This paper describes an objective statistical approach that can be used to decide between two alternate kinetic mechanisms of covalent enzyme inhibition from kinetic experiments based on the standard "kobs" method. The two alternatives are either a two-step kinetic mechanism, which involves a reversibly formed noncovalent intermediate, or a one-step kinetic mechanism, proceeding in a single bimolecular step. Recently published experimental data [Hopper et al. (2020) J. Pharm. Exp. Therap. 372, 331-338] on the irreversible inhibition of Bruton tyrosine kinase (BTK) and tyrosine kinase expressed in hepatocellular carcinoma (TEC) by ibrutinib (PCI-32765) and acalabrutinib are used as an illustrative example. The results show that the kinetic mechanism of inhibition was misdiagnosed in the original publication for at least one of the four enzyme/inhibitor combinations. In particular, based on the available kobs data, ibrutinib behaves effectively as a one-step inhibitor of the TEC enzyme, which means that it is not possible to reliably determine either the inhibition constant Ki or the inactivation rate constant kinact, but only the covalent efficiency constant keff = kinact/Ki. Thus, the published values of Ki and kinact for this system are not statistically valid.

中文翻译：

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在“ kobs”数据的基础上决定一步和两步不可逆抑制机制：一种统计方法

本文介绍了一种客观的统计方法，该方法可用于从基于标准“ kobs”方法的动力学实验中确定共价酶抑制的两种替代动力学机制。两种选择要么是涉及可逆形成的非共价中间体的两步动力学机理，要么是在单个双分子步骤中进行的一步动力学机理。最近发表的实验数据[Hopper等。（2020年）J. Pharm。经验 治疗。[372，331-338]中关于依鲁替尼（PCI-32765）和acalabrutinib对肝细胞癌（TEC）中表达的不可逆地抑制Bruton酪氨酸激酶（BTK）和酪氨酸激酶的作用作为例证。结果表明，在原始出版物中对四种酶/抑制剂组合中的至少一种误解了抑制的动力学机制。特别是，根据现有的kobs数据，依鲁替尼可作为TEC酶的一步抑制剂有效发挥作用，这意味着不可能可靠地确定抑制常数Ki或失活速率常数kinact，而只能确定共价键。效率常数keff = kinact / Ki。因此，该系统的Ki和kinact的公开值在统计上无效。但只有共价效率常数keff = kinact / Ki。因此，该系统的Ki和kinact的公开值在统计上无效。但只有共价效率常数keff = kinact / Ki。因此，该系统的Ki和kinact的公开值在统计上无效。