Inactivation of Escherichia coli elongation factor Tu by the arginine-specific reagent butanedione.

Elongation factor T, (EF-T,) is inactivated by the arginine-specific reagent 2,3-butanedione. Excess elongation factor T, (EF-T,), the protein substrate for EFT,, protects EF-T, from this inactivation. Borate ion affects the inactivation kinetics in a manner consistent with the formation of an arginine residueebutanedione l borate complex. The butanedione inactivation is rapidly reversible in the absence of borate ion by simple dilution, Sephadex G-25 gel filtration, or dialysis, but is irreversible in the presence of millimolar borate concentrations. Butanedione-containing solutions of EF-T, rapidly establish an equilibrium between free, active EF-T, and inactive EF-T,=butanedione complex that is described by a dissociation constant of 3.3 + 0.7 mM at pH 8.0. The addition of a borate molecule to the butanedione-inactivated protein showed a dissociation constant of 120 rf: 50 PM. A reaction order for butanedione uptake by active EF-T, of 0.8 f 0.1 mol of reagent/ mol of EF-T, was derived under pseudo-first order conditions from inactivation rate constants. The pH profile of these rate constants showed that the protein active site becomes increasingly available for attack at more alkaline pH values irrespective of the presence of borate. Amino acid analysis of partially inactivated EF-T. showed the loss of 2.3 + 0.2 arginine residues/molecule of EF-T, when extrapolated to 100% loss of enzymatic activity. On the other hand, inactivation of EF-T. by butanedione does not affect the reactivity toward Nethylmaleimide of the cysteine of EF-T, which is the only other residue known to be essential to its interaction with EF-T,. We conclude that EF-T. contains 2 reactive a&nine residues, one of which is essential to and protected by its interaction with EF-T,.