SUBSTITUTION OF HISTIDINE-84 AND THE GTPASE MECHANISM OF ELONGATION FACTOR-TU

Mutation of His84, a residue situated in one of the loops forming the guanine nucleotide binding pocket, was introduced in the G domain, the isolated N-terminal half molecule of bacterial elongation factor Tu (EF-Tu), in order to investigate the role of this residue on the basic activities of EF-Tu: the interaction with GDP and GTP and the hydrolysis of GTP. Substitution of His84 by Gly reduces the GTPase activity of the G domain to 5%; this activity can still be stimulated by raising the KCI concentration as the activity of wild-type G domain or the intact molecule. Since the affinites of the mutant protein for GDP and GTP are essentially the same as those of the wild-type G domain, His84 is apparently not involved in the binding of the substrates. Calculations of the change in free energy of activation of the GTPase reaction following substitution of His84 by Gly point to the disruption of a weak hydrogen bond, involved in the catalytic reaction. This probably concerns an interaction via a water molecule. The possible mechanism underlying the GTPase reaction is discussed in light of the three-dimensional structure of EF-Tu, taking into account the situation of Ha-ras p21.