title: Studies on Phe-228 and Leu-307 recombinant mutants of porcine kidney D-amino acid oxidase: expression, purification, and characterization.
abstract: Two recombinant mutants of porcine kidney D-amino acid oxidase [EC 1.4.3.3, DAO], in which Tyr(228) and His(307) are replaced with Phe and Leu, respectively, have been expressed in Escherichia coli and purified to apparent homogeneity. The molecular size and amino-terminal sequence of the two mutants were the same as those of the native DAO. Kinetic analysis revealed that the Michaelis constants of the Phe-228 and Leu-307 mutants for D-alanine were 71- and 10-fold and the inhibition constants for benzoate, a potent competitive inhibitor, were 1,189- and 18-fold greater than those of the native DAO, respectively. The maximum velocities of the Phe-228 and Leu-307 mutants were 66 and 58% that of the native DAO. The kinetically estimated dissociation constant of the Leu-307 mutant for FAD was 28-fold greater than that of the native DAO, whereas the value of the Phe-228 mutant was comparable to that of the native DAO. The Leu-307 mutant and the recombinant wild-type DAO were inactivated by D-propargylglycine (D-PG), a suicide substrate. However, the Phe-228 mutant was resistant to the inactivation. Absorption peaks of the Phe-228 mutant were blue-shifted about 10 nm from the corresponding peaks of the wild-type DAO, and the oxidized form was fully reduced by D-alanine without appearance of the purple intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, National Cardiovascular Center Research Institute, Osaka.
authors.forename: M
authors.initials: M
authors.lastname: Miyano
authors.forename: K
authors.initials: K
authors.lastname: Fukui
authors.forename: F
authors.initials: F
authors.lastname: Watanabe
authors.forename: S
authors.initials: S
authors.lastname: Takahashi
authors.forename: M
authors.initials: M
authors.lastname: Tada
authors.forename: M
authors.initials: M
authors.lastname: Kanashiro
authors.forename: Y
authors.initials: Y
authors.lastname: Miyake
mesh_terms: D000818:Animals; D001059:Apoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003605:D-Amino-Acid Oxidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine
chemical_list: D001059:Apoproteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D004247:DNA; D003605:D-Amino-Acid Oxidase