Substitution of proline 82 by threonine induces autophosphorylating activity in GTP-binding domain of elongation factor Tu.

Mutation of Pro82 into Thr, a residue situated in the second element (D80CPG83) of the consensus sequence proposed to interact with GTP/GDP in GTP-binding proteins was introduced via site-directed mutagenesis in the isolated guanine nucleotide-binding domain (G domain) of elongation factor Tu. G domainPT82 displays virtually no GTPase activity. As a major change, the apparent inhibition of the GTPase reaction is associated with the appearance of autophosphorylating activity, as in ras product p21 in the case of mutation Ala59----Thr, corresponding to 82 in elongation factor Tu. Dependence of this reaction on mono- and divalent cation concentration and on pH is essentially the same as for the GTPase of wild-type G domain. The autokinase reaction follows an apparent first order rate, suggesting an intermolecular mechanism. Analysis of amino acid and peptide composition of the 32P-labeled G domainPT82, as well as Edman degradation of the tryptic peptide containing the covalently bound 32P, shows that Thr82 is the phosphorylated residue. Taken together, these results point out that Thr82 is in close proximity to the gamma-phosphate of GTP, as in the case of Thr59 in p21. These results are in agreement with the observations derived from x-ray diffraction analysis that the tertiary structure of the GTP-binding domain of elongation factor Tu and that of p21 are similar.