Tryptic digestion of NADH dehydrogenase from alkalophilic Bacillus.
The alkalophile NADH dehydrogenase (NADH: 2,6-dichlorophenolindophenol oxidoreductase) [EC 1.6.99.3] consists of two identical subunits of 65 kDa, and each subunit contains the catalytic and liposome-binding regions. On treatment with trypsin, the polypeptide exhibiting the liposome-binding property in one of the subunits was digested to form an enzymatically active hetero-dimer (40 and 65 kDa), and then the polypeptide in the other subunit was digested to form an active homo-dimer (40 and 40 kDa). The hetero-dimer bound to liposomes, but the homo-dimer did not. Kinetic analysis showed that removal of one or two of the polypeptides in the enzyme slightly affects its kinetic parameters. For all the enzyme species, NAD inhibited competitively with respect to NADH and non-competitively with respect to 2,6-dichlorophenolindophenol. The partially determined amino acid sequence of this alkalophile enzyme suggested that (i) a long random-coiled peptide (58 amino acid residues) or a portion of the peptide is located between the polypeptides with liposome-binding and catalytic properties, (ii) the polypeptide exhibiting liposome-binding property is in the amino terminal region of the enzyme, (iii) the amino acid sequences around the subtilisin and trypsin cleavage sites of the peptide are hydrophilic and on the surface of the protein molecule and therefore are susceptible to digestion, and (iv) the FAD-binding site is located near the amino terminal region of the catalytic region.
Xu
X M
XM
Department of Life Science, Faculty of Science, Tokyo Institute of Technology, Kanagawa.
Kanaya
S
S
Koyama
N
N
Sekiguchi
T
T
Nosoh
Y
Y
Ohashi
S
S
Tsuda
K
K
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D002268:Carboxypeptidases; D002851:Chromatography, High Pressure Liquid; D003579:Cytochrome Reductases / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005612:Freeze Drying; D006868:Hydrolysis; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009245:NADH Dehydrogenase / Q000032:analysis*; D010455:Peptides / Q000032:analysis; D012967:Sodium Dodecyl Sulfate; D014357:Trypsin
D008081:Liposomes; D046911:Macromolecular Substances; D010455:Peptides; D012967:Sodium Dodecyl Sulfate; D003579:Cytochrome Reductases; D009245:NADH Dehydrogenase; D002268:Carboxypeptidases; C022633:serine carboxypeptidase; D014357:Trypsin