Human PBGS Quaternary Structure Dynamics, Drugs, and Half-of-the-sites Reactivity

描述（由申请人提供）：人卟啉原合成酶（PBGS； E.C. 4.2.1.24）在血红素生物合成途径中催化基本生物单吡素——卟啉原的古老而必需的形成。它是一种锌金属酶，是铅中毒的靶酶。人类PBGS参与了一个包括高活性八聚体、低活性六聚体和二聚体两种构象的四元结构平衡，每一种构象都决定了进一步组装的化学计量。向低活性六聚体方向扰乱四级结构平衡的突变与ALAD卟啉症有关，这是一种罕见但严重的疾病状态。用左levan进行皮肤病变的光动力治疗需要PBGS活性。提出了两个不同的目标。目的1研究药物和环境污染物是否可以调节人PBGS的四级结构平衡，从而抑制或激活酶的活性。批准的药物库和已知的环境污染物将通过原生的PAGE迁移率转移测定来评估，以确定哪些成分可以将人类PBGS的四级结构平衡转移到低或高活性状态。已建立的方法将用于验证命中作为抑制剂或活化剂。抑制剂是铅中毒或卟啉症患者应避免使用的分子；活化剂可以缓解铅中毒的某些症状，作为ALAD卟啉症的治疗方法，或作为优化光动力治疗结果的辅助剂。再加上对人类等位基因变异如何影响组装状态平衡的理解，这些研究为四级结构在个性化医疗中的应用提供了新的途径。它们为脱靶副作用提供了可能的解释，也提出了标签外治疗的建议。AIM 2使用人类PBGS作为模型系统来理解半位点反应性（HSR）的结构基础。HSR类似于变构调节，在蛋白质的一个位置的结合现象深刻地影响在同一蛋白质的远端的结合/反应性行为。高铁需要亚基间的通讯，一般来说，所需通讯的分子基础知之甚少。我们对人类PBGS结构和机制的理解使我们能够调节个体亚基间接口，确定哪一个负责所需的通信，并探测个体残基以确定通信途径。定量希夫碱捕获将被用于诊断高铁。我们已经确定，当活性位点盖子关闭时发生的亚基间相互作用是维持活性八聚体所必需的。我们认为底物/产物结合对于活性位点盖闭合是必不可少的，因此对于维持八聚体也是必不可少的，并决定了对高铁的要求。拟议的研究将提高我们对交替的四级结构组合在人类疾病中的作用的理解，并将增强对HSR和某些形式的变构所必需的亚基间通信的一般知识。

DESCRIPTION (provided by applicant): Human porphobilinogen synthase (PBGS; E.C. 4.2.1.24) catalyzes the ancient and essential formation of the fundamental biological monopyrrole, porphobilinogen in the heme biosynthetic pathway. It is a zinc metalloenzyme and the target enzyme in lead poisoning. Human PBGS participates in a quaternary structure equilibrium that includes high activity octamers, low activity hexamers, and two conformations of a dimer, each of which dictates the stoichiometry of further assembly. Mutations that perturb the quaternary structure equilibrium toward the low activity hexamer are associated with ALAD porphyria, a rare but serious disease state. Photodynamic therapy of dermatologic lesions with the drug Levulan requires PBGS activity. Two distinct aims are proposed. AIM 1 investigates whether drugs and environmental contaminants can modulate the human PBGS quaternary structure equilibrium and thus inhibit or activate enzyme activity. Libraries of approved drugs and known environmental contaminants will be evaluated by a native PAGE mobility shift assay to determine which components can shift the human PBGS quaternary structure equilibrium toward either the low or high activity states. Established methods will be used to validate hits as inhibitors or activators. Inhibitors are molecules that should be avoided in patients with lead poisoning or porphyria; activators may alleviate some symptoms of lead poisoning, serve as a therapeutic for ALAD porphyria, or as adjuvant to optimize the outcome of photodynamic therapy. Coupled with the understanding of how human allelic variation affects the equilibrium of assembly states, these studies introduce a new approach to the application of quaternary structure to personalized medicine. They provide a possible explanation for off-target side effects, and also suggest off-label therapies. AIM 2 uses human PBGS as a model system for understanding the structural basis of half-of-the-sites reactivity (HSR). HSR is similar to allosteric regulation in that binding phenomena at one location in the protein profoundly affect the binding/reactivity behavior at a distant site in the same protein. Intersubunit communication is required for HSR and, in general, the molecular basis for the required communication is poorly understood. Our understanding of human PBGS structure and mechanism allows us to modulate the individual intersubunit interfaces, determine which is responsible for the required communication, and probe individual residues to determine the communication pathway. Quantitative Schiff base trapping will be used as a diagnostic for HSR. We have established that intersubunit interactions that occur when the active site lid is closed are required for maintenance of the active octamer. We propose that substrate/product binding, which is essential for active site lid closure, is therefore also essential for maintenance of the octamer and dictates the requirement for HSR. The proposed studies will improve our understanding of the role of alternate quaternary structure assemblies in human disease and will enhance general knowledge about intersubunit communication essential for HSR and some forms of allostery. PUBLIC HEALTH RELEVANCE: Understanding protein structure-function relationships is essential to understanding the mechanism of action of myriad therapeutic agents. Investigation of porphobilinogen synthase, which participates in a newly discovered regulation mechanism involving quaternary structure dynamics, provides a novel structural basis for understanding the role of alternate protein assemblies in human disease, provides a mechanism for understanding drug side effects, and suggests new therapies. Inactivation of human PBGS is involved in the human conditions of ALAD porphyria and lead poisoning, the latter of which remains a pervasive public health problem in the United States and around the world.