Background and Purpose: Peptide P4 was described as a dimerization disruptor of trypanothione reductase (TryR), a homodimeric enzyme essential for survival of trypanosomatids. Determination of the true inhibitory constant (K_i) for P4 was not achieved because reaction rates continuously decreased with time, even when substrate concentration was kept constant. The aim of this study was to find a suitable kinetic model that could allow characterization of the complex pattern of TryR inhibition caused by P4.Experimental Approach: After showing the slow‐binding and pseudoirreversible activity of P4 against Leishmania infantum trypanothione reductase (Li‐TryR), analysis of the curvatures of the reaction progress curves at different inhibitor concentrations allowed us to define the apparent inhibitory constants (K_i^app) at five different substrate concentrations. Analysis of the changes in K_i^app values allowed precise definition of the type of inhibition.Key Results: Li‐TryR inhibition by P4 requires two sequential steps that involve rapid generation of a reversible enzyme–inhibitor complex followed by a pseudoirreversible slow inactivation of the enzyme. Recovery of enzyme activity after inhibitor dissociation is barely detectable. P4 is a non‐competitive pseudoirreversible inhibitor of Li‐ TryR that displays an overall inhibition constant (K_i^*) smaller than 0.02 μM.Conclusion and Implications: Li‐TryRdimer disruption by peptide P4 is a pseudoirreversible time‐dependent process which is non‐competitive with respect to the oxidized trypanothione (TS₂) substrate. Therefore, unlike reversible Li‐TryR competitive inhibitors, enzyme inhibition by P4 is not affected by the TS₂ accumulation observed during oxidant processes such as the oxidative burst in host macrophages.

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