The Glu219 and Asp225 Residues of the Rice Serine Racemase Involve in Magnesium (II) Binding

Abstract

We constructed the Glu219Ala/Asp225Ala serine racemase (E219A/D225A SerR) by site-directed mutagenesis, and compared the effects of Mg²⁺ on the catalytic efficiency and the structure with those of the wild-type protein. The studies on the effects of Mg²⁺ on both enzyme activities in the Glu219Ala/Asp225Ala SerR revealed that the catalytic efficiencies (kcat/Km) of both serine racemase and dehydratase reactions in the E219A/D225A SerR were not affected by the addition of 1 mM Mg²⁺, and Glu219 and Asp225 of the SerR are the essential amino acids residues for Mg²⁺ to affect both enzyme activities. Therefore, these amino acids residues are important for the SerR to form the Mg²⁺-binding site. Judging from the difference of the Keq values between the E219A/D225A SerR and the SerR, Mg²⁺ might effect on the equilibrium states in the racemase reaction. The fluorescence quenching analysis of the E219A/D225A SerR showed that Mg²⁺ probably bound to Glu219 and Asp 225 of the SerR causes a structural change in the ternary structure of the SerR, and this is one of the reasons for Mg²⁺ to increase a serine racemase activity and decrease a serine dehydratase activity of the SerR.