Functional role of the "aromatic cage" in human monoamine oxidase B: Structures and catalytic properties of Tyr435 mutant proteins
Functional role of the "aromatic cage" in human monoamine oxidase B: Structures and catalytic properties of Tyr435 mutant proteins
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DOI：
10.1021/bi051847g
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发表时间：
2006-04-18
期刊：
影响因子：
2.9
通讯作者：
Edmondson, DE
中科院分区：
文献类型：
--
作者：
Li, M;Binda, C;Edmondson, DE
目前几种依赖于黄素的胺氧化酶的结构结果表明，芳香族氨基酸残基与黄素环大致垂直，这表明它在催化中具有功能作用。在人MAO B的情况下，在共价黄素环表面的底物结合部位发现两个酪氨酸残基(Y398和Y435)[Binda et a]。(2002)J.Biol.化学。277,23973-23976]为了探讨这种结构的功能意义，用毕赤酵母表达的突变蛋白Phe、His、Leu或Trp突变MAO B中的Tyr435，并将其纯化为均一。每个突变蛋白都含有共价FAD，并显示出高度的催化功能。将它们各自的晶体结构与WT酶的晶体结构进行比较，发现它们的活性中心结构没有发生重大变化。各突变体酶的相对k(Cat)/K-m值分别为Y435&Gt；Y435F=Y435L=Y435H>；Y435W。膜结合形式的MAO A和MAO B也有类似的行为(MAO A Y444突变酶在膜提取时被发现是不稳定的)。研究发现，对硝基苄胺是一种较差的底物，而对硝基苯乙胺是所有WT和MAO B突变形式的良好底物。对这些动力学和结构数据的分析表明，MAO中的“芳香笼子”的功能是包括底物结合和黄素辅酶的立体作用，并增加底物胺部分的亲核性。这些结果与提出的催化胺氧化的亲核机理是一致的。
Current structural results of several flavin-dependent amine oxidizing enzymes including human monoamine oxidases A and B (MAO A and MAO B) show aromatic amino acid residues oriented approximately perpendicular to the flavin ring, suggesting a functional role in catalysis. In the case of human MAO B, two tyrosyl residues (Y398 and Y435) are found in the Substrate binding site on the re face of the covalent flavin ring [Binda et a]. (2002) J. Biol. Chem. 277, 23973-23976]. To probe the functional significance of this structure, Tyr435 in MAO B was mutated with the amino acids Phe, His, Leu, or Trp, the mutant proteins expressed in Pichia pastoris, and purified to homogeneity. Each mutant protein contains covalent FAD and exhibits a high level of catalytic functionality. No major alterations in active site structures are detected on comparison, of their respective crystal structures with that of WT enzyme. The relative k(cat)/K-m values for each Mutant enzyme show Y435 > Y435F = Y435L = Y435H > Y435W. A similar behavior is also observed with the membrane-bound forms of MAO A and MAO B (MAO A Y444 mutant enzymes are found to be unstable on membrane extraction). p-Nitrobenzylamine is found to be a poor substrate while p-nitrophenethylamine is found to be a good substrate for all WT and mutant forms of MAO B. Analysis of these kinetic and structural data suggests the function of the "aromatic cage" in MAO to include a steric role in substrate binding and access to the flavin coenzyme and to increase the nucleophilicity of the substrate amine moiety. These results are consistent with a proposed polar nucleophilic mechanism for catalytic amine oxidation.