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

Publication date: February 2016 Source:Journal of Molecular Catalysis B: Enzymatic, Volume 124 Author(s): Barbara Krajewska 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 KM 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°C. The determined values of pK as and of the enthalpies of ionization Δ H ion o allowed the ionizable groups to have the pK as assigned. Unlike previously assumed to have pK a ≈6.5, the determined acidic pK 2 ≈5.0 was assigned to a histidine residue (most likely His320 by Klebsiella aerogenes numbering). By contrast, the basic pK 1 ≈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 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 ...
Source: Journal of Molecular Catalysis B: Enzymatic - Category: Biochemistry Source Type: research