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J Biol Chem. 2008 Jan 11;283(2):756-65. Epub 2007 Nov 12.

Kinetic characterization of catalysis by the chemotaxis phosphatase CheZ. Modulation of activity by the phosphorylated CheY substrate.

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  • 1Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina 27599-7290, USA. silversr@med.unc.edu

Abstract

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 10(7) M (-1) s(-1)) relative to wild type CheZ (k(assoc) = 5.6 x 10(6) M(-1) s(-1)). Dissociation of the CheZ.CheYBeF(3) 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.

PMID:
17998207
[PubMed - indexed for MEDLINE]
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