Jack bean urease (EC 3.5.1.5). V. On the mechanism of action of urease on urea, formamide, acetamide, N-methylurea, and related compounds

Abstract

Acetamide and N-methylurea was substrates for jack bean urease. In the enzymatic hydrolysis of urea, formamide, acetamide, and N-methylurea at pH 7.0 and 38.degree. C, kcat has the values 5870, 85, 0.55 and 0.075 s-1, respectively. The urease-catalyzed hydrolysis of all these substrates involves the active-site Ni ion(s). Enzymatic hydrolysis of the following compounds was not detected: phenyl formate, p-nitroformanilide, trifluoroacetamide, p-nitrophenyl carbamate, thiourea and O-methylisouronium ion. In the enzymatic hydrolysis of urea, the pH dependence of kcat between pH 3.4 and 7.8 indicates that at least 2 prototropic forms are active. Enzymatic hydrolysis of urea in the presence of methanol gave no detectable methyl carbamate. A mechanism of action for urease is proposed which involves initially an O-bonded complex between urea and an active-site Ni2+ ion and subsequently an O-bonded carbamato-enzyme intermediate.