Urease Catalysis

Abstract

Urease has long been considered to be an absolutely specific enzyme (1, 2).However, during elaboration of an assay method for hydroxyurea, utilizing the Fearon carbamido reaction (3), Davidson and Winter noted that hydroxyurea levels declined following treatment with urease (4).That this decline represents true enzymic catalysis, and not adsorption or nonspecific degradation, is here documented fully.The kinetics of the decomposition of this substrate is compared with that of urea degradation.Early in the course of this work, it became clear that the reaction with hydroxyurea resulted in a striking inhibition of urease.Details of the inhibition by substrate, product, and analogue are reserved for the following paper (5).A preliminary report of these findings has been published (6).EXPERIMENTAL PROCEDURE Materials-Hydroxyurea was obtained through the Cancer Chemotherapy National Service Center (NSC No. 32065) and the Squibb Institute for Medical Research.It is the stable form of two isomers (7) with a melting point of 140", and has the hydroxyl group as a substituent on one of the nitrogen atoms (8-10).The crystalline material shows a single component at RF of 0.52 when chromatographed in butanol-ethanol-water (4:l :I) and stained with Ehrlich's reagent (urea, RF = 0.4).Crystalline jackbean urease preperations in three grades of purity were obtained from Sigma Chemical Company.These are designated by roman numerals, as shown below, along with their activities in modified Sumner units (assayed at 30") : I, 3.4 units per mg; II, 7.8 units per mg; III, 48 units per mg.Gel electrophoresis of these preparations at 20" in 1% ion agar with 0.05 M barbital buffer, pH 8.2 (II), suggests a purity of about 50% for urease III.Other enzymes used were obtained from the same source, in the lyophilized or crystalline state.Acetohydroxamic acid was prepared by slowly dripping 0.1 mole of ethyl acetate into a chilled flask fitted with magnetic stirrer, containing 0.1 mole of hydroxylamine hydrochloride and 0.15 mole of NaOH in 30 ml of 30y0 ethanol.After 1 hour, the solution was brought to neutral pH with HCl and evaporated under reduced pressure (15 mm of Hg) at 50".The residue was extracted with 300 ml of boiling ethyl acetate.The precipitat,e which formed on cooling was filtered, washed with ethyl acetate and ether, and air dried.After desiccation in a vacuum,