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J. Biol. Chem., Vol. 283, Issue 2, 756-765, January 11, 2008

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Kinetic Characterization of Catalysis by the Chemotaxis Phosphatase CheZ

MODULATION OF ACTIVITY BY THE PHOSPHORYLATED CheY SUBSTRATE^*

Ruth E. Silversmith¹, Matthew D. Levin², Elmar Schilling³, and Robert B. Bourret

From the Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina 27599-7290 and Department of Physiology, Development, and Neuroscience, University of Cambridge, Cambridge CB2 3EJ, United Kingdom

CheZ catalyzes the dephosphorylation of the response regulator CheY in the two-component regulatory system that mediates chemotaxis in Escherichia coli. CheZ is a homodimer with two active sites for dephosphorylation. To gain insight into cellular mechanisms for the precise regulation of intracellular phosphorylated CheY (CheYp) levels, we evaluated the kinetic properties of CheZ. The steady state rate of CheZ-mediated dephosphorylation of CheYp displayed marked sigmoidicity with respect to CheYp concentration and a k_cat of 4.9 s^–1. In contrast, the gain of function mutant CheZ-I21T with an amino acid substitution far from the active site gave hyperbolic kinetics and required far lower CheYp for half-saturation but had a similar k_cat value as the wild type enzyme. Stopped flow fluorescence measurements demonstrated a 6-fold faster CheZ/CheYp association rate for CheZ-I21T (k_assoc = 3.4 x 10⁷ M ^–1 s^–1) relative to wild type CheZ (k_assoc = 5.6 x 10⁶ M^–1 s^–1). Dissociation of the CheZ·CheYBeF₃ complex was slow for both wild type CheZ (k_dissoc = 0.040 s^–1) and CheZ-I21T (k_dissoc = 0.023 s^–1) and, when taken with the k_assoc values, implied K_d values of 7.1 and 0.68 nM, respectively. However, comparison of the k_dissoc and k_cat values implied that CheZ and CheYp are not at binding equilibrium during catalysis and that once CheYp binds, it is almost always dephosphorylated. The rate constants were collated to formulate a kinetic model for CheZ-mediated dephosphorylation that includes autoregulation by CheYp and allowed prediction of CheZ activities at CheZ and CheYp concentrations likely to be present in cells.

Received for publication, May 29, 2007 , and in revised form, October 19, 2007.

^* This work was supported by National Institutes of Health Grant GM 050860 (to R. B. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

² Supported by National Institutes of Health Grant GM 064713 (to D. Bray).

³ Current address: Dept. of Hematology and Oncology, Regensburg University Medical Center, Regensburg, 93042 Germany.

¹ To whom correspondence should be addressed. Tel.: 919-966-2679; Fax: 919-962-8103; E-mail: silversr{at}med.unc.edu.

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