Jack bean (Canavalia ensiformis) urease. Probing acid–base groups of the active site by pH variation

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 pKas equal to 5.3 ± 0.2, 6.6 ± 0.2 and 9.1 ± 0.4. Based on the appearance of the ionization step with pKa = 5.3 in vmax-pH, KM-pH and Ki-pH profiles, we assigned this group as participating both in the substrate binding and catalytic reaction. As shown by its presence in vmax-pH and KM-pH curves, the obvious role of the group with pKa = 9.1 is the participation in the catalytic reaction. One function of the group featuring pKa = 6.6, which was derived from a two-maxima vmax-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 pKa = 6.6 is a merger of two pKas 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.