PMID:10852878
| Citation |
Sullivan, SM, Mishra, R, Neubig, RR and Maddock, JR (2000) Analysis of guanine nucleotide binding and exchange kinetics of the Escherichia coli GTPase Era. J. Bacteriol. 182:3460-6 |
|---|---|
| Abstract |
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. |
| Links | |
| Keywords |
Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins; GTP-Binding Proteins/genetics; GTP-Binding Proteins/isolation & purification; GTP-Binding Proteins/metabolism; Guanosine Diphosphate/analogs & derivatives; Guanosine Diphosphate/metabolism; Guanosine Triphosphate/analogs & derivatives; Guanosine Triphosphate/metabolism; Histidine/chemistry; Hydrolysis; Kinetics; RNA-Binding Proteins; Time Factors; ortho-Aminobenzoates/metabolism |
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