Substitution of aspartic acid-80, a residue involved in coordination of magnesium, weakens the GTP binding and strongly enhances the GTPase of the G domain of elongation factor Tu.

The functional role of Asp80, a residue involved in the coordination of the Mg(2+).guanine nucleotide complex in elongation factor Tu (EF-Tu), has been investigated by its substitution with Asn in the isolated N-terminal domain (G domain). The G domain D80N is characterized by a strong decrease in binding affinity for GTP and magnesium, whereas the affinity for GDP is unchanged. This effect can be mimicked in wild-type G domain by the addition of EDTA. In contrast to this, EDTA does not essentially influence the selective effects of the mutation on the GTP and GDP binding of G domain D80N, indicating that the action of Asp80 is mainly mediated by the GTP-coordinated magnesium ion. The GTPase activity of the G domain D80N is very unstable, but can be markedly stabilized by the addition of glycerol without essentially modifying the specific effects of the mutation. In the absence of glycerol G domain D80N can express a short-lived GTPase activity. The presence of glycerol transforms this evanescent activity into a linear multiple-round activity that under optimal conditions can be almost 2 orders of magnitude higher than the GTPase of wild-type G domain. This enhanced catalytic activity represents the most striking consequence of the mutation and stresses the key role of Asp80 in the GTPase of EF-Tu.(ABSTRACT TRUNCATED AT 250 WORDS)