PROTEIN ENGINEERING OF HISTIDINE DECARBOXYLASE

这项拟议研究的总体目标是进一步了解 酶的作用机制，并帮助定义蛋白质的规则， 设计 研究对象是组氨酸脱羧酶（HDC）， 乳酸杆菌30 a。 这种不寻常的蛋白质经历了一个自动激活步骤 形成其异戊酰辅因子。 它还表现出协同动力学， 似乎拥有一个矢量基板流系统的基板和 产品 将在HDC中进行五类位点定向突变，并且将 基于我们对酶的结构和动力学的理解。 第一章 已知活性位点残基如Ile 59、Tyr 62和Asp 63相互作用 与基板，并将被改变，以量化的重要性， 交互. 这些和其他残基也可能参与 HDC中观察到的协同动力学，这一现象最初由 另一个“跨界”残基Glu 66的诱变。 此外，更多 戏剧性的变化，和成对的变化，将给予一个更清晰的 关键残基及其相互作用的观点。 2)据猜测 阳离子组氨酸被引导到中心活性位点， 通过静电场效应催化三聚体。 这将由以下人员进行测试： 扰乱了环绕井的几种羧酸盐， 创建字段。 最初酰胺将取代羧酸盐，但 也可以引入阳性残基。 3)x射线结构表明 该HDC实际上可以拥有底物流动系统，其中底物 从上述中心孔进入，在异戊酰位点反应， 产品通过一个充满水的隧道存在， 催化位点的背面，穿过三聚体壁到外部。 突变将通过阻断通道来测试这一假设。 各种方式 4)将作出努力，以改变地形的 活性位点裂。 将进行突变，其目的是改变 HDC的底物特异性，可能使其作用于鸟氨酸， 赖氨酸或天冬酰胺。 此外，将努力调整催化剂， 通过转化Tyr容纳新α-酮丁酰基辅因子的位点 262到更小的Leu残基。 5)将进行突变以改变HDC 构象 一种会破坏六聚体结构， 三聚体。 另一个目标是减轻折叠压力， 自动激活

The overall goal of this proposed research is to further our understanding of the mechanisms of enzyme action, and to help define rules for protein design. The object of study is histidine decarboxylase (HDC) from Lactobacillus 30a. This unusual protein undergoes an autoactivation step forming its pyruvoyl cofactor. It also exhibits cooperative kinetics and appears to possess a vectorial substrate flow system for substrate and product. Five classes of site directed mutations will be made in HDC, and will be based on our understanding of the structure and kinetics of the enzyme. 1) Active site residues like Ile 59, Tyr 62, and Asp 63 are known to interact with substrate and will be altered to quantify the importance of those interactions. These and other residues may also participate in the cooperative kinetics seen for HDC, a phenomenon investigated initially by mutagenesis of another "cross boundary" residue, Glu 66. In addition more dramatic alterations, and pairs of changes, will be made to give a clearer view of key residues and their interactions. 2) It is suspected that cationic histidine is guided into a central active site well in the catalytic trimer by an electrostatic field effect. This will be tested by perturbing several carboxylates ringing the well which are suspected to create the field. Initially amides will replace the carboxylates, but positive residues may also be introduced. 3) The x-ray structure suggests that HDC may in fact possess a substrate flow system, in which substrate enters from the central well mentioned above, react at the pyruvoyl site, and the product exists through a water filled tunnel which runs from the back of the catalytic site, through the trimer wall to the outside. Mutations will be made to test this hypothesis by blocking the tunnel in various ways. 4) Efforts will be made to alter the topography of the active site cleft. Mutations will be made which will aim to alter substrate specificity of HDC, perhaps allowing it to act on ornithine, lysine or asparagine. Also, efforts will be made to adjust the catalytic site to accommodate a novel alpha-ketobutyroyl cofactor by converting Tyr 262 to a smaller Leu residue. 5) Mutations will be made to alter the HDC conformation. One kind will disrupt the hexameric structure and produce trimers. Another will aim to relieve the folding strain which drive autoactivation.