Purification of mammalian homogentisate oxidase and evidence for the existence of ferrous mercaptans in the active center.

Previous studies on homogentisate oxidase by Knox and Edwards (Z), Crandall (3), and Schepartz (4) confirmed and extended the original demonstration of a specific ferrous iron requirement by Suda and Takeda (5). Qualitative evidence for the displacement of iron by hydrogen ions and by mercurials from a postulated enzymatic ferrous mercaptan (6) was obtained with relatively crude enzyme preparations by Crandall (3). Recently Tokuyama (7-9) has reported on extensive kinetic studies of a highly purified preparation of this enzyme and upon its activation and inhibition by a variety of agents and claims to have ruled out the possibility that enzymatic iron is bound to sulfhydryl (8). In this report we also describe the preparation of a highly purified enzyme and present several lines of evidence for the existence of ferrous mercaptans on the active center and evidence for their combination with osygen during the homogentisate oxidase reaction.