Abstract
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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
1. Revised Figure 1 to clarify differences between Ki and KI. 2. Added 'Appendix A' with explanation of symbols. 3. Revised Discussion section, next to last paragraph. 4. Added reference [9]. 5. Reformatted manuscript to single-column.