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Originally published In Press as doi:10.1074/jbc.M001045200 on July 25, 2000

J. Biol. Chem., Vol. 275, Issue 42, 33102-33109, October 20, 2000
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The Transport/Phosphorylation of N,N'-Diacetylchitobiose in Escherichia coli
CHARACTERIZATION OF PHOSPHO-IIBChb AND OF A POTENTIAL TRANSITION STATE ANALOGUE IN THE PHOSPHOTRANSFER REACTION BETWEEN THE PROTEINS IIAChb AND IIBChb*

Nemat O. KeyhaniDagger , Kirsten Bacia§, and Saul Roseman

From the Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218

Enzyme II permeases of the phosphoenolpyruvate:glycose phosphotransferase system comprise one to five separately encoded polypeptides, but most contain similar domains (IIA, IIB, and IIC). The phosphoryl group is transferred from one domain to another, with histidine as the phosphoryl acceptor in IIA and cysteine as the acceptor in certain IIB domains. IIBChb is a phosphocarrier in the uptake/phosphorylation of the chitin disaccharide, (GlcNAc)2 by Escherichia coli and is unusual because it is separately encoded and soluble. Both the crystal and solution structures of a IIBChb mutant (C10S) have been reported. In the present studies, homogeneous phospho-IIBChb was isolated, and the phosphoryl-Cys linkage was established by 31P NMR spectroscopy. Rate constants for the hydrolysis of phospho-IIBChb plotted versus pH gave the same shape peak reported for the model compound, butyl thiophosphate, but was shifted about 4 pH units. Evidence is presented for a stable complex between homogeneous Cys10SerIIBChb (which cannot be phosphorylated) and phospho-IIAChb, but not with IIAChb. The complex (a tetramer (3)) contains equimolar quantities of the two proteins and has been chemically cross-linked. It appears to be an analogue of the transition state complex in the reaction: phospho-IIAChb + IIBChb left-right-arrow IIAChb + phospho-IIBChb. This is apparently the first report of the isolation of a transition state analogue in a protein-protein phosphotransfer reaction.

* This work was supported by Grant 38759 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Dept. of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611.

§ Present address: Zentrum Biochemie, OE 4310, Medizinische Hochschule Hannover, D-30623 Hannover, Germany.

To whom correspondence should be addressed: Dept. of Biology and the McCollum-Pratt Inst., Johns Hopkins University, Mudd Hall, Rm. 214, 3400 N. Charles St., Baltimore, MD 21218.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.


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