Kinetic analysis for suicide-substrate inactivation of microperoxidase-11: A modified model for bisubstrate enzymes in the presence of reversible inhibitors

Abstract

Kinetics of microperoxidase-11 (MP-11) as a heme–peptide enzyme model in oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied in the presence of amino acids, taking into account the inactivation of MP-11 during reaction by its suicide substrate, H 2O 2. Reliability of the kinetic equation was evaluated by non-linear mathematical fitting. Fitting of experimental data into a new integrated kinetic relation showed a close match between the kinetic model and the experimental data. Indeed, it was found that the mechanism of suicide-peroxide inactivation of MP-11 in the presence of amino acids is different from MP-11 and/or horseradish peroxidase. In this mechanism, amino acids compete with hydrogen peroxide for the sixth co-ordination position of iron atom in the heme group through a competitive inhibition mechanism. The proposed model can successfully determine the kinetic parameters including inactivation by hydrogen peroxide as well as the inhibitory rate constants by the amino acid inhibitor. Kinetic parameters of inactivation including the initial activity of MP-11, α 0, the apparent inactivation rate constant, k i and the apparent inhibition rate constant for cysteine, k I were obtained 0.282 ± 0.006 min −1, 0.497 ± 0.013 min −1 and 1.374 ± 0.007 min −1 at [H 2O 2] = 1.0 mM, 27 °C, phosphate buffer 5.0 mM, pH 7.0. Results showed that inactivation and inhibition of microperoxidase as a peroxidase model enzyme occurred simultaneously even at low concentrations of hydrogen peroxide (0.4 mM). This kinetic analysis based on the suicide-substrate inactivation of microperoxidase-11, provides a tool and model for studying peroxidase models in the presence of reversible inhibitors. The introduced inhibition procedure can be used in designing activity tunable and specific protected enzyme models in the hidden and reversibly inhibited forms, which do not undergo inactivation.