Sm. Sullivan et al., Analysis of guanine nucleotide binding and exchange kinetics of the Escherichia coli GTPase Era, J BACT, 182(12), 2000, pp. 3460-3466
Era is an essential Escherichia coli guanine nucleotide binding protein tha
t appears to play a number of cellular roles. Although the kinetics of Era
guanine nucleotide binding and hydrolysis have been described, guanine nucl
eotide exchange rates have never been reported. Here we describe a kinetic
analysis of guanine nucleotide binding, exchange, and hydrolysis by Era usi
ng the fluorescent mant (N-methyl-3'-O-anthraniloyl) guanine nucleotide ana
logs. 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 u
nmodified 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 M
gCl2 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 r
apidly (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.