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 mu M and 3.6 +/- 0.80 mu M, 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 MgCl2 and 5.6 +/- 0.3 mmol of mGTP hydrolyzed/min/mol in the presence of 0.2 mM MgCl2. Moreover, Era associates with and exchanges guanine nucleotide rapidly (on the order of seconds) in both the presence and absence of Mg2+. We suggest that models of Era function should reflect the rapid exchange of nucleotides in addition to the GTPase activity inherent to Era.