COMPLETE ASSIGNMENT OF DISULFIDE BONDS IN BOVINE DOPAMINE-BETA-HYDROXYLASE

Peptide mapping, chemical sequencing, microbore HPLC/electrospray ioni zation mass spectrometry (LC/ESI/MS),(1) and matrix-assisted laser des orption mass spectrometry (MALDI/MS) were used to identify the sites o f intra- and intermolecular disulfide linkages in bovine dopamine beta -hydroxylase. The enzyme contains 14 cysteines and seven disulfides pe r monomer. Edman sequencing of tryptic and peptic peptides determined linkages at positions Cys140-Cys582, Cys218-Cys269, Cys255-Cys281, Cys 452-Cys474, Cys514-Cys514, and Cys516-Cys516, where cysteines at posit ions 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 th e N-terminus. The remaining intramolecular disulfides occur along two separate internal regions of the protein. The density of histidine res idues in these two regions suggests binding sites for two Cu2+ atoms p er monomer. In addition, previously identified amino acids that react with mechanism-based inactivators occur in these two regions. We propo se that these five internal disulfide bonds define two Cu2+ binding do mains that make up the active site of a dopamine beta-hydroxylase mono mer. Considering previous data on the location of glycosylation sites, mechanism-based inactivation sites, and the disulfide linkages presen ted here, the data suggest an overall topology where the N- and C-term ini are in close proximity and are solvent exposed and where the Cu2binding sites are buried in two interior domains stabilized by five di sulfide bonds.