Department of Chemistry, University of Georgia, Athens, GA 30602, United States; Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, United States.
Arch Biochem Biophys. 2014 Feb 15;544:69-74. doi: 10.1016/j.abb.2013.10.020. Epub 2013 Nov 4.
The kynurenine pathway is the major pathway of l-tryptophan catabolism in eukaryotes and some bacteria. In this pathway, kynureninase catalyzes the hydrolysis of l-kynurenine (in bacteria) or 3-hydroxy-l-kynurenine (in eukaryotes) to give anthranilic acid or 3-hydroxyanthranilic acid, respectively, and l-alanine. Kynureninase is a member of the aminotransferase superfamily and contains pyridoxal-5'-phosphate (PLP) as cofactor. The enzyme is a dimer of two identical subunits, with the active site containing residues contributed from both subunits. The reaction of kynureninase is formally a retro-Claisen reaction, and thus requires extensive acid-base catalysis. The pH dependence of the reaction of Pseudomonas fluorescens kynureninase shows two pKa's, a base with 6.5 and an acid with 8.8, on kcat/Km, and one pKa of 6.8 on kcat. The effects of mutagenesis of Tyr-226 and (31)P NMR results suggest that the basic group with pKa of 6.5 is the phosphate group of the PLP, which accepts a proton from the amino acid substrate zwitterion to initiate transaldimination. The external aldimine of kynurenine and PLP is then deprotonated by the ε-amino group of Lys-227 to give a quinonoid intermediate, which is reprotonated at C-4' to give a ketimine. Addition of water to the γ-carbonyl, assisted by Lys-227, then gives a gem-diol, which undergoes Cβ-Cγ cleavage to give the first product, anthranilic acid, and an enamine intermediate. The enamine is protonated at the β-carbon, resulting in a pyruvate ketimine. Deprotonation at C-4' and reprotonation of the α-carbon gives the external aldimine of l-alanine, which releases the second product, l-alanine. The reaction specificity of kynureninases is determined in part by active site residues, Trp64, Gly281, and Thr282 in P. fluorescens, and the homologous His102, Ser332, and Asn333 in human kynureninase. Asn333 can form a hydrogen bond to the 3-OH of 3-hydroxykynurenine in the human enzyme. Halogenation of kynurenine at C-5 increases activity with both enzymes, but halogenation at C-3 only increases activity for human kynureninase. The effect of halogenation at C-5 may be due to hydrophobic or van der Waals effects, and the effect of halogenation at C-3 for the human enzyme may be due to halogen bonding.
犬尿酸途径是真核生物和某些细菌中 l-色氨酸分解代谢的主要途径。在该途径中，犬尿酸酶催化 l-犬尿酸（在细菌中）或 3-羟基-l-犬尿酸（在真核生物中）的水解，分别生成邻氨基苯甲酸或 3-羟基邻氨基苯甲酸和 l-丙氨酸。犬尿酸酶属于氨基转移酶超家族成员，含有吡哆醛-5'-磷酸（PLP）作为辅因子。该酶是两个相同亚基的二聚体，活性位点包含来自两个亚基的残基。犬尿酸酶的反应是一个正式的反-Claisen 反应，因此需要广泛的酸碱催化。荧光假单胞菌犬尿酸酶反应的 pH 依赖性在 kcat/Km 上显示出两个 pKa，一个碱基为 6.5，一个酸为 8.8，在 kcat 上显示出一个 pKa 为 6.8。Tyr-226 的突变和 (31)P NMR 结果表明，pKa 为 6.5 的碱性基团是 PLP 的磷酸基团，它从氨基酸底物两性离子接受一个质子，启动转醛缩合反应。然后，外亚胺的犬尿酸和 PLP 被 Lys-227 的 ε-氨基去质子化，生成醌亚胺中间体，在 C-4'处质子化生成酮亚胺。在 Lys-227 的协助下，γ-羰基加水，然后生成一个偕二醇，它经历 Cβ-Cγ 裂解，生成第一个产物邻氨基苯甲酸和烯胺中间产物。烯胺在 β-碳上质子化，生成丙酮酸酮亚胺。C-4'去质子化和 α-碳质子化生成 l-丙氨酸的外亚胺，释放第二个产物 l-丙氨酸。犬尿酸酶的反应特异性部分由活性位点残基决定，荧光假单胞菌中的 Trp64、Gly281 和 Thr282，以及人源犬尿酸酶中的同源 His102、Ser332 和 Asn333。Asn333 可以与人源犬尿酸酶中的 3-羟基犬尿酸的 3-OH 形成氢键。犬尿酸 C-5 上的卤化作用会增加两种酶的活性，但 C-3 上的卤化作用仅增加人源犬尿酸酶的活性。C-5 上卤化作用的影响可能是由于疏水性或范德华力的影响，而 C-3 上卤化作用对人源酶的影响可能是由于卤键的作用。
