Site‐Directed Mutagenesis of Rice Serine Racemase: Evidence That Glu219 and Asp225 Mediate the Effects of Mg2+ on the Activity

Abstract

We succeeded in constructing the Glu219Ala/Asp225Ala (i.e., E219A/D225A) serine racemase (SerR) by site-directed mutagenesis, and the effects of Mg(2+) on the catalytic efficiency and the structure were compared between the E219A/D225A-SerR and the wild-type protein. This is the first example of a serine racemase whose amino acid residues in the Mg(2+)-binding site were replaced with other amino acids by site-directed mutagenesis. Neither the serine racemase nor the dehydratase activities of the E219A/D225A-SerR were affected by the addition of Mg(2+), and Glu219 and Asp225 of the SerR are the essential amino acid residues for Mg(2+) to affect both kinds of enzyme activities. Therefore, Glu219 and Asp225 mediate the effects of Mg(2+) on the activity and are important for the SerR to form the Mg(2+)-binding site. Judging from the difference of the K(eq) values between the E219A/D225A-SerR and the SerR, Mg(2+) might affect the equilibrium states in the racemase reaction. The fluorescence quenching analysis of the E219A/D225A-SerR showed that Mg(2+) bound to Glu219 and Asp225 of the SerR probably causes a conformational change in the ternary structure of the SerR.