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.
Highlights
CheZ catalyzes the dephosphorylation of the response regulator CheY in the two-component regulatory system that mediates chemotaxis in Escherichia coli
Analysis of presteady state time courses that monitor the CheZ-induced shift in the equilibrium between CheYp and CheY suggested that CheZ catalytic activity exhibits positive cooperativity with respect to CheYp concentration
Analysis of the raw data as described under “Experimental Procedures” first involved calculation of the concentration of CheYp present under each steady state reaction condition (Fig. 2B), which led to the relationship between CheZ activity and CheYp concentration (Fig. 2C)
Summary
The gain of function mutant CheZ-R54C displays enhanced phosphatase activity at low CheYp concentrations, suggesting that the substitution resulted in a constitutively activated state [22]. These observations suggest CheZ activity might be regulated in a manner that could potentially play an important role in control of CheYp levels in the cell. Results from measurements made on both wild type CheZ and the gain of function mutant CheZ-I21T were combined to formulate a kinetic model for CheZ-mediated dephosphorylation that includes positive cooperativity with respect to CheYp. The model was used in computer simulations to predict CheZ activities at concentrations of CheZ and CheYp present in the cell.
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