Koontz W A, Shiman R
J Biol Chem. 1976 Jan 25;251(2):368-77.
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.
牛肾3-羟基邻氨基苯甲酸加氧酶已被纯化至同质。它是一种单亚基蛋白质，Mr = 34,000 +/- 2,000，摩擦系数（f/f0）约为1.1。该酶很容易聚集形成显然无活性的高分子量寡聚体。对测定过程中酶活性的快速丧失进行了广泛分析。发现这是由于底物3-羟基邻氨基苯甲酸使酶失活，与酶的周转或结合铁的氧化无关。活性丧失显示为一级衰减过程，并给出了在所有条件下获得准确初始反应速率的方法。有证据表明，该酶在pH 3.4时呈现催化无活性的构象，在pH 6.5时仅相对缓慢地重排为活性形式；底物的存在可阻止这种重排。我们发现，酶活性所需的Fe2+在酶反应过程中即使在存在饱和3-羟基邻氨基苯甲酸的情况下也能与酶自由平衡，尽管速度较慢。在测定条件下，Fe2+的表观解离常数为0.04 mM。发现该酶在β，β-二甲基戊二酸缓冲液和可力丁缓冲液中的动力学性质有显著差异；测定过程中的活性丧失速率以及底物Km和Vmax均受到影响。
