Haem peptide–protein interactions. Part 3.—The kinetics and mechanism of the interaction between human placental glutathione S-transferase π and the non-substrate ligand microperoxidase-8 (MP-8)

Abstract

The kinetics of the reaction between the haem-peptide microperoxidase-8 and human placental glutathione S-transferase π have been followed using the quenching of the MP-8 Soret absorbance, which is observed on mixing the two species. Under pseudo-first-order conditions ([MP-8]≫[GST]) and over a concentration range in which the MP-8 is monomeric in aqueous solution, the binding curves are biphasic. The rapid phase of binding is associated with a hydrophobic site at or close to the binding site for the co-substrate 1-chloro-2,4-dinitrobenzene (CDNB), while the slow-binding phase reflects hydrophobic interaction with or spatially close to the bilirubin binding site on the enzyme. The mechanism for the interaction has been shown to be of the random sequential form with perturbation due to solvational elimination of the fast site. These studies recommend MP-8 as a useful non-substrate ligand model for monomeric haemin with which to study the kinetics and mechanism of ligand binding to the glutathione S-transferases.