Hydrogen peroxide-induced inactivation of urease: Mechanism, kinetics and inhibitory potency

Evidence has been collected that H2O2 resulting from redox reactions taking place in some inhibitor systems, is responsible for the inactivation of urease. Accordingly, in this study the inactivation by H2O2 exogenously added to urease (jack bean) was investigated. The reaction accountable for the inactivation was the oxidation of the enzyme thiol groups. The reaction was studied at pHs over 6.2–8.2. At each pH, the first-order kinetics was obeyed, the resulting dependences of kobs on H2O2 concentration being hyperbolic. Analyzed with Kitz-Wilson method, the kinact appeared to be invariant with pH, while the inhibitor binding constant KI decreased with increasing pH. Consequently, the process grew faster with an increase in pH, the second-order rate constants increasing from 0.0032 M−1 s−1 at pH 6.2 to 0.0615 M−1 s−1 at pH 8.2, consistent with higher susceptibility of thiolate anions to oxidation by H2O2 compared to reduced thiols. The reactions always resulted in irreversible inactivation of urease, indicative of the oxidation of thiol groups to either sulfinic or sulfonic acid, independent of the extent of inactivation. Analysis of the numbers of urease thiol groups modified with H2O2 revealed that for the complete inactivation, out of 36 thiols/molecule available under non-denaturating conditions, the rapid oxidation of 30 highly reactive –SH groups was responsible for a 50% loss in enzyme activity, and the slower oxidation of the remaining six –SH groups, for the other 50%. The enzyme was protected by active-site binding inhibitors, boric acid and fluoride, from the inactivation, suggesting that the six thiols are the active-site flap Cys-592s. For comparison with other urease inhibitors, the IC50 was determined for 20 min incubation with H2O2, its value changing from 242 mM at pH 6.2 to 11 mM at pH 8.2.