Inactivation of Dopamine β-Hydroxylase by p-Cresol: Isolation and Characterization of Covalently Modified Active Site Peptides

Walter E. DeWolf, Steven A. Carr, Angela Varrichio, Paula J. Goodhart, Mary A. Mentzer, Gerald D. Roberts, Christopher Southan, Roland E. Dolle, Lawrence I. Kruse

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63 Scopus citations

Abstract

Recently, p-cresol has been shown to be a mechanism-based inhibitor of dopamine β-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-Val-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 />-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.

Original languageEnglish
Pages (from-to)9093-9101
Number of pages9
JournalBiochemistry
Volume27
Issue number26
DOIs
StatePublished - Dec 1 1988

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