A two-domain structure for the two subunits of NAD(P)H:quinone acceptor oxidoreductase.
NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is a FAD-containing reductase that catalyzes a unique 2-electron reduction of quinones. It consists of 2 identical subunits. In this study, it was found that the carboxyl-terminal portion of the 2 subunits can be cleaved by various proteases, whereas the amino-terminal portion cannot. It was also found that proteolytic digestion of the enzyme can be blocked by the prosthetic group FAD, substrates NAD(P)H and menadione, and inhibitors dicoumarol and phenindione. Interestingly, chrysin and Cibacron blue, 2 additional inhibitors, cannot protect the enzyme from proteolytic digestion. The results obtained from this study indicate that the subunit of the quinone reductase has a 2-domain structure, i.e., an amino-terminal compact domain and a carboxyl-terminal flexible domain. A structural model of the quinone reductase is generated based on results obtained from amino-terminal and carboxyl-terminal protein sequence analyses and electrospray mass spectral analyses of hydrolytic products of the enzyme generated by trypsin, chymotrypsin, and Staphylococcus aureus protease. Furthermore, based on the data, it is suggested that the binding of substrates involves an interaction between 2 structural domains.
Chen
S
S
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Deng
P S
PS
Bailey
J M
JM
Swiderek
K M
KM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002918:Chymotrypsin / Q000378:metabolism; D001728:Dicumarol / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D006868:Hydrolysis; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D009243:NAD / Q000494:pharmacology; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000737:chemistry* / Q000378:metabolism; D009249:NADP / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010630:Phenindione / Q000494:pharmacology; D051381:Rats; D014357:Trypsin / Q000378:metabolism; D014812:Vitamin K / Q000494:pharmacology
D010446:Peptide Fragments; D009243:NAD; D014812:Vitamin K; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D010630:Phenindione; D001728:Dicumarol; D016660:NAD(P)H Dehydrogenase (Quinone); D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus