The kinetics of concurrent instantaneous and time dependent inhibition of alkaline phosphatase by urea and related compounds

Urea, thiourea and guanidine-HCl greatly inhibit bone Alkaline Phosphatase activity. The inhibition displays saturation kinetics, fitting a model for the reaction sequence in which formation of complexes with increasing affinity occurs. The time dependent inactivation by urea and guanidine follows pseudo-first order kinetics, while thiourea behaviour is better described by a biexponential equation. The pH variation strongly affects the activity, the instantaneous inhibition and the time dependent inactivation. At the pH optimum the three compounds display their strongest effect. Data analysis suggests a three-stage model for the kinetics of Alkaline Phosphatase inhibition by urea and related compounds, involving a consecutive binding process with several sites of the protein and the production of different and interchanging inhibitor-enzyme complexes, leading to irreversibly inactivated forms.