Site-directed mutagenesis of rice serine racemase: evidence that Glu219 and Asp225 mediate the effects of Mg2+ on the activity.
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.
Gogami
Yoshitaka
Y
Department of Life Science and Biotechnology, Faculty of Chemistry, Materials, and Bioengineering, Kansai University, 3-3-35 Yamate-Cho, Suita, Osaka-Fu 564-8680, Japan.
Kobayashi
Ai
A
Ikeuchi
Toshihiko
T
Oikawa
Tadao
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry*; D001665:Binding Sites; D055162:Biocatalysis; D018698:Glutamic Acid / Q000737:chemistry*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012275:Oryza / Q000201:enzymology*; D019998:Racemases and Epimerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D000099300:Serine Racemase
D001224:Aspartic Acid; D018698:Glutamic Acid; D008274:Magnesium; D019998:Racemases and Epimerases; D000099300:Serine Racemase