tRNA.cntdot.elongation factor Tu.cntdot.GTP complex with acetyl-Phe-tRNA polyuridylate-programmed ribosomes were measured. The GTPase reaction is second-order up to reactant concentrations of 0.2 .mu.M, and has a rate constant of 5 .times. 106 M-1 s-1 at 5.degree. C and 5 mM Mg2+, pH 7.2. The formation of peptide shows a lag phase and has a rate constant of 0.4 s-1 under these conditions. The results of a series of experiments between 5.degree. C and 25.degree. C show that GTP hydrolysis and peptide formation have Arrhenius activation energies of 13.1 and 15.3 kcal mol-1, respectively. The results indicate that these reactions proceed in vitro at rates comparable to those observed for protein biosynthesis in vivo, and that peptide bond formation occurs after GTP hydrolysis.