Complete assignment of disulfide bonds in bovine dopamine beta-hydroxylase.
Peptide mapping, chemical sequencing, microbore HPLC/electrospray ionization mass spectrometry (LC/ESI/MS), and matrix-assisted laser desorption mass spectrometry (MALDI/MS) were used to identify the sites of intra- and intermolecular disulfide linkages in bovine dopamine beta-hydroxylase. The enzyme contains 14 cysteines and seven disulfides per monomer. Edman sequencing of tryptic and peptic peptides determined linkages at positions Cys140-Cys582, Cys218-Cys269, Cys255-Cys281, Cys452-Cys474, Cys514-Cys514, and Cys516-Cys516, where cysteines at positions 514 and 516 on one monomer disulfide pair with their homologs on a second monomer. These linkages were confirmed by LC/ESI/MS and MALDI/MS. Further analysis by LC/ESI/MS and MALDI/MS identified linkages at positions Cys376-Cys489 and Cys380-Cys551. Cysteines 140 and 582 form a disulfide linkage that folds the C-terminus back in proximity to the N-terminus. The remaining intramolecular disulfides occur along two separate internal regions of the protein. The density of histidine residues in these two regions suggests binding sites for two Cu2+ atoms per monomer. In addition, previously identified amino acids that react with mechanism-based inactivators occur in these two regions. We propose that these five internal disulfide bonds define two Cu2+ binding domains that make up the active site of a dopamine beta-hydroxylase monomer. Considering previous data on the location of glycosylation sites, mechanism-based inactivation sites, and the disulfide linkages presented here, the data suggest an overall topology were the N- and C-termini are in close proximity and are solvent exposed and where the Cu2+ binding sites are buried in two interior domains stabilized by five disulfide bonds.
Robertson
J G
JG
Department of Chemistry, Pennsylvania State University, University Park 16802.
Adams
G W
GW
Medzihradszky
K F
KF
Burlingame
A L
AL
Villafranca
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004299:Dopamine beta-Hydroxylase / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D004220:Disulfides; D010446:Peptide Fragments; D004299:Dopamine beta-Hydroxylase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D003545:Cysteine