Williams LaKenya, Nguyen Tinh, Li Yingchun, Porter Tamiko N, Raushel Frank M
Department of Chemistry, P.O. Box 30012, Texas A&M University, College Station, Texas 77842-3012, USA.
Biochemistry. 2006 Jun 20;45(24):7453-62. doi: 10.1021/bi060531l.
Uronate isomerase, a member of the amidohydrolase superfamily, catalyzes the isomerization of D-glucuronate and D-fructuronate. During the interconversion of substrate and product the hydrogen at C2 of D-glucuronate is transferred to the pro-R position at C1 of the product, D-fructuronate. The exchange of the transferred hydrogen with solvent deuterium occurs at a rate that is 4 orders of magnitude slower than the interconversion of substrate and product. The enzyme catalyzes the elimination of fluoride from 3-deoxy-3-fluoro-D-glucuronate. These results have been interpreted to suggest a chemical reaction mechanism in which an active site base abstracts the proton from C2 of D-glucuronate to form a cis-enediol intermediate. The conjugate acid then transfers this proton to C1 of the cis-enediol intermediate to form D-fructuronate. The loss of fluoride from 3-deoxy-3-fluoro-D-glucuronate is consistent with a stabilized carbanion at C2 of the substrate during substrate turnover. The slow exchange of the transferred hydrogen with solvent water is consistent with a shielded conjugate acid after abstraction of the proton from either D-glucuronate or D-fructuronate during the isomerization reaction. This conclusion is supported by the competitive inhibition of the enzymatic reaction by D-arabinaric acid and the monohydroxamate derivative with Ki values of 13 and 670 nM, respectively. There is no evidence to support a hydride transfer mechanism for uronate isomerase. The wild type enzyme was found to contain 1 equiv of zinc per subunit. The divalent cation could be removed by dialysis against the metal chelator, dipicolinate. However, the apoenzyme has the same catalytic activity as the Zn-substituted enzyme and thus the divalent metal ion is not required for enzymatic activity. This is the only documented example of a member in the amidohydrolase superfamily that does not require one or two divalent cations for enzymatic activity.
糖醛酸异构酶是酰胺水解酶超家族的成员之一，催化D - 葡萄糖醛酸和D - 果糖醛酸的异构化反应。在底物和产物的相互转化过程中，D - 葡萄糖醛酸C2位的氢转移至产物D - 果糖醛酸C1位的前手性R位置。转移的氢与溶剂氘的交换速率比底物和产物的相互转化速率慢4个数量级。该酶催化从3 - 脱氧 - 3 - 氟 - D - 葡萄糖醛酸中消除氟。这些结果被解释为表明一种化学反应机制，其中活性位点碱从D - 葡萄糖醛酸的C2位提取质子以形成顺式烯二醇中间体。共轭酸然后将该质子转移至顺式烯二醇中间体的C1位以形成D - 果糖醛酸。在底物周转期间，3 - 脱氧 - 3 - 氟 - D - 葡萄糖醛酸中氟的损失与底物C2位稳定的碳负离子一致。在异构化反应过程中，转移的氢与溶剂水的缓慢交换与从D - 葡萄糖醛酸或D - 果糖醛酸提取质子后被屏蔽的共轭酸一致。D - 阿拉伯糖酸和单异羟肟酸衍生物对酶促反应的竞争性抑制分别以13和670 nM的Ki值支持了这一结论。没有证据支持糖醛酸异构酶的氢化物转移机制。发现野生型酶每个亚基含有1当量的锌。通过用金属螯合剂二吡啶甲酸盐透析可以去除二价阳离子。然而，脱辅基酶具有与锌取代酶相同的催化活性，因此二价金属离子对于酶活性不是必需的。这是酰胺水解酶超家族中唯一一个不需要一个或两个二价阳离子来进行酶活性的记录实例。
