A thermodynamic study on the binding of mercury and silver ions to urease

A pH-variation study of jack bean (Canavalia ensiformis) urease steady-state kinetic parameters and of the inhibition constant of boric acid, a urease competitive inhibitor, was performed using both noninhibitory organic (MES, HEPES and CHES) and inhibitory inorganic (phosphate) buffers, in an effort to elucidate the functions exercised in the catalysis by the ionizable groups of the enzyme active site. The results obtained are consistent with the requirement for three groups utilized by urease with pK(a)s equal to 5.3+/-0.2, 6.6+/-0.2 and 9.1+/-0.4. Based on the appearance of the ionization step with pK(a)=5.3 in v(max)-pH, K(M)-pH and K(i)-pH profiles, we assigned this group as participating both in the substrate binding and catalytic reaction. As shown by its presence in v(max)-pH and K(M)-pH curves, the obvious role of the group with pK(a)=9.1 is the participation in the catalytic reaction. One function of the group featuring pK(a)=6.6, which was derived from a two-maxima v(max)-pH profile obtained upon increasing phosphate buffer concentration, an effect the first time observed for urease-phosphate systems, is the substrate binding, another possible function being modulation of the active site structure controlled by the ionic strength. It is also possible that the pK(a)=6.6 is a merger of two pK(a)s close in value. The study establishes that regular bell-shaped activity-pH profiles, commonly reported for urease, entail more complex pH-dependent behavior of the urease active site ionizable groups, which could be experimentally derived using species interacting with the enzyme, in addition to changing solution pH and ionic strength. Show more

In their inhibition-inducing interactions with enzymes, quinones primarily utilize two mechanisms, arylation and oxidation of enzyme thiol groups. In this work, we investigated the interactions of 1,4-naphthoquinone with urease in an effort to estimate the contribution of the two mechanisms in the enzyme inhibition. Jack bean urease, a homohexamer, contains 15 thiols per enzyme subunit, six accessible under non-denaturing conditions, of which Cys592 proximal to the active site indirectly participates in the enzyme catalysis. Unlike by 1,4-benzoquinone, a thiol arylator, the inactivation of urease by 1,4-naphthoquinone under aerobic conditions was found to be biphasic, time- and concentration-dependent with a non-linear residual activity-modified thiols dependence. DTT protection studies and thiol titration with DTNB suggest that thiols are the sites of enzyme interactions with the quinone. The inactivated enzyme had approximately 40% of its activity restored by excess DTT supporting the presence of sulfenic acid resulting from the oxidation of enzyme thiols by ROS. Furthermore, the aerobic inactivation was prevented in approximately 30% by catalase, proving the involvement of hydrogen peroxide in the process. When H2O2 was directly applied to urease, the enzyme showed susceptibility to this inactivation in a time- and concentration-dependent manner with the inhibition constant of H2O2 Ki = 3.24 mM. Additionally, anaerobic inactivation of urease was performed and was found to be weaker than aerobic. The results obtained are consistent with a double mode of 1,4-naphthoquinone inhibitory action on urease, namely through the arylation of the enzyme thiol groups and ROS generation, notably H2O2, resulting in the oxidation of the groups. Show more