Site-specific mutagenesis of lysine-204, tyrosine-224, tyrosine-228, and histidine-307 of porcine kidney D-amino acid oxidase and the implications as to its catalytic function.
In order to evaluate the possible contributions of Lys-204, Tyr-224, Tyr-228, and His-307 in porcine kidney D-amino acid oxidase [EC 1.4.3.3] (DAO) to its catalytic function, we constructed four point mutant cDNAs encoding enzymes possessing Glu-204, Phe-224, Phe-228, and Leu-307 by oligonucleotide-directed in vitro mutagenesis. The four mutant cDNAs and the wild type cDNA could be expressed in vitro with similar efficiencies and about 200 ng of each enzyme protein was produced from 5 micrograms of the respective capped RNA. The electrophoretic mobilities of the in vitro synthesized mutant enzymes on SDS-polyacrylamide gel were almost identical with that of the wild type DAO, and the molecular weight was calculated to be 38,000. The Glu-204 and Phe-224 mutant DAOs showed comparable enzyme activities to that of the wild type enzyme, and were inhibited strongly by sodium benzoate, a potent competitive inhibitor of DAO. The kinetic parameters of the two mutant DAOs were also comparable to those of the wild type DAO. On the other hand, the Phe-228 and Leu-307 mutant DAOs showed no detectable activity. The results indicate that Tyr-228 and His-307 play important roles as to the constitution of the active site or participate in the reaction directly, while Lys-204 and Tyr-224 are not essential in the enzyme reaction.
Watanabe
F
F
Department of Biochemistry, National Cardiovascular Center Research Institute, Osaka.
Fukui
K
K
Momoi
K
K
Miyake
Y
Y
D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D003605:D-Amino-Acid Oxidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D006639:Histidine / Q000032:analysis; D007668:Kidney / Q000201:enzymology*; D008239:Lysine / Q000032:analysis; D009154:Mutation; D010761:Phosphorus Radioisotopes; D013552:Swine; D014158:Transcription, Genetic; D014443:Tyrosine / Q000032:analysis
D010761:Phosphorus Radioisotopes; D014443:Tyrosine; D006639:Histidine; D004247:DNA; D003605:D-Amino-Acid Oxidase; D008239:Lysine