Héctor de Lucio1, Miguel A Toro2, María-José Camarasa3, Sonsoles Velázquez3, Federico Gago4, Antonio Jiménez-Ruiz1. 1. Área de Bioquímica y Biología Molecular, Departamento de Biología de Sistemas, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain. 2. Centro Nacional de Secuenciación Genómica-CNSG, Universidad de Antioquia, Medellin, Antioquia, Colombia. 3. Departamento de Biomiméticos para el descubrimiento de Fármacos, Instituto de Química Médica (IQM-CSIC), Madrid, Spain. 4. Área de Farmacología, Departamento de Ciencias Biomédicas, Unidad Asociada Al IQM-CSIC, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain.
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 (Ki app ) at five different substrate concentrations. Analysis of the changes in Ki 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 (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.
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 infantumtrypanothione reductase (Li-TryR), analysis of the curvatures of the reaction progress curves at different inhibitor concentrations allowed us to define the apparent inhibitory constants (Ki app ) at five different substrate concentrations. Analysis of the changes in Ki 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 (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.
Authors: Antonio Ariza; Tim J Vickers; Neil Greig; Kirsten A Armour; Mark J Dixon; Ian M Eggleston; Alan H Fairlamb; Charles S Bond Journal: Mol Microbiol Date: 2006-02 Impact factor: 3.501
Authors: Miguel A Toro; Pedro A Sánchez-Murcia; David Moreno; Marta Ruiz-Santaquiteria; Juan Fernando Alzate; Ana Negri; María-José Camarasa; Federico Gago; Sonsoles Velázquez; Antonio Jiménez-Ruiz Journal: Chembiochem Date: 2013-06-06 Impact factor: 3.164
Authors: Mohammad Akhoundi; Tim Downing; Jan Votýpka; Katrin Kuhls; Julius Lukeš; Arnaud Cannet; Christophe Ravel; Pierre Marty; Pascal Delaunay; Mohamed Kasbari; Bruno Granouillac; Luigi Gradoni; Denis Sereno Journal: Mol Aspects Med Date: 2017-01-31