Kinetics and thermodynamics of the interaction of elongation factor Tu with elongation factor Ts, guanine nucleotides, and aminoacyl-tRNA.
The exchange of elongation factor Tu (EF-Tu)-bound GTP in the presence and absence of elongation factor Ts (EF-Ts) was monitored by equilibrium exchange kinetic procedures. The kinetics of the exchange reaction were found to be consistent with the formation of a ternary complex EF-Tu X GTP X EF-Ts. The equilibrium association constants of EF-Ts to the EF-Tu X GTP complex and of GTP to EF-Tu X EF-Ts were calculated to be 7 X 10(7) and 2 X 10(6) M-1, respectively. The dissociation rate constant of GTP from the ternary complex was found to be 13 s-1. This is 500 times larger than the GTP dissociation rate constant from the EF-Tu X GTP complex (2.5 X 10(-2) s-1). A procedure based on the observation that EF-Tu X GTP protects the aminoacyl-tRNA molecule from phosphodiesterase I-catalyzed hydrolysis was used to study the interactions of EF-Tu X GTP with Val-tRNAVal and Phe-tRNAPhe. Binding constants of Phe-tRNAPhe and Val-tRNAVal to EF-Tu X GTP of 4.8 X 10(7) and 1.2 X 10(7)M-1, respectively, were obtained. The exchange of bound GDP with GTP in solution in the presence of EF-Ts was also examined. The kinetics of the reaction were found to be consistent with a rapid equilibrium mechanism. It was observed that the exchange of bound GDP with free GTP in the presence of a large excess of the latter was accelerated by the addition of aminoacyl-tRNA. On the basis of these observations, a complete mechanism to explain the interactions among EF-Tu, EF-Ts, guanine nucleotides, and aminoacyl-tRNA has been developed.
D006150:Guanine Nucleotides / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D010444:Peptide Elongation Factor Tu; D010445:Peptide Elongation Factors / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012996:Solutions; D013816:Thermodynamics
D006150:Guanine Nucleotides; D010445:Peptide Elongation Factors; D012346:RNA, Transfer, Amino Acyl; D012996:Solutions; C023512:elongation factor Ts; D010444:Peptide Elongation Factor Tu