title: Mechanism-based inhibition of enzyme I of the Escherichia coli phosphotransferase system. Cysteine 502 is an essential residue.
abstract: 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 K(m) values of 0.08 and 0.12 mm, respectively, very similar to the K(m) of 0.14 mm measured for PEP, and k(cat) 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 K(I) 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 K(m)(inact) 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.
authors.affiliation: Departement für Chemie und Biochemie and the Mass-Spectrometry Laboratory, Universität Bern, Freiestrasse 3, Bern CH-3012, Switzerland. garcia@ibc.unibe.ch
authors.forename: Luis Fernando
authors.initials: LF
authors.lastname: Garcia-Alles
authors.forename: Karin
authors.initials: K
authors.lastname: Flükiger
authors.forename: Johannes
authors.initials: J
authors.lastname: Hewel
authors.forename: Regula
authors.initials: R
authors.lastname: Gutknecht
authors.forename: Christian
authors.initials: C
authors.lastname: Siebold
authors.forename: Stefan
authors.initials: S
authors.lastname: Schürch
authors.forename: Bernhard
authors.initials: B
authors.lastname: Erni
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry* / Q000378:metabolism; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors
chemical_list: D000596:Amino Acids; D004791:Enzyme Inhibitors; D010770:Phosphotransferases; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase; D003545:Cysteine