Inhibition of chitosan-immobilized urease by slow-binding inhibitors: Ni2+, F- and acetohydroxamic acid

The inhibitions by Ni2+ and F- ions and by acetohydroxamic acid of jack bra n urease covalently immobilized on chitosan membrane was studied (pH 7.0, 2 5 degreesC) and compared with those of the native enzyme. The reaction prog ress curves of the immobilized urease-catalyzed hydrolysis of urea were rec orded in the absence and presence of the inhibitors. They revealed that the inhibitions are of the competitive slow-binding type similar to those of n ative urease. The immobilization weakened the inhibitory effect of the inhi bitors on urease as measured by the inhibition constants K-i(*). The increa se in their values: 17.9-fold for Ni2+, 26.5-fold for F- and 1.7-fold for a cetohydroxamic acid, was accounted for by environmental effects generated b y heterogeneity of the urease-chitosan system: (1) mass transfer limitation s imposed on substrate and reaction product in the external solution, and ( 2) the increase in local pH on the membrane produced by both the enzymatic reaction and the electric charge of the support. By relating the K-M/K-i(*) ratio to the electrostatic potential of chitosan it was found that while t he reduced Ni2+ inhibition is mainly brought about by the potential, inhibi tion by acetohydroxamic acid is independent of the potential, and the acid inhibits urease in its non-ionic form. The reduction in F- inhibition was a scribed to the increased pH in the local environment of the immobilized enz yme, (C) 2001 Elsevier Science B.V. All rights reserved.