MECHANISM OF ACTION OF INORGANIC PYROPHOSPHATASES

无机焦磷酸酶的作用机制

• 批准号: 3225003
• 负责人: BARRY S. COOPERMAN
• 金额: $ 16万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-01-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3225003
• 关键词: Escherichia coli; Saccharomyces cerevisiae; acidity /alkalinity; active sites; biochemical evolution; chemical binding; chemical kinetics; circular dichroism; conformation; crystallization; divalent metal; electron spin resonance spectroscopy; enzyme mechanism; hydrolysis; hydropathy; magnesium; manganese; molecular rearrangement; nuclear magnetic resonance spectroscopy; phosphates; protein sequence; protein structure function; pyrophosphatase; pyrophosphates; site directed mutagenesis; stereochemistry; thermostability

Our major goal is to use site-specific mutagenesis together with rate, equilibrium, and structural studies to understand the basis for PPase catalysis of PPi hydrolysis by fully characterizing the active site structure and catalytic mechanism of S. cerevisiae PPase (Y1-PPase) and E. coli PPase (E-PPase). Phosphoryl transfer enzymes form one of the largest classes of enzymes in Nature, yet their mechanisms of action remain incompletely understood. Success in achieving our goal will provide a detailed understanding of the mechanism of action of an important member of this class and allow us to test the proposition that the catalytic mechanism and active site of soluble PPases are largely conserved evolutionarily. This work will involve the study of Y1-PPase and E-PPase variants mutated at putative site residues including: a) kinetic, binding, and stereochemical investigations directed toward determination of kinetic schemes, microscopic rate constants, pH-rate profiles, ligand [M2+, inorganic phosphate (Pi)] binding constants and stoichiometry; b) structural studies, including circular dichroism, dye binding, and thermal stability directed toward determining whether effects seen on mutation are limited to changes in the active site or rather reflect larger structural changes in the enzyme; c) NMR and EPR experiments directed toward measuring M2+:phosphoryl ligand and M2+:M2+ interactions at the enzyme active site; d) the preparation of crystals of E-PPase and Y1-PPase variants suitable for X-ray crystallographic analysis. The results of such studies, interpreted in the light of ongoing studies of the three-dimensional structures of both enzymes, will permit us to determine how site-specific mutations affect PPase. They also will reveal whether mutations at residues that are identical in Y1-PPase and E-PPase have the same relative effects on function and/or structure, thus permitting an important test of the notion of a conserved active site and mechanism for PPases. Successful completion of the work herein proposed will have three important consequences. First, it will provide a model for the mechanisms of other phosphoryl transfer enzymes that use several metal ions at the active site. Second, by exploring the differences in detail between the mechanism of procaryotic PPases, represented by E-PPase, and of eucaryotic PPases, represented by Y1-PPase, it may allow for the development of therapeutic agents that exploit these differences. Third, it could have important consequences for the treatment of calcium pyrophosphate dihydrate (CPPD) crystal deposition disease. A disorder of PPi metabolism is suspected in the above-mentioned disease. Recent work on bovine retinal PPase makes it likely that the human PPA clone will soon be available. The availability of this clone would provide an important tool for the further study and/or treatment of CPPD crystal deposition disease e.g., by gene therapy. Our detailed studies on Y1-PPase might suggest human PPase variants of heightened therapeutic effectiveness (e.g., by manipulation of pH optima, of Km values).

我们的主要目标是使用位点特异性诱变和速率， 平衡和结构研究以了解 PPase 的基础 通过充分表征活性位点催化 PPi 水解 酿酒酵母PPase (Y1-PPase)和E.的结构和催化机制。 大肠杆菌 PPase（E-PPase）。 磷酰基转移酶是最大的酶之一 自然界中的酶种类，但它们的作用机制仍然存在 不完全理解。 成功实现我们的目标将提供 详细了解重要成员的作用机制 这堂课让我们测试催化的命题 可溶性 PPase 的机制和活性位点在很大程度上是保守的 进化地。 这项工作将涉及 Y1-PPase 和 E-PPase 变体突变的研究 在推定位点的残基包括：a) 动力学、结合和 针对动力学测定的立体化学研究 方案、微观速率常数、pH 速率曲线、配体 [M2+、 无机磷酸盐 (Pi)] 结合常数和化学计量； b) 结构研究，包括圆二色性、染料结合和热学 稳定性旨在确定对突变的影响是否是 仅限于活性位点的变化或更确切地说反映了更大的结构 酶的变化； c) NMR 和 EPR 实验旨在测量 M2+：磷酰基配体和M2+：M2+在酶活性位点相互作用； d) 合适的E-PPase和Y1-PPase变体晶体的制备 用于 X 射线晶体学分析。 此类研究的结果根据正在进行的研究进行解释 两种酶的三维结构，将使我们能够 确定位点特异性突变如何影响 PPase。 他们还将揭晓 Y1-PPase 和 E-PPase 中是否存在相同残基的突变 对功能和/或结构具有相同的相对影响，因此 允许对保守活性位点的概念进行重要测试，并且 PPase 机制。 成功完成本文提出的工作将具有三个重要意义 结果。 首先，它将为其他机制提供一个模型。 磷酰基转移酶在活性位点使用多个金属离子。 其次，通过探讨两者机制的细节差异 以E-PPase为代表的原核PPase和真核PPase， 以 Y1-PPase 为代表，它可能有助于开发治疗药物 利用这些差异的代理。 第三，它可能具有重要意义 二水焦磷酸钙 (CPPD) 治疗的后果 晶体沉积病。 怀疑 PPi 代谢紊乱 上述疾病。 最近对牛视网膜 PPase 的研究使其 人类 PPA 克隆可能很快就会面世。 可用性 该克隆的研究将为进一步研究和/或 CPPD晶体沉积病的治疗，例如通过基因疗法。 我们的 对 Y1-PPase 的详细研究可能表明人类 PPase 变体 提高治疗效果（例如，通过控制最佳 pH 值， Km 值）。