A combined temperature-pH study of urease kinetics. Assigning pKa values to ionizable groups of the active site involved in the catalytic reaction

• KMand vmax of urease measured between 15 and 35 °C at pHs between 5.0 and 8.3.
• pKa and ΔHiono of the ionizable groups of urease active site involved in the reaction determined.
• pK2 ≈ 5 and pK1 ≈ 8.8 assigned to a histidine residue and to the Ni–Ni bridging hydroxyl ion, respectively.
• The most favorable conditions for the substrate binding at pH 5.5–6; for the catalytic step at pH 6.5–7.

A combined temperature-pH study of urease kinetics was performed with the primary objective to assign the observed pKa values to the ionizable groups of the enzyme active site involved in the reaction. This was done in view of the fact that the identity of the groups detected by pH studies reported in the literature has never been conclusively resolved. Accordingly, herein the urease kinetic parameters KM and vmax were measured at eight pHs between 5.0 and 8.3 in noninteracting biological buffers (MES and HEPES), at each pH at five temperatures between 15 and 35 °C. The determined values of pKas and of the enthalpies of ionizationΔHiono allowed the ionizable groups to have the pKas assigned. Unlike previously assumed to have pKa ≈ 6.5, the determined acidic pK2 ≈ 5.0 was assigned to a histidine residue (most likely His320 by Klebsiella aerogenes numbering). By contrast, the basic pK1 ≈ 8.8 was assigned to the Ni–Ni bridging hydroxyl ion, a feature that supports the mechanism operative in the urease activity in which this hydroxyl is a general acid. Further, the pH-dependent analysis of the obtained KM and vmax values revealed that even though both the thermodynamic and activation parameters of the urease reaction were little pH-dependent, their values indicated that the most favorable conditions for the substrate binding are at pH 5.5–6.0, and those for the catalytic step, at pH 6.5–7.0. Taken together, the work brings in new mechanistic information of significance to the understanding of the activity of urease.

Figure optionsDownload as PowerPoint slide