Modulatory Effects of Mg2+ and Zn2+ Ions on Monoesterase Activity of Wild-type and Mutant E. coli Alkaline Phosphatases

Abstract

Metal cofactors and arginine-166 residue are active site participants in alkaline phosphatase catalysis. However, the mechanism by which the metal cofactors coordinate with arginine-166 residue during alkaline phosphatase catalysis is elusive. This study investigated the effects of Mg²⁺ and Zn²⁺ on monoesterase activity of wild-type and mutant E. coli alkaline phosphatases (ECAPs). The intact arginine-166 residue of wild-type ECAP was replaced by alanine and serine in the mutant ECAPs, R166A and R166S, respectively. Monoesterase activity of ECAP was measured by monitoring the rate of hydrolysis of para-nitrophenyl phosphate (pNPP). The monoesterase activity of wild-type ECAP was approximately 2-fold higher than the mutant ECAPs. Mg²⁺ (0.1-10mM) increased the activities of wild-type and mutant enzymes in a concentration-dependent manner. Zn²⁺ (0.05-0.1mM) slightly increased the activities of wild-type and mutants ECAPs. In the absence and in the presence of 10mM Mg²⁺ or 0.1mM Zn²⁺, the maximum reaction rate of wild-type ECAP was higher than those of the mutant ECAPs while its Michaelis constant was lower than those of the mutant ECAPs. Findings in this study revealed that monoesterase activity of ECAP was greatly reduced by the loss of arginine-166 residue but its modulation by Mg²⁺ and Zn²⁺ ions was independent of arginine-166 residue.