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Journal of Bacteriology, June 2000, p. 3460-3466, Vol. 182, No. 12
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

S. M. Sullivan,1 R. Mishra,1 R. R. Neubig,2 and J. R. Maddock1,*

Department of Biology1 and Departments of Pharmacology and Internal Medicine/Hypertension,2 University of Michigan, Ann Arbor, Michigan 48109

Received 20 December 1999/Accepted 26 March 2000

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 (KD) 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 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.

* Corresponding author. Mailing address: Department of Biology, University of Michigan, 830 North University, Ann Arbor, MI 48109-1048. Phone: (734) 936-8068. Fax: (734) 647-0884. E-mail: maddock{at}umich.edu.

Journal of Bacteriology, June 2000, p. 3460-3466, Vol. 182, No. 12
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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