REPROTONATION KINETICS OF ASPARTIC PROTEASES

天冬氨酸蛋白酶的重质子化动力学

• 批准号: 2184186
• 负责人: DEXTER B NORTHROP
• 金额: $ 11.68万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-01-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2184186
• 关键词: acidity /alkalinity; chemical kinetics; enzyme activity; enzyme mechanism; enzyme substrate; mass spectrometry; nonradiation isotope effect; pepsin; protease inhibitor; protonation; stable isotope double label; stop flow technique; synthetic peptide

The purpose of this research is to test a new kinetic mechanism for porcine pepsin. The central feature of this mechanism is a rate-limiting reprotonation of the active site aspartic carboxyl groups, Asp-32 and Asp-215. Preliminary evidence in support of this mechanism include a solvent deuterium isotope effect on the maximal velocity, with none on V/K, when using a substrate whose V/K approaches diffusion control. Experiments designed to detect a rate-limiting reprotonation include: solvent deuterium isotope effects as a function of buffers of different concentration and pKa, product inhibition kinetics, steady-state isotopic exchange, and stopped-flow kinetics. A second important feature of the proposed mechanism is that the equilibrium for reprotonation lies far to the right; hence, the immediate form of enzyme after product release but before reprotonation is expected to be kinetically competent to synthesize peptide bonds. Experiments designed to detect synthetic competence involve back-exchange of labeled products into peptide substrates during catalytic turnover, but not from the products and free enzyme alone. The significance this proposal derives from the family of enzymes to which pepsin belongs, the aspartic proteases. Studies on pepsin serve as models to a series of clinically-significant enzymes, including most notably the HIV protease and renal renin. Similar solvent isotope effects have been reported for renin, suggesting that a ratelimiting reprotonation may be a common feature of the aspartic proteases. If that is so, then this mechanism will have an important relevance to the design of inhibitors; these should be designed to bind to the form of enzyme that is present in the greatest concentration in vivo, which this proposal holds is a form of free enzyme that has not yet undergone reprotonation.

本研究的目的是测试一种新的猪的动力学机制， 胃蛋白酶 该机制的核心特征是速率限制 活性位点天冬氨酸羧基的再质子化，Asp-32和 天冬氨酸215 支持这一机制的初步证据包括 溶剂氘同位素对最大速度有影响，对V/K无影响， 当使用V/K接近扩散控制的衬底时， 实验 设计用于检测速率限制的重质子化包括：溶剂氘 同位素效应作为不同浓度缓冲液的函数， pKa、产物抑制动力学、稳态同位素交换，以及 停流动力学 第二个重要特点是， 其机制是，责备的均衡点远偏右; 因此，在产品释放之后但在产品释放之前， 预期再质子化在动力学上能够合成肽 债券 旨在检测合成能力的实验包括 在催化过程中标记产物交换回肽底物 营业额，但不是从产品和游离酶单独。 的 重要的是，这个建议来自酶家族， 胃蛋白酶属于天冬氨酸蛋白酶。 以胃蛋白酶为模型的研究 一系列具有临床意义的酶，包括最值得注意的 HIV蛋白酶和肾肾素。类似的溶剂同位素效应已经被 报告的肾素，这表明一个ratelimiting reprotoning可能是一个 天冬氨酸蛋白酶的共同特征。 如果是这样的话， 机理对缓蚀剂的设计具有重要意义; 这些应该被设计成与存在于细胞中的酶的形式结合， 体内的最大浓度，这一建议认为是一种形式， 还没有经历再质子化的游离酶。