Site-directed mutagenesis of Arg58 and Asp86 of elongation factor Tu from Escherichia coli: effects on the GTPase reaction and aminoacyl-tRNA binding

Elongation factor Tu from Escherichia coli was mutated separatelyat positions Asp86 and Arg58, in order to shed light both onthe GTPase mechanism of elongation factor Tu and on the bindingof aminoacyl-tRNA. In addition, the binding of guanine nucleotideswas investigated by determination of the dissociation and associationrate constants. The results imply that Arg58 is unimportantfor the intrinsic GTPase mechanism and the binding of guaninenucleotides, whereas it is strongly involved in the bindingof aminoacyl-tRNA and of the ribosome. Asp86 appears to be essentialfor the regulation of guanine-nucleotide affinities, and itmay also play a role in the intrinsic GTPase mechanism.