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

• Published in: Biochemistry (Easton) 1988-12, Vol.27 (26), p.9093-9101
• Main Authors: DeWolf, Walter E, Carr, Steven A, Varrichio, Angela, Goodhart, Paula J, Mentzer, Mary A, Roberts, Gerald D, Southan, Christopher, Dolle, Roland E, Kruse, Lawrence I
• Format: Article
• Language: English
• Subjects: Alkylation; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Binding Sites; Biological and medical sciences; Chromatography, High Pressure Liquid; Cresols - pharmacology; Dopamine beta-Hydroxylase - analysis; Dopamine beta-Hydroxylase - antagonists & inhibitors; Enzyme Activation - drug effects; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Mass Spectrometry; Models, Chemical; Molecular Sequence Data; Peptide Fragments - isolation & purification; Peptide Mapping
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00426a005

Recently, p-cresol has been shown to be a mechanism-based inhibitor of dopamine beta-hydroxylase (DBH; EC 1.14.17.1) [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.