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revised on 04.08.2020 and posted on 06.08.2020by Petr Kuzmic
This report describes an algebraic equation for the
time course of irreversible enzyme inhibition following a two-step mechanism. In the first step, the enzyme and the
inhibitor associate reversibly to form a non-covalent complex. In the second step, the noncovalent complex
is irreversibly converted to the final covalent conjugate. Importantly, the algebraic derivation was
performed under the steady-state approximation. Under the previously
invoked rapid-equilibrium approximation [Kitz & Wilson (1962) J.
Biol. Chem.237, 3245] it is by definition assumed that the rate
constant for the reversible dissociation of the initial noncovalent complex is
very much faster than the rate constant for the irreversible inactivation step.
In contrast, the steady-state algebraic
equation reported here removes any restrictions on the relative magnitude of
microscopic rate constants. The resulting formula was used in heuristic
simulations designed to test the performance of the standard rapid-equilibrium
kinetic model. The results show that if the inactivation rate constant is
significantly higher than the dissociation rate constant, the conventional
“kobs” method for evaluating the potency of covalent inhibitors in drug
discovery is incapable of correctly distinguishing between the two-step
inhibition mechanism and a simpler one-step variant, even for inhibitors that
have very high binding affinity in the reversible noncovalent step.
1. Revised Figure 1 to clarify differences between Ki and KI.
2. Added 'Appendix A' with explanation of symbols.
3. Revised Discussion section 'One-step kinetics of high-affinity covalent inhibitors'.
4. Added references  through  and .
5. Reformatted manuscript to single-column.