Oxygen transfer by the homogentisate oxidase of rat liver.

In the net reaction catalyzed by homogentisate oxidase of rat liver, two atoms of osygen are added to the double bond between carbon atoms 1 and 2 of homogentisate (2, 3) resulting in the formation of maleylacetoacetate (Fig. 1). The enzyme is located in the soluble phase of the liver cell; its activity is totally independent of the hydrogen and electron transport system. Its properties and mode of action seem to be very similar to those of pyrocatechase and 3-hydroxyanthranilate oxidase (4), both of which have been shown to incorporate molecular oxygen directly into their substrates (5, 6). In view of these considerations, it is probable that the homogentisate oxidase reaction also involves the direct incorporation of molecular oxygen into positions 1 and 2 of the substrate. This report describes our attempt to determine the source of these two oxygen atoms by incubation of the oxidase with its substrate in the presence of O&8 + Hz0 and, conversely, 02 + Hz0’8. As shown in Fig. 1, the two atoms of oxygen which are added to the substrate must of necessity be located in the terminal carboxyl group and in the P-carbonyl group of the product. It follows that, if the source of this oxygen (02 or HzO) is labeled with 018, the resulting labeled maleylacetoacetate should give rise to labeled fumarylacetoacetate (7), which in turn should give rise by enzymatic hydrolysis (2) to carboxyl-labeled fumarate and carbonyl-labeled acetoacetate, thus permitting the sources of both oxygen atoms to be investigated individually. Were it not for the tendency of carbonyl oxygen to undergo spontaneous exchange with water as shown by Cohn and Urey (8), the experimental plan outlined above would have yielded conclusive data. However, nonenzymatic exchange of the carbonyl oxygens of maleylacetoacetate and fumarylacetoacetate with water has proved to be sufficiently rapid to invalidate our attempt to determine the source of the /3-carbonyl oxygen of maleylacetoacetate.