Analysis of Guanine Nucleotide Binding and Exchange Kinetics of the Escherichia coli GTPase Era

Era is an essential Escherichia coli guanine nucleotide binding protein that appears to play a number of cellular roles. Although the kinetics of Era guanine nucleotide binding and hydrolysis have been described, guanine nucleotide exchange rates have never been reported. Here we describe a kinetic analysis of guanine nucleotide binding, exchange, and hydrolysis by Era using the fluorescent mant (N-methyl-3′-O-anthraniloyl) guanine nucleotide analogs. The equilibrium binding constants (K(D)) for mGDP and mGTP (0.61 ± 0.12 μM and 3.6 ± 0.80 μM, respectively) are similar to those of the unmodified nucleotides. The single turnover rates for mGTP hydrolysis by Era were 3.1 ± 0.2 mmol of mGTP hydrolyzed/min/mol in the presence of 5 mM MgCl(2) and 5.6 ± 0.3 mmol of mGTP hydrolyzed/min/mol in the presence of 0.2 mM MgCl(2). Moreover, Era associates with and exchanges guanine nucleotide rapidly (on the order of seconds) in both the presence and absence of Mg(2+). We suggest that models of Era function should reflect the rapid exchange of nucleotides in addition to the GTPase activity inherent to Era.