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Title of Journal: Amino Acids

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Abbravation: Amino Acids

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Springer Vienna

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DOI

10.1007/s002870050100

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1438-2199

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Role of the active site residues arginine216 and

Authors: Masumi Katane Yasuaki Saitoh Kazuhiro Maeda Toshihiko Hanai Masae Sekine Takemitsu Furuchi Hiroshi Homma
Publish Date: 2010/06/22
Volume: 40, Issue: 2, Pages: 467-476
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Abstract

dAspartate oxidase DDO and damino acid oxidase DAO are flavin adenine dinucleotidecontaining flavoproteins that catalyze the oxidative deamination of damino acids Unlike DAO which acts on several neutral and basic damino acids DDO is highly specific for acidic damino acids Based on molecular modeling and simulated annealing docking analyses a recombinant mouse DDO carrying two substitutions Arg216 to Leu and Arg237 to Tyr was generated R216LR237Y variant This variant and two previously constructed singlepoint mutants of mouse DDO R216L and R237Y variants were characterized to investigate the role of Arg216 and Arg237 in the substrate specificity of mouse DDO The R216LR237Y and R216L variants acquired a broad specificity for several neutral and basic damino acids and showed a considerable decrease in activity against acidic damino acids The R237Y variant however did not show any additional specificity for neutral or basic damino acids and its activity against acidic damino acids was greatly reduced The kinetic properties of these variants indicated that the Arg216 residue is important for the catalytic activity and substrate specificity of mouse DDO However Arg237 is apparently only marginally involved in substrate recognition but is important for catalytic activity Notably the substrate specificity of the R216LR237Y variant differed significantly from that of the R216L variant suggesting that Arg237 has subsidiary effects on substrate specificity Additional experiments using several DDO and DAO inhibitors also suggested the involvement of Arg216 in the substrate specificity and catalytic activity of mouse DDO and that Arg237 is possibly involved in substrate recognition by this enzyme Collectively these results indicate that Arg216 and Arg237 play crucial and subsidiary roles respectively in the substrate specificity of mouse DDOThe authors would like to thank Prof Ryuichi Konno International University of Health and Welfare for his kind gift of the mouse DAO expression plasmid and Mr Yutaka Taniguchi and Ms Mayumi Tetsuka Midori Nitta and Suno Masakawa for their technical assistance This work was supported in part by a GrantinAid for Scientific Research 21590071 from the Ministry of Education Culture Sports Science and Technology of Japan as well as by a Kitasato University Research Grant for Young Researchers to MK

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