STRUCTURE-FUNCTION OF PYRUVOYL & PLP-DEPENDENT ENZYMES

碱性氨基酸脱羧酶的结构与功能关系 将通过X射线衍射和蛋白质工程技术进行研究。 大多数脱羧酶都需要磷酸吡哆醛(PLP)作为必需的 辅因子，而其他利用共价结合的a-酮酸。建议数 研究涉及每种辅因子类型的脱羧酶：丙酮酰- 依赖组氨酸脱羧酶(PRV-HDC)和依赖PLP的鸟氨酸 脱羧酶(PLP-ODC)，均来自革兰氏阳性乳杆菌30pha 存在于哺乳动物肠道中的细菌。相应的哺乳动物 酶不太适合进行X射线衍射研究。 组氨酸的脱羧基是唯一已知的 组胺的生物合成。组胺的生理作用包括 血管扩张、低血压、胃酸分泌、过敏反应和 调停过敏反应。来自L.30pha的PRV-HDC是一种 可诱导性酶，也是一类不寻常的 从体内的氨基酸产生自己的修复基团的酶 残留物。我们测定了低pH(双曲线)的X-射线结构 动力学)形式的PRV-HDC，并提出了一种作用机制 产品和缓蚀剂复合体的基础。HDC基因已被克隆 在乔恩·罗伯特斯博士和几个定点突变体的实验室里 准备好了。我们已经分离并结晶了几个这样的突变体 结晶学研究此外，我们还建议生产稳定的proHDC 突变株(S82a)研究该酶的激活特性，还 检查一种较高pH(S型动力学)的HDC。高分辨率数据 我们的哈姆林/徐州地区探测器将收集这些系统中的 系统和结构在大学的克雷超级计算机上得到了改进。 鸟氨酸脱羧酶(ODC)是一种依赖于PLP的酶，它能将 鸟氨酸到腐胺，腐胺是多胺精胺和 亚精胺。ODC在所有细胞中都有发现，但在 快速分裂细胞。它已被用作治疗癌症的靶点。 化疗和抑制剂α-DL-二氟甲基鸟氨酸已经 成功地用于治疗锥虫感染。这种酶是 通常以其与哺乳动物酶的快速周转速度而闻名 具有大约1小时的稳定半衰期。相比之下，来自L. 30α稳定，是大亚基(80 KDa)PLP的代表 脱羧酶。我们的ODC晶体衍射率超过2.4A分辨率 潜在的重原子衍生物已经被收集起来。我们建议 确定ODC的晶体结构，克隆ODC基因并测序， 并将其表达用于其活性的定点突变研究 地点。

The structure-function relationships of basic amino acid decarboxylases will be studied by X-ray diffraction and protein engineering techniques. Most decarboxylases require pyridoxal phosphate (PLP) as an essential cofactor while others utilize a covalently bound a-keto acid. The proposed studies involve a decarboxylase of each cofactor type: a pyruvoyl- dependent histidine decarboxylase (Prv-HDC) and a PLP-dependent ornithine decarboxylase (PLP-ODC), both from Lactobacillus 30alpha, a Gram-positive bacterium resident in the mammalian gut. The corresponding mammalian enzymes are less amenable for X-ray diffraction studies. The decarboxylation of histidine is the only known mechanism for the biosynthesis of histamine. Physiological effects of histamine include vasodilation, hypotension, gastric HCl secretion, anaphylaxis, and mediation of the allergic response. The Prv-HDC from L. 30alpha is an inducible enzyme, and the most thoroughly studied of an unusual class of enzymes that produce their own prosthetic group from an internal amino acid residue. We have determined the X-ray structure of a low pH (hyperbolic kinetics) form of Prv-HDC and have proposed a mechanism of action on the basis of product and inhibitor complexes. The gene for HDC has been cloned in the laboratory of Dr. Jon Robertus and several site-specific mutants prepared. We have isolated and crystallized several such mutants for crystallographic study. In addition, we propose to produce a stable proHDC mutant (S82A) to study the activation properties of this enzyme and also examine a higher pH (sigmoidal kinetics) form of HDC. High resolution data of these systems will be collected with our Hamlin/Xuong area detector system and the structures refined on the University's Cray supercomputer. Ornithine decarboxylase (ODC) is a PLP-dependent enzyme that converts ornithine to putrescine which is a precursor of the polyamines spermine and spermidine. ODC is found in all cells but in particularly high activity in rapidly dividing cells. It has been used as a target for cancer chemotherapy and the inhibitor alpha-DL-difluoromethylornithine has been used successfully to treat Trypanosomal infections. The enzyme is generally noted for its rapid turnover rate with the mammalian enzyme having a stable half-life of only about 1 hour. In contrast, ODC from L. 30alpha is stable and representative of large subunit (80kDa) PLP decarboxylase. Our ODC crystals diffract to beyond 2.4A resolution and two potential heavy atom derivatives have been collected. We propose to determine the crystal structure of ODC, to clone and sequence the ODC gene, and later express it for site-directed mutagenesis studies on its active site.