REPROTONATION KINETICS OF ASPARTIC PROTEASES

本研究的目的是记录和表征iso- 天冬氨酸蛋白酶的机制。 建议的主要特点 等离子体机理是部分限速再水合和再质子化 天冬氨酸羧基的活性部位。 这方面的证据 实验室支持天冬氨酸蛋白酶（和其他 酶）是多种多样的，包括：（a）溶剂同位素对最大 速度，（B）溶剂同位素对抑制起始的影响，（c） 溶剂同位素对抑制常数的影响，（d）产物抑制 动力学模式，（e）死端抑制模式，（f）进展曲线 分析，和（g）修改和传统的诱导运输动力学 布里顿（即，转肽作用和同位素交换）。 本提案所述的原始实验设计包括： (a)速率常数和同位素效应对回收率的比较 胃蛋白酶从时间依赖性抑制胃蛋白酶抑制剂引起的 底物的存在和不存在，（B）底物的稳态动力学 胃蛋白酶抑制剂对胃蛋白酶初始抑制作用与底物的关系 浓度和身份，（c）反应性变化的影响 水对等机制动力学，（d）质子库存对 转肽反应，（e）转肽的动力学能力 在羧基或氨基末端封闭的肽，和（f） 多肽瞬态动力学中猝发现象的研究 使用高灵敏度pH指示剂测定进行水解。 意义 该建议来源于胃蛋白酶所属的酶家族， 天冬氨酸蛋白酶 以胃蛋白酶为模型的一系列研究 具有临床意义的酶，包括最著名的艾滋病毒 蛋白酶和肾肾素。 类似的同位素效应也有报道， 这表明限速斥责可能是 天冬氨酸蛋白酶 如果是真的，那么这个动力学机制 这对抑制剂的设计有重要意义。 这些应该是 被指定为结合酶的形式，存在于最大的 浓度在体内，这一建议认为是一种形式的游离酶 还没有经过再水化和再生。

The purpose of this research is to document and characterize iso- mechanisms in aspartic proteases. The central features of the proposed iso-mechanism are partially rate-limiting rehydration and reprotonation of the aspartic carboxyl groups in the active site. Evidence from this laboratory in support of iso-mechanisms for aspartic proteases (and other enzymes) is diverse and includes: (a) solvent isotope effects on maximal velocities, (b) solvent isotope effects on onsets of inhibition, (c) solvent isotope effects on inhibition constants, (d) product inhibition kinetic patterns, (e) dead-end inhibition patterns, (f) progress curve analysis, and (g) modified and traditional induced transport kinetics of Britton (i.e., transpeptidation and isotopic exchange, respectively). Original experimental designs described in the current proposal include: (a) comparisons of rate constants and isotope effects on the recovery of pepsin from time-dependent inhibition by pepstatin incurred in the presence and absence of substrates, (b) steady-state kinetics of the initial inhibition of pepsin by pepstatin as a function of substrate concentration and identity, (c) effects of changes in the reactivity of water on iso-mechanism kinetics, (d) proton inventories on transpeptidation reactions, (e) kinetic competency for transpeptidation of peptides blocked at either the carboxyl or amino terminus, and (f) a search for burst phenomena in transient-state kinetics of peptide hydrolysis using highly sensitive pH indicator assays. 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 isotope effects have been reported, suggesting that a rate-limiting reprotonation may be a common feature of the aspartic proteases. If true, then this kinetic mechanism will have an important relevance to the design of inhibitors. These should be designated 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 rehydration and reprotonation.