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 (ΔH° = −14 kJ mol−1) and the Gibbs free energy ( $$ \Delta G_{298}^{^\circ } $$ = −14 kJ mol−1) for the substrate binding were negative, proving favorable for an exothermic and spontaneous reaction. With the standard entropy change ΔS° 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 (ΔH° = −42 kJ mol−1, $$ \Delta G_{298}^{^\circ } $$ = −23 kJ mol−1) than the substrate and the other two inhibitors. Those for 2-mercaptoethanol were ΔH° = −20 kJ mol−1 and $$ \Delta G_{298}^{^\circ } $$ = −17 kJ mol−1, while for phosphate buffer pH 6.45, ΔH° = −17 kJ mol−1, and $$ \Delta G_{298}^{^\circ } $$ = −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 is. Remarkably, the inhibitors showed negative values of ΔS° (to various degrees) proving that their binding to the enzyme, like that of the substrate, is driven by enthalpy.