Pseudoirreversible slow-binding inhibition of trypanothione reductase by a protein-protein interaction disruptor

ABSTRACT: 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 (Ki ) 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 (Kiapp ) at five different substrate concentrations. Analysis of the changes in Kiapp 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 (Ki* ) 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.

• Autores: Toro Londoño, Miguel Ángel; de Lucio, Héctor; Camarasa, María José; Velázquez, Sonsoles; Gago, Federico; Jiménez Ruiz, Antonio
• Tipo de recurso: Article of investigation
• Fecha de publicación: 2020
• Institución: Universidad de Antioquia
• Repositorio: Repositorio UdeA
• Idioma: eng
• OAI Identifier: oai:bibliotecadigital.udea.edu.co:10495/44162
• Acceso en línea: https://hdl.handle.net/10495/44162
• Palabra clave: Dimerización; Dimerization; Inhibidores Enzimáticos; Enzyme Inhibitors; Leishmania infantum; NADH NADPH Oxidorreductasas; NADH, NADPH Oxidoreductases; Trypanosomatina; https://id.nlm.nih.gov/mesh/D019281; https://id.nlm.nih.gov/mesh/D004791; https://id.nlm.nih.gov/mesh/D018314; https://id.nlm.nih.gov/mesh/D009247; https://id.nlm.nih.gov/mesh/D014351
• Rights: openAccess
• License: https://creativecommons.org/licenses/by-nc-nd/2.5/co/