PROTEIN ENGINEERING OF HISTIDINE DECARBOXYLASE

这项拟议研究的总体目标是加深我们对 酶的作用机制，并帮助定义蛋白质的规则 设计。本研究的研究对象是人参皂苷脱羧酶 乳杆菌30a。这种不同寻常的蛋白质经历了一个自动激活的步骤 形成了它的丙酮酰辅因子。它还表现出合作动力学和 似乎拥有一套用于衬底的矢量衬底流动系统 产品。 将在HDC中进行五类定点突变，并将 基于我们对酶的结构和动力学的理解。1) 已知Ile 59、Tyr 62和Asp 63等活性位点残基相互作用 并将被改变以量化这些因素的重要性 互动。这些残基和其他残基也可能参与 HDC的合作动力学，这是一种最初由 另一个“跨界”残基Glu 66的诱变。此外，更多 戏剧性的变化，以及成对的变化，将给出一个更清晰的 关键残基及其相互作用的观点。2)怀疑 阳离子组氨酸被引导到一个中心活性部位 静电场效应催化三聚体。这将通过以下方式进行测试 扰乱了环绕油井的几种羧酸盐，怀疑 创建场。最初，酰胺将取代羧酸盐，但 也可以引入阳性残留物。3)X射线结构表明 HDC实际上可能具有衬底流动系统，其中衬底 从上面提到的中心井进入，在丙酮酰位置反应， 产品通过一个充满水的隧道存在，该隧道从 催化部位的背面，穿过三聚体的壁向外。 为了验证这一假说，将进行突变，方法是阻止隧道进入 不同的方式。4)将努力改变该地区的地形 活动部位裂开。将进行突变，目的是改变 HDC的底物特异性，可能允许它作用于鸟氨酸， 赖氨酸或天门冬氨酸。此外，还将努力调整催化剂 通过转化轮胎来容纳一种新的α-酮丁酰辅因子的位点 262到较小的亮氨酸残留物。5)将进行突变以改变HDC 构象。一种会破坏六聚体结构并产生 三聚体。另一项将旨在缓解驱动 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.