Inactivation of dopamine .beta.-hydroxylase by .beta.-ethynyltyramine: kinetic characterization and covalent modification of an active site peptide

Abstract

beta-Ethynyltyramine has been shown to be a potent, mechanism-based inhibitor of dopamine beta-hydroxylase (DBH). This is evidenced by pseudo-first-order, time-dependent inactivation of enzyme, a dependence of inactivation on the presence of ascorbate and oxygen cosubstrates, the ability of tyramine (substrate) and 1-(3,5-difluoro-4-hydroxybenzyl)imidazole-2-thione (competitive multisubstrate inhibitor) to protect against inactivation, and a high affinity of beta-ethynyltyramine for enzyme. Inactivation of DBH by beta-ethynyltyramine is accompanied by stoichiometric, covalent modification of the enzyme. Analysis of the tryptic map following inactivation by [3H]-beta-ethynyltyramine reveals that the radiolabel is associated with a single, 25 amino acid peptide. The sequence of the modified peptide is shown to be Cys-Thr-Gln-Leu-Ala-Leu-Pro-Ala-Ser-Gly-Ile-His-Ile-Phe-Ala-Ser-Gln-Leu- His*- Thr-His-Leu-Thr-Gly-Arg, where His* corresponds to a covalently modified histidine residue. In studies using the separated enantiomers of beta-ethynyltyramine, we have found the R enantiomer to be a reversible, competitive inhibitor versus tyramine substrate with a Ki of 7.9 +/- 0.3 microM. The S enantiomer, while also being a competitive inhibitor (Ki = 33.9 +/- 1.4 microM), is hydroxylated by DBH to give the expected beta-ethynyloctopamine product and also efficiently inactivates the enzyme [kinact(app) = 0.18 +/- 0.02 min-1; KI(app) = 57 +/- 8 microM]. The partition ratio for this process is very low and has been estimated to be about 2.5. This establishes an approximate value for kcat of 0.45 min(-1) and reveals that (S)-beta-ethynyltyramine undergoes a slow turnover relative to that of tyramine (kcat approximately 50 s(-1), despite the nearly 100-fold higher affinity of the inactivator for enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence similarity between dopamine β‐hydroxylase and peptide α‐amidating enzyme: Evidence for a conserved catalytic domain

“…This idea is supported by the location of all 3 modified residues within the putative catalytic domain described above. Characterisation of the attachment site peptides [4,5] together with extensive partial sequence data (unpublished observations) indicate approximately 95°7o sequence identity between human and bovine DBH throughout this domain. Several consensus features in this section of the PAM sequences from 3 species are also present in DBH ( fig.3).…”

“…Independent evidence for an extended catalytic domain in DBH comes from attachment sites of mechanism-based inhibitors. Three of these have recently been identified, Tyr 216 [4] and Tyr 357 for p-cresol (unpublished results) and His 398 for fl-ethinyltyramine [5]. The chemistry of inactivation for these compounds indicates that covalent incorporation should be restricted to the vicinity of the active site.…”

“…The attachment site for ~-ethinyltyramine at His 398 was isolated as a 25 residue tryptic peptide that included a putative copper binding site [5]. A search of the protein sequence data banks with this peptide revealed, in addition to a match with the corresponding section from human DBH, a similarity with peptide C-terminal ce-amidating enzyme (PAM; EC 1.14.17.3) from Xenopus laevis [6].…”

“…In the course of our active-site mapping studies of bovine DBH we have located attachment sites for two mechanism-based inhibitors [4,5] and have determined approximately 85°70 of the primary structure (unpublished results). The attachment site for ~-ethinyltyramine at His 398 was isolated as a 25 residue tryptic peptide that included a putative copper binding site [5]. A search of the protein sequence data banks with this peptide revealed, in addition to a match with the corresponding section from human DBH, a similarity with peptide C-terminal ce-amidating enzyme (PAM; EC 1.14.17.3) from Xenopus laevis [6].…”

Molecular Cloning, Structure, and Expression of Dopamine‐β–Hydroxylase from Bovine Adrenal Medulla

“…DeWolf et al (1988,1989) have reported the isolation of two bovine DBH tryptic peptides which are labelled by substrate analogs-APDVLIPGZZTT-YWCYVTELPDGFPR, labelled by p-cresol , and CTQLALPASGIHIFASQLHTH-LTGR, labelled by P-ethinyltyramine (DeWolf et al, 1989). These peptides are contained within the bovine DBH primary structure (Figs.…”