| Abstract: | Cyanase is an inducible enzyme in Escherichia coli that catalyzes the hydrolysis of cyanate. Bicarbonate is required for activity, perhaps as a substrate, and the initial product of the reaction is carbamate, which spontaneously breaks down to ammonia and bicarbonate [Anderson, P. M. (1980) Biochemistry 19, 2882]. The purpose of this study was to characterize the kinetic properties of cyanase. Initial velocity studies showed that both cyanate and bicarbonate act as competitive substrate inhibitors. A number of monovalent anions act as inhibitors. Azide and acetate appear to act as competitive inhibitors with respect to cyanate and bicarbonate, respectively. Chloride, bromide, nitrate, nitrite, and formate also inhibit, apparently as the result of binding at either substrate site. Malonate and several other dicarboxylic dianions at very low concentrations display "slow-binding", reversible inhibition which can be prevented by saturating concentrations of either substrate. The results are consistent with a rapid equilibrium random mechanism in which bicarbonate acts as a substrate, bicarbonate and cyanate bind at adjacent anion-binding sites, and both substrates can bind at the other substrate anion binding site to give a dead-end complex. |
