Mutational analysis of the active site residues of a D: -psicose 3-epimerase from Agrobacterium tumefaciens.
D-Psicose 3-epimerase from Agrobacterium tumefacience catalyzes the conversion of D: -fructose to D-psicose. According to mutational analysis, the ring at position 112, the negative charge at position 156, and the positive charge at position 215 were essential components for enzyme activity and for binding fructose and psicose. The surface contact area and distance to the bound substrate by molecular modeling suggest that the positive charge of Arg215 was involved in stabilization of cis-endiol intermediate. The distances between the catalytic residues (Glu150 and Glu244) and Mn(2+) are critical to the catalysis, and the negative charges of the metal-binding residues are important for interaction with metal ion. The kinetic parameters of the D183E and H209A mutants for metal-binding residues with substrate and the near-UV circular dichroism spectra indicate that the metal ion bound to Asp183 and His209 is involved not only in catalysis but also in substrate binding.
Department of Bioscience and Biotechnology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, South Korea.
Hye-Jung
HJ
Kim
Soo-Jin
SJ
Yeom
Kwangsoo
K
Kim
Sangkee
S
Rhee
Dooil
D
Kim
Deok-Kun
DK
Oh
D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D001665:Binding Sites; D003198:Computer Simulation; D004252:DNA Mutational Analysis / Q000379:methods*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011485:Protein Binding; D019998:Racemases and Epimerases / Q000737:chemistry* / Q000235:genetics*
D019998:Racemases and Epimerases