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 Mg2+.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. Our results strongly suggest that the catalytically active conformation of EF-Tu.GTP is favored by a relief of the constraints imposed by the interactions between Asp80, Thr25, and Mg2+ and that the activators of the EF-Tu GTPase (ribosomes, kirromycin, and monovalent cations) act by influencing the coordination of Mg2+.GTP. This work illustrates the complexity of the structures controlling the binding and the cleavage of the gamma-phosphate of GTP and emphasizes the central role of Asp80 in the GTPase activity and in maintaining the structural integrity of the G domain.