Non-classical inhibition of uricase by cyanide

Abstract

The interactions of Aspergillus flavus uricase with the substrates O2 and urate and the inhibitors xanthine, cyanide, periodate and hydroxylamine were investigated. Under equilibrium conditions O2 does not bind directly to the enzyme, and the absence of O2 had no effect on either the binding stoicheiometry or binding constant for xanthine, as measured by equilibrium dialysis and microcalorimetry. Cyanide, periodate and hydroxylamine inhibit uricase in a non-classical manner. A decrease in initial velocity to a steady-state inhibited velocity can be observed on a time scale of minutes. The time-dependence, which is unaltered by prior incubation with the inhibitors, is consistent with a first-order transition. Rate constants for induction of inhibition are linearly dependent on inhibitor concentration, but independent of urate and O2 concentrations. Radioactively labelled urate forms a stable but reversible complex with uricase in the presence of cyanide and O2. These results were used to deduce the nature of enzyme-bound intermediates and thus for the proposal of a novel mechanism for cyanide inhibition.