Beef kidney 3-hydroxyanthranilic acid oxygenase. Purification, characterization, and analysis of the assay.
Beef kidney 3-hydroxyanthranilic acid oxygenase has been purified to homogeneity. It is a single subunit protein of Mr = 34,000 +/- 2,000 with a frictional coefficient (f/f0) of about 1.1. The enzyme readily aggregates to form, apparently inactive, higher molecular weight oligomers. The very rapid loss of enzyme activity during the assay was analyzed extensively. It was found to be due to inactivation of the enzyme by the substrate, 3-hydroxyanthranilate, and unrelated to enzyme turnover or oxidation of bound iron. The loss of activity was shown to be a first order decay process, and methods are given for obtaining accurate initial reaction rates under all conditions. Evidence was presented that the enzyme assumes a catalytically inactive conformation at pH 3.4, which only relatively slowly rearranges to an active form at pH 6.5; the rearrangement can be blocked by the presence of substrate. We have found that Fe2+, which is required for enzymatic activity, can equilibrate freely, albeit slowly, with the enzyme during the course of the enzyme reaction even in the presence of saturating 3-hydroxanthranilate. Under assay conditons, the Fe2+ has an apparent dissociation constant of 0.04 mM. The kinetic properties of the enzyme were found to be dramatically different in beta,beta-dimethylglutarate buffer and collidine buffer; both the rate of loss of activity during the assay and the substrate Km and Vmax were affected.
D000818:Animals; D002417:Cattle; D004355:Drug Stability; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D010105:Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation
D007501:Iron; D010105:Oxygenases