Grimsby J, Zentner M, Shih J C
University of Southern California, Department of Molecular Pharmacology and Toxicology, School of Pharmacy, Los Angeles, CA 90033, USA.
Life Sci. 1996;58(9):777-87. doi: 10.1016/0024-3205(95)02356-9.
Monoamine oxidase (MAO) A and B are flavoenzymes that catalyze the oxidative deamination of biogenic and xenobiotic amines. To search for domains that confer substrate and inhibitor selectivities, two chimeric proteins were constructed and expressed in yeast. The kinetic constants and IC50 values were determined for these chimeric enzymes using MAO-A/B selective substrates and inhibitors. Replacement of MAO-A amino acids 161-375 with the corresponding region of MAO-B, termed AB(161-375)A, converted MAO-A catalytic properties to MAO-B like ones. The specificity constants (k(cat)/K(m))for the oxidation of beta-phenylethylamine (PEA) (1.6 x 10(5) s-1 M-1) and benzylamine (2.4 x 10(4) s-1 M-1) by AB (161-375)A were similar to wild-type MAO-B (PEA, 8 x 10(5)s(-1) M(-1); benzylamine, 4.9 x 10(4) s(-1) M(-1). Serotonin (5-HT), a preferred substrate for MAO-A, was not oxidized by AB(161-375)A or wild-type MAO-B. Furthermore, (AB161-375)A was more sensitive to the MAO-B specific inhibitor deprenyl (IC50 2.7 +/- 0.4 x 10(-8) M) than to the MAO-A specific inhibitor clorgyline (IC50 5.4 +/- 0.8 x 10(-7) M). However, the reciprocal chimera in which a MAO-B segment was replaced with the corresponding region of MAO-A, termed ++(+BA152-366B), lacked catalytic activity. The lack of catalytic activity was not due to aberrant expression but rather an inactive protein as demonstrated by Western blot analysis. These results demonstrate that MAO-B amino acids 152-366 contain a domain(s) that confers substrate and inhibitor selectivity.
单胺氧化酶（MAO）A和B是黄素酶，可催化生物胺和外源性胺的氧化脱氨反应。为了寻找赋予底物和抑制剂选择性的结构域，构建了两种嵌合蛋白并在酵母中表达。使用MAO-A/B选择性底物和抑制剂测定了这些嵌合酶的动力学常数和IC50值。用MAO-B的相应区域替换MAO-A的161-375位氨基酸，即AB(161-375)A，可将MAO-A的催化特性转变为类似MAO-B的特性。AB(161-375)A氧化β-苯乙胺（PEA）（1.6×10⁵ s⁻¹ M⁻¹）和苄胺（2.4×10⁴ s⁻¹ M⁻¹）的特异性常数（kcat/Km）与野生型MAO-B相似（PEA为8×10⁵ s⁻¹ M⁻¹；苄胺为4.9×10⁴ s⁻¹ M⁻¹）。5-羟色胺（5-HT）是MAO-A的优选底物，不能被AB(161-375)A或野生型MAO-B氧化。此外，AB(161-375)A对MAO-B特异性抑制剂司来吉兰（IC50 2.7±0.4×10⁻⁸ M）比对MAO-A特异性抑制剂氯吉兰（IC50 5.4±0.8×10⁻⁷ M）更敏感。然而，将MAO-B片段替换为MAO-A相应区域的反向嵌合体，即++(+BA152-366B)，缺乏催化活性。催化活性的缺乏并非由于异常表达，而是如蛋白质免疫印迹分析所示为无活性蛋白。这些结果表明，MAO-B的152-366位氨基酸包含赋予底物和抑制剂选择性的结构域。
