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Journal of Bacteriology, September 1999, p. 5825-5832, Vol. 181, No. 18
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

The Caulobacter crescentus CgtA Protein Displays Unusual Guanine Nucleotide Binding and Exchange Properties

Bin Lin, Kelly L. Covalle, and Janine R. Maddock*

Department of Biology, University of Michigan, Ann Arbor, Michigan 48109-1048

Received 26 April 1999/Accepted 30 June 1999

The Caulobacter crescentus CgtA protein is a member of the Obg-GTP1 subfamily of monomeric GTP-binding proteins. In vitro, CgtA specifically bound GTP and GDP but not GMP or ATP. CgtA bound GTP and GDP with moderate affinity at 30°C and displayed equilibrium binding constants of 1.2 and 0.5 µM, respectively, in the presence of Mg2+. In the absence of Mg2+, the affinity of CgtA for GTP and GDP was reduced 59- and 6-fold, respectively. N-Methyl-3'-O-anthranoyl (mant)-guanine nucleotide analogs were used to quantify GDP and GTP exchange. Spontaneous dissociation of both GDP and GTP in the presence of 5 to 12 mM Mg2+ was extremely rapid (kd = 1.4 and 1.5 s-1, respectively), 103- to 105-fold faster than that of the well-characterized eukaryotic Ras-like GTP-binding proteins. The dissociation rate constant of GDP increased sevenfold in the absence of Mg2+. Finally, there was a low inherent GTPase activity with a single-turnover rate constant of 5.0 × 10-4 s-1 corresponding to a half-life of hydrolysis of 23 min. These data clearly demonstrate that the guanine nucleotide binding and exchange properties of CgtA are different from those of the well-characterized Ras-like GTP-binding proteins. Furthermore, these data are consistent with a model whereby the nucleotide occupancy of CgtA is controlled by the intracellular levels of guanine nucleotides.

* 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}biology.lsa.umich.edu.

Journal of Bacteriology, September 1999, p. 5825-5832, Vol. 181, No. 18
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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