Inactivation of dopamine beta-hydroxylase by p-cresol: isolation and characterization of covalently modified active site peptides

Biochemistry. 1988 Dec 27;27(26):9093-101. doi: 10.1021/bi00426a005.


Recently, p-cresol has been shown to be a mechanism-based inhibitor of dopamine beta-hydroxylase (DBH; EC [Goodhart, P. J., DeWolf, W. E., Jr., & Kruse, L. I. (1987) Biochemistry 26, 2576-2583]. This inactivation was suggested to result from alkylation of an active site residue by an aberrant 4-hydroxybenzyl radical intermediate. In support of this hypothesis, we report here the isolation and characterization of two modified tryptic peptides from DBH inactivated by p-cresol. Using a combination of automated Edman sequencing, mass spectroscopy (MS), and tandem MS, we have determined the sequence of the putative active site peptides, identified the site of attachment of p-cresol, and defined the chemical nature of the adduct formed. Both modified peptides are the same primary sequence: Ala-Pro-Asp-Val-Leu-Ile-Pro-Gly-Gln-Gln-Thr-Thr-Tyc-Trp-Cys-Tyr-Va l-Thr-Glu- Leu-Pro-Asp-Gly-Phe-Pro-Arg, where Tyc is an amino acid residue with the in-chain mass of a cresol-Tyr adduct (106 + 163 Da). Gas-phase deuterium exchange studies (employing N2H3-DCI MS) of the isolated phenylthiohydantoin (Pth) derivatives of modified residue 13 demonstrate that p-cresol forms two chemically distinct covalent adducts and support the hypothesis that a (4-hydroxyphenyl)methyl radical is generated during catalysis. Rearrangement to a (4-methylphenyl)oxy radical may also occur prior to inactivation.

MeSH terms

  • Alkylation
  • Amino Acid Sequence
  • Binding Sites
  • Chromatography, High Pressure Liquid
  • Cresols / pharmacology*
  • Dopamine beta-Hydroxylase / analysis
  • Dopamine beta-Hydroxylase / antagonists & inhibitors*
  • Enzyme Activation / drug effects
  • Mass Spectrometry
  • Models, Chemical
  • Molecular Sequence Data
  • Peptide Fragments / isolation & purification
  • Peptide Mapping


  • Cresols
  • Peptide Fragments
  • 4-cresol
  • Dopamine beta-Hydroxylase