STRUCTURE/FUNCTION OF PYRUVOYL AND PLP DEPENDENT ENZYMES

本研究的目的是了解其结构和功能 氨基酸脱羧酶的相互关系和作用机制 磷酸吡哆醛(PLP)依赖酶的主要类别 可获得的结构性信息相对较少。脱羧酶是 重要的治疗靶点，产生生物胺，如 组胺、多巴胺和多胺。关于的结构信息 了解脱羧酶及其抑制物复合体是必要的 不同的PLP酶的足够详细，以帮助药物设计 以其特定的酶活性为靶标。 PLP的X射线结构、基因序列和效应性质-- 依赖的鸟氨酸脱羧酶(ODC)已被测定。我们建议 使用X射线结晶学，辅以现场测试技术- 定向诱变和稳态动力学，以检查 参与作用机制和效应器激活的关键残基 来自L.30a的ODC缓蚀剂复合体的结构将会回答 关于ODC的“封闭”和“开放”形式以及ODC的性质和 催化中间体的立体化学。X射线结构和动力学 定点突变体的特性将使特定的 对底物专一性和氨基酸残基的作用 结合、催化机制、GTP效应作用和亚基 互动。 基于L.30a中ODC结构的序列比较使我们得到了 表明至少有两个不同的结构族 脱羧酶。已经为两个人生产了X射线质量的晶体 第二类脱羧酶的例子，小鼠ODC(非常 与人类和锥虫ODCs密切相关)和生物合成 精氨酸脱羧酶(BADC)。这些化合物的X射线结构 酶的测定和机理研究将类似于 L.30a中为ODC提出的建议将扩展到这些系统。 自杀抑制剂小鼠ODC-DFMO(二氟甲基鸟氨酸)的晶体 用于治疗非洲昏睡病的ODC也已被 获得。这些酶代表了一种新的高度调控的， 用于X射线结构分析的治疗靶点。它们还共享一个 抗酶结合失活的新模型及条件 为了产生脱羧酶-抗酶复合体晶体将被筛选。

The goal of this research is to understand the structure-function relationships and mechanisms of action of amino acid decarboxylases, a major class of pyridoxal phosphate (PLP)-dependent enzymes for which relatively little structural information is available. Decarboxylases are important therapeutic targets, generating biogenic amines such as histamine, dopamine, and polyamines. Structural information on decarboxylases and their inhibitor complexes are necessary to understand different PLP enzymes in sufficient detail to aid in the design of drugs targeted against their specific enzymatic activities. The X-ray structure, gene sequence and effector properties of a PLP- dependent ornithine decarboxylase (ODC) have been determined. We propose to use X-ray crystallography, supplemented with the techniques of site- directed mutagenesis and steady state kinetics, to examine the roles of key residues involved in the mechanism of action and effector activation of ODC from L.30a. Structures of inhibitor complexes will answer questions about "closed" and "open" forms of ODC and the nature and stereochemistry of catalytic intermediates. X-ray structures and kinetic properties of site-directed mutants will enable the assignment of specific roles to amino acid residues responsible for substrate specificity and binding, catalytic mechanism, GTP effector action, and subunit interactions. Sequence comparisons based on the structure of ODC from L.30a led us to suggest that there are at least two distinct structural families of decarboxylases. X-ray quality crystals have been produced for two examples of second class of decarboxylases, mouse ODC (which is very closely related to the human and trypanosomal ODCs) and biosynthetic arginine decarboxylase (bADC) from E. coli. The X-ray structures of these enzymes will be determined and mechanistic studies similar to those proposed for the ODC from L.30a will be extended to these systems. Crystals of mouse ODC-DFMO (difluoromethyl ornithine), a suicide inhibitor of ODC used in the treatment of African sleeping sickness, have also been obtained. These enzymes represent a new class of highly regulated, therapeutic targets for X-ray structural analysis. They also share a novel model of inactivation resulting from antizyme binding and conditions to produce decarboxylase-antizyme complex crystals will be screened.