Fluoride inhibition of Klebsiella aerogenes urease: Mechanistic implications of a pseudo-uncompetitive, slow-binding inhibitor

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 ra te studies yield parallel double-reciprocal plots; however, we show that fl uoride interaction with urease is not compatible with classical uncompetiti ve inhibition. Rather, we propose that fluoride binds to an enzyme state (E *) that is in equilibrium with resting enzyme (E) and produced during catal ysis. Fluoride binding rates are directly proportional to inhibitor concent ration. Substrate reduces both the rate of fluoride binding to urease and t he rate of fluoride dissociation from the complex, consistent with urea bin ding to E* and E*.F in addition to E. Fluoride inhibition is pH-dependent d ue to a protonation event linked to fluoride dissociation. Fluoride binding is pH-independent, suggesting that fluoride anion, not HF, is the actual i nhibitor. We assess the kinetic results in terms of the known protein cryst al structure and evaluate possible molecular interpretations for the struct ure of the E* state, the site of fluoride binding, and the factors associat ed with fluoride release. Finally, we note that the apparent uncompetitive inhibition by fluoride as reported for several other metalloenzymes may nee d to be reinterpreted in terms of fluoride interaction with the correspondi ng E* states.