title: Fluoride inhibition of Klebsiella aerogenes urease: mechanistic implications of a pseudo-uncompetitive, slow-binding inhibitor.
abstract: Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze the hydrolysis of urea. Here, we describe the steady-state and pre-steady-state kinetics of urease inhibition by fluoride. Urease is slowly inhibited by fluoride in both the presence and absence of substrate. Steady-state rate studies yield parallel double-reciprocal plots; however, we show that fluoride interaction with urease is not compatible with classical uncompetitive inhibition. Rather, we propose that fluoride binds to an enzyme state (E) that is in equilibrium with resting enzyme (E) and produced during catalysis. Fluoride binding rates are directly proportional to inhibitor concentration. Substrate reduces both the rate of fluoride binding to urease and the rate of fluoride dissociation from the complex, consistent with urea binding to E and E.F in addition to E. Fluoride inhibition is pH-dependent due to a protonation event linked to fluoride dissociation. Fluoride binding is pH-independent, suggesting that fluoride anion, not HF, is the actual inhibitor. We assess the kinetic results in terms of the known protein crystal structure and evaluate possible molecular interpretations for the structure of the E state, the site of fluoride binding, and the factors associated with fluoride release. Finally, we note that the apparent uncompetitive inhibition by fluoride as reported for several other metalloenzymes may need to be reinterpreted in terms of fluoride interaction with the corresponding E states.
authors.affiliation: Departments of Biochemistry and Microbiology, Michigan State University, East Lansing, Michigan 48824, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Todd
authors.forename: R P
authors.initials: RP
authors.lastname: Hausinger
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007887:Fabaceae / Q000201:enzymology; D005459:Fluorides / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D010946:Plants, Medicinal; D011485:Protein Binding; D014508:Urea / Q000737:chemistry; D014510:Urease / Q000037:antagonists & inhibitors* / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D014508:Urea; D014510:Urease; D005459:Fluorides