Schneck Jessica L, Villa James P, McDevitt Patrick, McQueney Michael S, Thrall Sara H, Meek Thomas D
Department of Biological Reagents and Assay Development and Discovery Technology Group, GlaxoSmithKline Pharmaceuticals, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426-0989, USA.
Biochemistry. 2008 Aug 19;47(33):8697-710. doi: 10.1021/bi8007627. Epub 2008 Jul 26.
Cathepsin C, or dipeptidyl peptidase I, is a lysosomal cysteine protease of the papain family that catalyzes the sequential removal of dipeptides from the free N-termini of proteins and peptides. Using the dipeptide substrate Ser-Tyr-AMC, cathepsin C was characterized in both steady-state and pre-steady-state kinetic modes. The pH(D) rate profiles for both log k cat/ K m and log k cat conformed to bell-shaped curves for which an inverse solvent kinetic isotope effect (sKIE) of 0.71 +/- 0.14 for (D)( k cat/ K a) and a normal sKIE of 2.76 +/- 0.03 for (D) k cat were obtained. Pre-steady-state kinetics exhibited a single-exponential burst of AMC formation in which the maximal acylation rate ( k ac = 397 +/- 5 s (-1)) was found to be nearly 30-fold greater than the rate-limiting deacylation rate ( k dac = 13.95 +/- 0.013 s (-1)) and turnover number ( k cat = 13.92 +/- 0.001 s (-1)). Analysis of pre-steady-state burst kinetics in D 2O allowed abstraction of a normal sKIE for the acylation half-reaction that was not observed in steady-state kinetics. Since normal sKIEs were obtained for all measurable acylation steps in the presteady state [ (D) k ac = 1.31 +/- 0.04, and the transient kinetic isotope effect at time zero (tKIE (0)) = 2.3 +/- 0.2], the kinetic step(s) contributing to the inverse sKIE of (D)( k cat/ K a) must occur more rapidly than the experimental time frame of the transient kinetics. Results are consistent with a chemical mechanism in which acylation occurs via a two-step process: the thiolate form of Cys-234, which is enriched in D 2O and gives rise to the inverse value of (D)( k cat/ K a), attacks the substrate to form a tetrahedral intermediate that proceeds to form an acyl-enzyme intermediate during a proton transfer step expressing a normal sKIE. The subsequent deacylation half-reaction is rate-limiting, with proton transfers exhibiting normal sKIEs. Through derivation of 12 equations describing all kinetic parameters and sKIEs for the proposed cathepsin C mechanism, integration of both steady-state and pre-steady-state kinetics with sKIEs allowed the provision of at least one self-consistent set of values for all 13 rate constants in this cysteine protease's chemical mechanism. Simulation of the resulting kinetic profile showed that at steady state approximately 80% of the enzyme exists in an active-site cysteine-acylated form in the mechanistic pathway. The chemical and kinetic details deduced from this work provide a potential roadmap to help steer drug discovery efforts for this and other disease-relevant cysteine proteases.
组织蛋白酶C，即二肽基肽酶I，是木瓜蛋白酶家族的一种溶酶体半胱氨酸蛋白酶，可催化从蛋白质和肽的游离N末端顺序去除二肽。使用二肽底物Ser-Tyr-AMC，对组织蛋白酶C进行了稳态和预稳态动力学模式的表征。log kcat/Km和log kcat的pH(D)速率曲线均符合钟形曲线，其中(D)(kcat/Ka)的逆溶剂动力学同位素效应(sKIE)为0.71±0.14，(D)kcat的正常sKIE为2.76±0.03。预稳态动力学显示AMC形成呈单指数爆发，其中最大酰化速率(kac = 397±5 s(-1))比限速脱酰化速率(kdac = 13.95±0.013 s(-1))和周转数(kcat = 13.92±0.001 s(-1))大近30倍。在D2O中对预稳态爆发动力学的分析允许提取酰化半反应的正常sKIE，这在稳态动力学中未观察到。由于在预稳态的所有可测量酰化步骤中都获得了正常的sKIE[(D)kac = 1.31±0.04，零时刻的瞬态动力学同位素效应(tKIE(0)) = 2.3±0.2]，导致(D)(kcat/Ka)逆sKIE的动力学步骤必须比瞬态动力学的实验时间框架发生得更快。结果与一种化学机制一致，其中酰化通过两步过程发生：Cys-234的硫醇盐形式在D2O中富集并产生(D)(kcat/Ka)的逆值，攻击底物形成四面体中间体，该中间体在表达正常sKIE的质子转移步骤中继续形成酰基酶中间体。随后的脱酰化半反应是限速的，质子转移表现出正常的sKIE。通过推导描述所提出的组织蛋白酶C机制的所有动力学参数和sKIE的12个方程，将稳态和预稳态动力学与sKIE相结合，为该半胱氨酸蛋白酶化学机制中的所有13个速率常数提供了至少一组自洽的值。对所得动力学曲线的模拟表明，在稳态下，约80%的酶以机制途径中活性位点半胱氨酸酰化的形式存在。这项工作推导的化学和动力学细节为指导针对该蛋白酶及其他与疾病相关的半胱氨酸蛋白酶的药物发现工作提供了潜在路线图。
