Amino acid sequence of the nucleotide-binding site of D-(-)-beta-hydroxybutyrate dehydrogenase labeled with arylazido-beta-[3-3H]alanylnicotinamide adenine dinucleotide.
In the dark, arylazido-beta-alanylnicotinamide adenine dinucleotide (N3-NAD) can replace NAD as cofactor for D-(-)-beta-hydroxybutyrate dehydrogenase (BDH) purified from bovine heart mitochondria. When photoirradiated with visible light, N3-NAD forms a nitrene species that binds covalently to BDH and inhibits the enzyme. NAD(H) protects BDH against photolabeling and inhibition by N3-NAD [Yamaguchi, M., Chen, S., & Hatefi, Y. (1985) Biochemistry 24, 4912-4916]. In the present study, a tryptic peptide of purified BDH photolabeled with arylazido-beta-[3-3H] alanyl-NAD [( 3H]N3-NAD) was isolated and sequenced. The same tryptic peptide was also isolated from BDH not labeled with [3H]N3-NAD and sequenced. Both peptides indicated the sequence Met-Glu-Ser-Tyr-Cys-Thr-Ser-Gly-Ser-Thr-Asp-Thr-Ser-Pro-Val-Ile-Lys. The residue labeled with [3H]N3-NAD was Cys. This heptadecapeptide contains 14 uncharged residues and is marked by having in an undecapeptide segment 8 hydroxy amino acids located symmetrically around a central glycine.
D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D006884:Hydroxybutyrate Dehydrogenase / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology; D009243:NAD / Q000031:analogs & derivatives* / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D014316:Tritium; D014357:Trypsin
D001386:Azides; D010446:Peptide Fragments; C020606:arylazido-beta-alanyl-NAD; D009243:NAD; D014316:Tritium; D006884:Hydroxybutyrate Dehydrogenase; D014357:Trypsin