Effects of hydrogen bonds in association with flavin and substrate in flavoenzyme d-amino acid oxidase. The catalytic and structural roles of Gly313 and Thr317.
According to the three-dimensional structure of a porcine kidney D-amino acid oxidase-substrate (D-leucine) complex model, the G313 backbone carbonyl recognizes the substrate amino group by hydrogen bonding and the side-chain hydroxyl of T317 forms a hydrogen bond with C(2)=O of the flavin moiety of FAD [Miura et al. (1997) J. Biochem. 122, 825-833]. We have designed and expressed the G313A and T317A mutants and compared their enzymatic and spectroscopic properties with those of the wild type. The G313A mutant shows decreased activities to various D-amino acids, but the pattern of substrate specificity is different from that of the wild type. The results imply that the hydrogen bond between the G313 backbone carbonyl and the substrate amino group plays important roles in substrate recognition and in defining the substrate specificity of D-amino acid oxidase. The T317A mutant shows a decreased affinity for FAD. The steady-state kinetic measurements indicate diminished activities of T317A to substrate D-amino acids. The transient kinetic parameters measured by stopped-flow spectroscopy revealed that T317 plays key roles in stabilizing the purple intermediate, a requisite intermediate in the oxidative half-reaction, and in enhancing the release of the product from the active site, thereby optimizing the overall catalytic process of D-amino acid oxidase.
Setoyama
Chiaki
C
Department of Biochemistry, Kumamoto University School of Medicine, Honjo, Kumamoto 860-0811, Japan.
Nishina
Yasuzo
Y
Tamaoki
Haruhiko
H
Mizutani
Hisashi
H
Miyahara
Ikuko
I
Hirotsu
Ken
K
Shiga
Kiyoshi
K
Miura
Retsu
R
D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism; D003605:D-Amino-Acid Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005415:Flavins / Q000737:chemistry / Q000378:metabolism*; D005998:Glycine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D003067:Coenzymes; D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; D013912:Threonine; D003605:D-Amino-Acid Oxidase; D005998:Glycine