Beswick Tracy Clyne, Willert Erin K, Phillips Margaret A
Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 6001 Forest Park Boulevard, Dallas, Texas 75390-9041, USA.
Biochemistry. 2006 Jun 27;45(25):7797-807. doi: 10.1021/bi0603975.
S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl-dependent enzyme that catalyzes an essential step in polyamine biosynthesis. The polyamines are required for cell growth, and the biosynthetic enzymes are targets for antiproliferative drugs. The function of AdoMetDC is regulated by the polyamine-precursor putrescine in a species-specific manner. AdoMetDC from the protozoal parasite Trypanosoma cruzi requires putrescine for maximal enzyme activity, but not for processing to generate the pyruvoyl cofactor. The putrescine-binding site is distant from the active site, suggesting a mechanism of allosteric regulation. To probe the structural basis by which putrescine stimulates T. cruzi AdoMetDC we generated mutations in both the putrescine-binding site and the enzyme active site. The catalytic efficiency of the mutant enzymes, and the binding of the diamidine inhibitors, CGP 48664A and CGP 40215, were analyzed. Putrescine stimulates the k(cat)/K(m) for wild-type T. cruzi AdoMetDC by 27-fold, and it stimulates the binding of both inhibitors (IC(50)s decrease 10-20-fold with putrescine). Unexpectedly CGP 48664A activated the T. cruzi enzyme at low concentrations (0.1-10 microM), while at higher concentrations (>100 microM), or in the presence of putrescine, inhibition was observed. Analysis of the mutant data suggests that this inhibitor binds both the putrescine-binding site and the active site, providing evidence that the putrescine-binding site of the T. cruzi enzyme has broad ligand specificity. Mutagenesis of the active site identified residues that are important for putrescine stimulation of activity (F7 and T245), while none of the active site mutations altered the apparent putrescine-binding constant. Mutations of residues in the putrescine-binding site that resulted in reduced (S111R) and enhanced (F285H) catalytic efficiency were both identified. These data provide evidence for coupling between residues in the putrescine-binding site and the active site, consistent with a mechanism of allosteric regulation.
S-腺苷甲硫氨酸脱羧酶（AdoMetDC）是一种依赖于丙酮酸的酶，它催化多胺生物合成中的关键步骤。多胺是细胞生长所必需的，而生物合成酶是抗增殖药物的作用靶点。AdoMetDC的功能受到多胺前体腐胺的物种特异性调控。原生动物寄生虫克氏锥虫的AdoMetDC需要腐胺来实现最大酶活性，但不需要腐胺来生成丙酮酸辅因子。腐胺结合位点与活性位点相距较远，提示存在变构调节机制。为了探究腐胺刺激克氏锥虫AdoMetDC的结构基础，我们在腐胺结合位点和酶活性位点都进行了突变。分析了突变酶的催化效率以及双脒抑制剂CGP 48664A和CGP 40215的结合情况。腐胺可使野生型克氏锥虫AdoMetDC的k(cat)/K(m)提高27倍，并刺激两种抑制剂的结合（腐胺存在时IC(50)降低10 - 20倍）。出乎意料的是，CGP 48664A在低浓度（0.1 - 10 microM）时激活克氏锥虫酶，而在高浓度（>100 microM）时或存在腐胺时则观察到抑制作用。对突变数据的分析表明，该抑制剂同时结合腐胺结合位点和活性位点，这证明克氏锥虫酶的腐胺结合位点具有广泛的配体特异性。活性位点的诱变确定了对腐胺刺激活性很重要的残基（F7和T245），而活性位点的突变均未改变表观腐胺结合常数。还鉴定出了腐胺结合位点中导致催化效率降低（S111R）和提高（F285H）的残基突变。这些数据为腐胺结合位点和活性位点的残基之间的偶联提供了证据，这与变构调节机制一致。
