Functional and structural characterization of D-aspartate oxidase from porcine kidney: non-Michaelis kinetics due to substrate activation.
D-aspartate oxidase (DDO, EC 1.4.3.1) catalyzes dehydrogenation of D-aspartate to iminoaspartate and the subsequent re-oxidation of reduced FAD with O2 to produce hydrogen peroxide. In the mammalian neuroendocrine system, D-aspartate, a natural substrate, plays important roles in the regulation of the synthesis and secretion of hormones. To elucidate the kinetic and structural properties of native DDO, we purified DDO from porcine kidney to homogeneity, cloned the cDNA, and overexpressed the enzyme in Escherichia coli. The purified DDO was a homotetramer with tightly-bound FAD. The enzyme consisted of 341 amino acids and had GAGVMG as the dinucleotide binding motif and a C-terminal SKL peroxisomal-targeting signal sequence. Porcine DDO showed a strong affinity for meso-tartrate (Kd = 118 microM). The oxidase exhibited pronounced substrate activation at D-aspartate and D-glutamate concentrations, [S], higher than 0.2 and 4 mM, respectively, and the [S]/v versus [S] plot showed marked downward curvature (v, the initial velocity), whereas substrate inhibition occurred with N-methyl-D-aspartate. These kinetic properties of DDO suggested that at high substrate concentrations, the FAD-reduced form of the enzyme also catalyzes the reaction: the oxidative half-reaction precedes the reductive one. The present direct approach to the analysis of non-Michaelis kinetics is indispensable for understanding the functional properties of DDO.
Yamamoto
Atsushi
A
Department of Biochemistry and Molecular Biology, Shiga University of Medical Science, Seta, Ohtsu, Shiga 520-2192, Japan. atsu@belle.shiga-med.ac.jp
Tanaka
Hiroyuki
H
Ishida
Tetsuo
T
Horiike
Kihachiro
K
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001483:Base Sequence; D050804:D-Aspartate Oxidase / Q000737:chemistry / Q000378:metabolism*; D003998:Dicarboxylic Acids / Q000378:metabolism; D005260:Female; D018698:Glutamic Acid / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016202:N-Methylaspartate / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D016415:Sequence Alignment; D034421:Sus scrofa
D003998:Dicarboxylic Acids; D001224:Aspartic Acid; D018698:Glutamic Acid; D016202:N-Methylaspartate; D050804:D-Aspartate Oxidase; D010100:Oxygen