Purification and characterization of uricase, a nitrogen-regulated enzyme, from Neurospora crassa.

Abstract Uricase, a purine catabolic enzyme, is controlled by induction and by nitrogen catabolite repression in Neurospora . Uricase was purified nearly 1000-fold to homogeneity. Only a single protein band could be detected in analytical gels of the pure enzyme, and the protein band in each case corresponded exactly to the position of in situ staining for enzyme activity. The molecular weight of native uricase was estimated to be 123,000 ± 7000. The enzyme is a tetramer composed of identical or similar-sized subunits. The K m value of uricase for uric acid was estimated to be 4.2 × 10 −5 , m . Oxonic acid was shown to be a competitive inhibitor of uricase, with a K i value of 6.7 × 10 −7 , m . Uricase is a stable enzyme and is not subject to feedback inhibition by ammonia, glutamate, or glutamine in Neurospora . The regulation of uricase appears to occur primarily at the biosynthesis level. Uricase appears to be a metalloenzyme with no essential sulfhydryl groups.