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J. Biol. Chem., Vol. 281, Issue 17, 11450-11455, April 28, 2006

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Effects of Mutations and Truncations on the Kinetic Behavior of IIA^Glc, a Phosphocarrier and Regulatory Protein of the Phosphoenolpyruvate Phosphotransferase System of Escherichia coli^*

Norman D. Meadow, Regina S. Savtchenko, S. James Remington, and Saul Roseman¹

From the Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218 and the Department of Physics, University of Oregon, Eugene, Oregon 97403

IIA^Glc, a component of the glucose-specific phosphoenolpyruvate:phosphotransferase system (PTS) of Escherichia coli, is important in regulating carbohydrate metabolism. In Glc uptake, the phosphotransfer sequence is: phosphoenolpyruvate Enzyme I HPr IIA^Glc IICB^Glc Glc. (HPr is the first phosphocarrier protein of the PTS.) We previously reported two classes of IIA^Glc mutations that substantially decrease the P-transfer rate constants to/from IIA^Glc. A mutant of His⁷⁵ which adjoins the active site (His⁹⁰), (H75Q), was 0.5% as active as wild-type IIA^Glc in the reversible P-transfer to HPr. Two possible explanations were offered for this result: (a) the imidazole ring of His⁷⁵ is required for charge delocalization and (b) H75Q disrupts the hydrogen bond network: Thr⁷³, His⁷⁵, phospho-His⁹⁰. The present studies directly test the H-bond network hypothesis. Thr⁷³ was replaced by Ser, Ala, or Val to eliminate the network. Because the rate constants for phosphotransfer to/from HPr were largely unaffected, we conclude that the H-bond network hypothesis is not correct. In the second class of mutants, proteolytic truncation of seven residues of the IIA^Glc N terminus caused a 20-fold reduction in phosphotransfer to membrane-bound IICB^Glc from Salmonella typhimurium. Here, we report the phosphotransfer rates between two genetically constructed N-terminal truncations of IIA^Glc (7 and 16) and the proteins IICB^Glc and IIB^Glc (the soluble cytoplasmic domain of IICB^Glc). The truncations did not significantly affect reversible P-transfer to IIB^Glc but substantially decreased the rate constants to IICB^Glc in E. coli and S. typhimurium membranes. The results support the hypothesis (Wang, G., Peterkofsky, A., and Clore, G. M. (2000) J. Biol. Chem. 275, 39811–39814) that the N-terminal 18-residue domain "docks" IIA^Glc to the lipid bilayer of membranes containing IICB^Glc.

Received for publication, July 8, 2005 , and in revised form, December 5, 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.

¹ 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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