Role of Modified Lysine Residues on Enzyme Kinetic of Bromoperoxidase

Abstract

Bromoperoxidase (BPO) from Thai red seaweed Gracilaria sp. has been purified and chemically modified with iodoacetamide under defined experimental conditions yielding derivatives of native bromoperoxidase. The modification increased the catalytic activity of the enzyme at concentrations of iodoacetamide up to a level of 50 mM. In order to investigate the molecular mechanism of the activation, the differences changes between native and modified enzymes were studied using kinetics methodology. The modified BPO showed greater affinity and maximal velocity for different substrates than native BPO. It has been shown that this modification reagent reacts with the ε-amino groups of the 15 out of the total 46 lysine residues of bromoperoxidase. The substrate affinity of modified BPO increased with the increases of substituents at ε-amino groups of lysine of the enzyme. The chemical modification of BPO increased its affinity for substrate monochlorodimedone (MCD) and hydrogen peroxide (about 2-and 110-fold, respectively) and also increased the maximal velocity with MCD, KBr and hydrogen peroxide (about 3-, 2.7 and 21-fold, respectively). The improvements of catalytic properties are also related to the changes in the number of ε-amino groups modified. The modification might involve neutralization of positive charges residues in BPO. The results presented in this study indicate that bromoperoxidase may acquire some new and useful characteristics related to stability and activity upon modification of specific amino acid side chains.