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Four phosphoenolpyruvate (PEP) derivatives, carrying reactive or activable chemical functions in each of the three chemical regions of PEP, were assayed as alternative substrates of enzyme I (EI) of the Escherichia coli PEP:glucose phosphotransferase system. The Z- and E-isomers of 3-chlorophosphoenolpyruvate (3-Cl-PEP) were substrates, presenting Km values of 0.08 and 0.12 mM, respectively, very similar to the Km of 0.14 mM measured for PEP, and kcat of 40 and 4 min-1, compared with 2,200 min-1, for PEP. The low catalytic efficiency of these substrates permits the study of activity at in vivo EI concentrations. Z-Cl-PEP was a competitive inhibitor of PEP with a KI of 0.4 mM. E-Cl-PEP was not an inhibitor. Compounds 3 and 4, obtained by modification of the carboxylic and phosphate groups of PEP, were neither substrates nor inhibitors of EI, highlighting the importance of these functionalities for recognition by EI. Z-Cl-PEP is a suicide inhibitor. About 10-50 turnovers sufficed to inactivate EI completely. Such a property can be exploited to reveal and quantitate phosphoryl transfer from EI to other proteins at in vivo concentrations. Inactivation was saturatable in Z-Cl-PEP, with an apparent Kminact of 0.2-0.4 mM. The rate of inactivation increased with the concentration of EI, indicating a preferential or exclusive reaction with the dimeric form of EI. E-Cl-PEP inactivates EI much more slowly, and unlike PEP, it did not protect against inactivation by Z-Cl-PEP. This and the ineffectiveness of E-Cl-PEP as a competitive inhibitor have been related to the presence of two EI active species. Cys-502 of EI was identified by mass spectrometry as the reacting residue. The C502A EI mutant showed less than 0.06% wild-type activity. Sequence alignments and comparisons of x-ray structures of different PEP-utilizing enzymes indicate that Cys-502 might serve as a proton donor during catalysis.

Original publication

DOI

10.1074/jbc.M110067200

Type

Journal article

Journal

Journal of Biological Chemistry

Publication Date

01/03/2002

Volume

277

Pages

6934 - 6942