Thermodynamic study of competitive inhibitors’ binding to urease

Abstract

Three competitive inhibitors of urease: boric acid, 2-mercaptoethanol and phosphate buffer pH 6.45, were subjected to the studies of temperature effects on their binding to the enzyme. The first of their kind for these systems, the studies were carried out at temperatures between 15 and 35°C in HEPES buffers. The Michaelis K M and inhibition K i constants determined for the uninhibited and inhibited urease reactions, respectively, were found to increase with an increase in temperature, importantly, the inhibition remaining competitive irrespective of temperature. The inverse of the constants were further analyzed as equilibrium constants of substrate and inhibitor binding reactions resulting in the formation of ES and EI complexes. The reactions were thermodynamically analyzed with use of the van't Hoff equation. Found pH-independent in the pH range 6.45-7.0, the changes in the standard enthalpy (DH°= -14 kJ mol -1 ) and the Gibbs free energy (DG 298 = -14 kJ mol -1 ) for the substrate binding were negative, proving favorable for an exothermic and spontaneous reaction. With the standard entropy change DS°close to zero, the reaction is enthalpy driven. The thermodynamic functions of the inhibitor binding by contrast, were found strongly correlated with the inhibitory strengths of the inhibitors in the order: boric acid [ 2-mercaptoethanol [ phosphate buffer pH 6.45. Accordingly, boric acid demonstrated the changes in the thermodynamic functions considerably bigger (DH°= -42 kJ mol -1 , DG 298 = -23 kJ mol -1 ) than the substrate and the other two inhibitors. Those for 2-mercaptoethanol were DH°= -20 kJ mol -1 and DG 298 = -17 kJ mol -1 , while for phosphate buffer pH 6.45, DH°= -17 kJ mol -1 , and DG 298 = -13 kJ mol -1 , and although slightly bigger, they were found practically comparable in value with those of the substrate, features that apparently result from the comparable values of K M and K i s. Remarkably, the inhibitors showed negative values of DS°(to various degrees) proving that their binding to the enzyme, like that of the substrate, is driven by enthalpy.

Layer‐by‐layer self‐assembly of multifunctional enzymatic UF membranes

“…The reaction was carried out in 22 mM phosphate buffer solution of pH 7 at 25 °C in a constant shaker water bath. The concentration of the buffer solution was selected such that it had a negligible competitive inhibitory action on URE . In the case of free URE, reaction was initiated by the addition of 0.5 mL URE (1 mg/mL) into 4.5 mL of 50 mM urea.…”

“…The concentration of the buffer solution was selected such that it had a negligible competitive inhibitory action on URE. 28 In the case of free URE, reaction was initiated by the addition of 0.5 mL URE (1 mg/mL) into 4.5 mL of 50 mM urea. The amount of ammonia produced during 15 min of reaction was determined followed by addition of 2.5 mL acetic acid solution (10 v %) to stop the reaction.…”

Urease-aided calcium carbonate mineralization for engineering applications: A review

“…Its elucidation is crucial for the efficacious use of the enzyme and for the control of its activity. One way to achieve this control is by using inhibitors [5] , [25] , [26] , [27] , [28] , the primary among them being: amides and esters of phosphoric acid [22] , [29] , [30] , thiols [30] , [31] , [32] , hydroxamic acids [29] , [30] , [33] , [34] , phosphinic and thiophosphinic acids [35] , boric acid [29] , [32] , [36] , phosphate [30] , [32] , [37] , [38] , heavy metal ions [39] , [40] , bismuth compounds [41] , quinones [42] , [43] , [44] , and fluoride [29] , [45] .…”

In vitroinhibition ofHelicobacter pyloriand interaction studies of lichen natural products with jack bean urease

“…45 It is claimed that -SH groups blockade was achieved through a nucleophilic addition into the carbonyl bond at C1 of usnic acid (Figure 1), a condition that can be reversed in the presence of Cys or dithiothreitol. 46 The mechanism of partial inactivation and reactivation of urease by (S)-(-)-UA (2) seems to be similar to that of omeprazole. 44b Indeed, the kinetics and interaction profiles for (S)-(-)-UA (2) revealed that this lichen metabolite is a typical mixed inhibitor.…”

“…Finally, (R)-(+)-UA (1) interacts preferably with urease active site by electrostatic forces as demonstrated by kinetics/interaction assays in a manner compared with other already reported for known urease inhibitors (Figure 7). 46 and its interaction with hydroxyurea (b), 47 omeprazole after reaction with Cys residue (c) 44a or (R)-(+)-UA (1) (d; proposed model). W1-W3, water molecules in the active site; WB, bridging water (hydroxide form).…”