REPROTONATION KINETICS OF ASPARTIC PROTEASES

本研究的目的是测试一种新的猪体内代谢动力学机制。 胃酶。该机制的核心特征是速率限制 活性部位天冬氨酸羧基Asp-32和Asp-32的再质子化 ASP-215。支持这一机制的初步证据包括 最大速度受溶剂氚同位素的影响，而对V/K无影响。 当使用V/K接近扩散控制的基板时。实验 用于检测限速再质子化的设计包括：溶剂氘 作为不同浓度和不同缓冲液的函数的同位素效应 PKA、产物抑制动力学、稳态同位素交换和 停流动力学。建议的第二个重要特点是 其机制是，再质子化的平衡点非常靠右； 因此，在产品释放之后但在此之前，酶的直接形式 反质子化有望在动力学上合成多肽 债券。旨在检测合成能力的实验包括 催化过程中标记产物向多肽底物的反向交换 周转率，但不是来自产品和游离酶本身。这个 意义本建议源于酶家族，该酶家族 胃酶属于天冬氨酸蛋白酶。胃蛋白酶作为模型的研究 一系列具有临床意义的酶，包括最值得注意的 HIV蛋白水解酶和肾脏肾素。类似的溶剂同位素效应也是如此 报告为肾素，这表明限制再质子化的速率可能是 天冬氨酸蛋白酶的共同特征。如果是这样的话，那么这个 机理将对缓蚀剂的设计具有重要的相关性； 这些酶应该被设计成与存在于 活体内的最大浓度，这一提议认为是一种形式的 尚未进行再质子化的游离酶。

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.