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

A combined temperature-pH study of urease kinetics was performed with the primary objective to assign the observed pK(a) 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 K-M and v(max) 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 degrees C. The determined values of pK(a)s and of the enthalpies of ionization Delta H-ion(0) allowed the ionizable groups to have the pK(a)s assigned. Unlike previously assumed to have pK(a) approximate to 6.5, the determined acidic pK(2) approximate to 5.0 was assigned to a histidine residue (most likely His320 by Klebsiella aerogenes numbering). By contrast, the basic pK(1) approximate to 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 K-M and v(max) 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. (C) 2015 Elsevier B.V. All rights reserved.