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J. Biol. Chem., Vol. 281, Issue 26, 17579-17587, June 30, 2006

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Properties of the C-terminal Domain of Enzyme I of the Escherichia coli Phosphotransferase System^*

Himatkumar V. Patel¹, Kavita A. Vyas¹, Roshan L. Mattoo², Maurice Southworth, Francine B. Perler, Donald Comb, and Saul Roseman³

From the Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218 and New England Biolabs Inc., Ipswich, Massachusetts 01938

The bacterial phosphoenolpyruvate (PEP):glycose phosphotransferase system (PTS) mediates uptake/phosphorylation of sugars. The transport of all PTS sugars requires Enzyme I (EI) and a phosphocarrier histidine protein of the PTS (HPr). The PTS is stringently regulated, and a potential mechanism is the monomer/dimer transition of EI, because only the dimer accepts the phosphoryl group from PEP. EI monomer consists of two major domains, at the N and C termini (EI-N and EI-C, respectively). EI-N accepts the phosphoryl group from phospho-HPr but not PEP. However, it is phosphorylated by PEP(Mg²⁺) when complemented with EI-C. Here we report that the phosphotransfer rate increases 25-fold when HPr is added to a mixture of EI-N, EI-C, and PEP(Mg²⁺). A model to explain this effect is offered. Sedimentation equilibrium results show that the association constant for dimerization of EI-C monomers is 260-fold greater than the K_a for native EI. The ligands have no detectable effect on the secondary structure of the dimer (far UV CD) but have profound effects on the tertiary structure as determined by near UV CD spectroscopy, thermal denaturation, sedimentation equilibrium and velocity, and intrinsic fluorescence of the 2 Trp residues. The binding of PEP requires Mg²⁺. For example, there is no effect of PEP on the T_m, an increase of 7 °C in the presence of Mg²⁺, and 14 °C when both are present. Interestingly, the dissociation constants for each of the ligands from EI-C are approximately the same as the kinetic (K_m) constants for the ligands in the complete PTS sugar phosphorylation assays.

Received for publication, August 15, 2005 , and in revised form, March 17, 2006.

^* This work was supported by National Institutes of Health Grant GM38759. 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.

¹ Both authors contributed equally to this work.

² Current address: Dept. of Biochemistry & Biotechnology, University of Jammu, Jammu, J&K-180 006, India.

³ To whom correspondence should be addressed: Dept. of Biology, The Johns Hopkins University, 3400 North Charles St., Baltimore, MD 21218. Tel.: 410-516-7333; Fax: 410-516-5430; E-mail: roseman{at}jhu.edu.

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