Inactivation of dopamine beta-hydroxylase by p-cresol: isolation and characterization of covalently modified active site peptides.
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.
DeWolf
W E
WE
Department of Medicinal Chemistry, Smith Kline & French Laboratories, King of Prussia, Pennsylvania 19406.
Carr
S A
SA
Varrichio
A
A
Goodhart
P J
PJ
Mentzer
M A
MA
Roberts
G D
GD
Southan
C
C
Dolle
R E
RE
Kruse
L I
LI
D000478:Alkylation; D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003408:Cresols / Q000494:pharmacology*; D004299:Dopamine beta-Hydroxylase / Q000032:analysis / Q000037:antagonists & inhibitors*; D004789:Enzyme Activation / Q000187:drug effects; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping
D003408:Cresols; D010446:Peptide Fragments; C032538:4-cresol; D004299:Dopamine beta-Hydroxylase