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J. Biol. Chem., Vol. 280, Issue 51, 41872-41880, December 23, 2005

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Transient State Kinetics of Enzyme IICB^Glc, a Glucose Transporter of the Phosphoenolpyruvate Phosphotransferase System of Escherichia coli

EQUILIBRIUM AND SECOND ORDER RATE CONSTANTS FOR THE GLUCOSE BINDING AND PHOSPHOTRANSFER REACTIONS^*

From the Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218

During translocation across the cytoplasmic membrane of Escherichia coli, glucose is phosphorylated by phospho-IIA^Glc and Enzyme IICB^Glc, the last two proteins in the phosphotransfer sequence of the phosphoenolpyruvate:glucose phosphotransferase system. Transient state (rapid quench) methods were used to determine the second order rate constants that describe the phosphotransfer reactions (phospho-IIA^Glc to IICB^Glc to Glc) and also the second order rate constants for the transfer from phospho-IIA^Glc to molecularly cloned IIB^Glc, the soluble, cytoplasmic domain of IICB^Glc. The rate constants for the forward and reverse phosphotransfer reactions between IIA^Glc and IICB^Glc were 3.9 x 10⁶ and 0.31 x 10⁶ M^–1 s^–1, respectively, and the rate constant for the physiologically irreversible reaction between [P]IICB^Glc and Glc was 3.2 x 10⁶ M^–1 s^–1. From the rate constants, the equilibrium constants for the transfer of the phospho-group from His⁹⁰ of [P]IIA^Glc to the phosphorylation site Cys of IIB^Glc or IICB^Glc were found to be 3.5 and 12, respectively. These equilibrium constants signify that the thiophospho-group in these proteins has a high phosphotransfer potential, similar to that of the phosphohistidinyl phosphotransferase system proteins. In these studies, preparations of IICB^Glc were invariably found to contain endogenous, firmly bound Glc (estimated K'_D 10^–7 M). The bound Glc was kinetically competent and was rapidly phosphorylated, indicating that IICB^Glc has a random order, Bi Bi, substituted enzyme mechanism. The equilibrium constant for the binding of Glc was deduced from differences in the statistical goodness of fit of the phosphotransfer data to the kinetic model.

Received for publication, February 7, 2005 , and in revised form, October 3, 2005.

^* 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental data, including supplemental Fig. S1.

¹ Present address: Dana Farber Cancer Institute, Harvard Medical School, Smith 1040, 44 Binney St., Boston, MA 02115.

² To whom correspondence should be addressed: Dept. of Biology, The Johns Hopkins University, 3400 North Charles St., Baltimore, MD 21218. E-mail: roseman{at}jhu.edu.

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