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J. Biol. Chem., Vol. 281, Issue 26, 17570-17578, June 30, 2006

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The Monomer/Dimer Transition of Enzyme I of the Escherichia coli Phosphotransferase System^*

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

Enzyme I (EI) is the first protein in the phosphotransfer sequence of the bacterial phosphoenolpyruvate:glycose phosphotransferase system. This system catalyzes sugar phosphorylation/transport and is stringently regulated. Since EI homodimer accepts the phosphoryl group from phosphoenolpyruvate (PEP), whereas the monomer does not, EI may be a major factor in controlling sugar uptake. Previous work from this and other laboratories (e.g. Dimitrova, M. N., Szczepanowski, R. H., Ruvinov, S. B., Peterkofsky, A., and Ginsburg A. (2002) Biochem. 41, 906–913), indicate that K_a is sensitive to several parameters. We report here a systematic study of K_a determined by sedimentation equilibrium, which showed that it varied by 1000-fold, responding to virtually every parameter tested, including temperature, phosphorylation, pH (6.5 versus 7.5), ionic strength, and especially the ligands Mg²⁺ and PEP. This variability may be required for a regulatory protein. Further insight was gained by analyzing EI by sedimentation velocity, by near UV CD spectroscopy, and with a nonphosphorylatable active site mutant, EI-H189Q, which behaved virtually identically to EI. The singular properties of EI are explained by a model consistent with the results reported here and in the accompanying paper (Patel, H. V., Vyas, K. A., Mattoo, R. L., Southworth, M., Perler, F. B., Comb, D., and Roseman, S. (2006) J. Biol. Chem. 281, 17579–17587). We suggest that EI and EI-H189Q each comprise a multiplicity of conformers and progressively fewer conformers as they dimerize and bind Mg²⁺ and finally PEP. Mg²⁺ alone induces small or no detectable changes in structure, but large conformational changes ensue with Mg²⁺/PEP. This effect is explained by a "swiveling mechanism" (similar to that suggested for pyruvate phosphate dikinase (Herzberg, O., Chen, C. C., Kapadia, G., McGuire, M., Carroll, L. J., Noh, S. J., and Dunaway-Mariano, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2652–2657)), which brings the C-terminal domain with the two bound ligands close to the active site His¹⁸⁹.

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

^* This work was supported by National Institutes of Health Grant GM38759 and a grant from New England Biolabs generously made available by Dr. Donald Comb. 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 These authors contributed equally to this work.

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

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