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 microgM and 3.6 +/- 0.80 microM, 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.
Sullivan
S M
SM
Department of Biology, University of Michigan, Ann Arbor 48109, USA.
Mishra
R
R
Neubig
R R
RR
Maddock
J R
JR
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D016601:RNA-Binding Proteins; D013997:Time Factors; D062367:ortho-Aminobenzoates / Q000378:metabolism
D029968:Escherichia coli Proteins; D016601:RNA-Binding Proteins; C068027:era protein, E coli; D062367:ortho-Aminobenzoates; C065197:3'-(methylanthraniloyl)-2'-deoxy-guanosine diphosphate; D006153:Guanosine Diphosphate; D006639:Histidine; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins