Complete assignment of disulfide bonds in bovine dopamine beta-hydroxylase

Biochemistry. 1994 Sep 27;33(38):11563-75. doi: 10.1021/bi00204a019.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cattle
  • Cysteine / chemistry*
  • Disulfides / chemistry*
  • Dopamine beta-Hydroxylase / chemistry*
  • Mass Spectrometry / methods
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Peptide Mapping
  • Sequence Analysis
  • Serine Endopeptidases / metabolism
  • Trypsin / metabolism


  • Disulfides
  • Peptide Fragments
  • Dopamine beta-Hydroxylase
  • Serine Endopeptidases
  • glutamyl endopeptidase
  • Trypsin
  • Cysteine