Department of Chemistry, University of Georgia,Athens, Georgia 30602, USA.
Biochemistry. 2010 Sep 14;49(36):7913-9. doi: 10.1021/bi100955b.
Kynureninase is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the hydrolytic cleavage of l-kynurenine to give l-alanine and anthranilic acid. beta-Benzoyl-l-alanine, the analogue of l-kynurenine lacking the aromatic amino group, was shown to a good substrate for kynureninase from Pseudomonas fluorescens, and the rate-determining step changes from release of the second product, l-Ala, to formation of the first product, benzoate [Gawandi, V. B., et al. (2004) Biochemistry 43, 3230-3237]. In this work, a series of aryl-substituted beta-benzoyl-dl-alanines was synthesized and evaluated for substrate activity with kynureninase from P. fluorescens. Hammett analysis of k(cat) and k(cat)/K(m) for 4-substituted beta-benzoyl-dl-alanines with electron-withdrawing and electron-donating substituents is nonlinear, with a concave downward curvature. This suggests that there is a change in rate-determining step for benzoate formation with different substituents, from gem-diol formation for electron-donating substituents to C(beta)-C(gamma) bond cleavage for electron-withdrawing substituents. Rapid-scanning stopped-flow kinetic experiments demonstrated that substituents have relatively minor effects on formation of the quinonoid and 348 nm intermediates but have a much greater effect on the formation of the aldol product from reaction of benzaldehyde with the 348 nm intermediate. Since there is a kinetic isotope effect on its formation from beta,beta-dideuterio-beta-(4-trifluoromethylbenzoyl)-dl-alanine, the 348 nm intermediate is proposed to be a vinylogous amide derived from abortive beta-deprotonation of the ketimine intermediate. These results provide additional evidence for a gem-diol intermediate in the catalytic mechanism of kynureninase.
犬尿氨酸酶是一种依赖于吡哆醛 5'-磷酸的酶，能够催化 l-犬尿氨酸的水解裂解，生成 l-丙氨酸和邻氨基苯甲酸。β-苯甲酰-l-丙氨酸是 l-犬尿氨酸缺少芳香族氨基酸的类似物，被证明是荧光假单胞菌犬尿氨酸酶的良好底物，反应的速控步骤由第二个产物 l-丙氨酸的释放转变为第一个产物苯甲酸的形成[Gawandi, V. B., et al. (2004) Biochemistry 43, 3230-3237]。在这项工作中，合成了一系列芳基取代的β-苯甲酰-dl-丙氨酸，并评估了它们对荧光假单胞菌犬尿氨酸酶的底物活性。用 4-取代的β-苯甲酰-dl-丙氨酸进行 k(cat)和 k(cat)/K(m)的哈米特分析，发现电子给体和电子受体取代基的 k(cat)和 k(cat)/K(m)是非线性的，具有凹向下的曲率。这表明，对于具有不同取代基的苯甲酸形成，速控步骤发生了变化，对于给电子取代基，从偕二醇形成变为 C(beta)-C(gamma)键的断裂；对于吸电子取代基，则反之。快速扫描停流动力学实验表明，取代基对醌型和 348nm 中间体的形成影响相对较小，但对苯甲醛与 348nm 中间体反应生成的醛醇产物的形成影响较大。由于其β,β-二氘代-β-(4-三氟甲基苯甲酰)-dl-丙氨酸的形成存在动力学同位素效应，因此，348nm 中间体被提议为来自酮亚胺中间体的无效β-去质子化的乙烯基酰胺。这些结果为犬尿氨酸酶的催化机制中存在偕二醇中间体提供了额外的证据。
