The Copper-Enzyme Family of Dopamine β-Monooxygenase and Peptidylglycine α-Hydroxylating Monooxygenase: Resolving the Chemical Pathway for Substrate Hydroxylation

Abstract

Dopamine β-monooxygenase (DβM) 2The abbreviations used are: DβM, dopamine β-monooxygenase; PHM, peptidylglycine α-hydroxylating monooxygenase; DFPEA, β,β-difluorophenethylamine. 2The abbreviations used are: DβM, dopamine β-monooxygenase; PHM, peptidylglycine α-hydroxylating monooxygenase; DFPEA, β,β-difluorophenethylamine. and peptidylglycine α-hydroxylating monooxygenase (PHM) belong to a small class of copper proteins found exclusively in higher eukaryotes. These physiologically important enzymes catalyze the transformation of dopamine to norepinephrine (DβM) (Equation 1) and C-terminal glycine-extended peptides to their α-hydroxylated products (PHM) (Equation 2). Although their substrate specificities are grossly different, these enzymes greatly resemble each other in many other respects.EQUATION 2View Large Image Figure ViewerDownload Hi-res image Download (PPT) Both enzymes are localized in subcellular compartments: the chromaffin vesicles of the adrenal gland or synaptic vesicles of the sympathetic nervous system (DβM) (1Stewart L.C. Klinman J.P. Annu. Rev. Biochem. 1988; 57: 551-591Crossref PubMed Google Scholar) and the secretory vesicles of the pituitary gland (PHM) (2Eipper B.A. Stoffers P.A. Mains R.E. Annu. Rev. Neurosci. 1992; 15: 57-85Crossref PubMed Scopus (559) Google Scholar). Although DβM and PHM exist in soluble and membrane-bound forms within their vesicular compartments (3Winkler H. Carmichael S.W. Poisner A.M. Trifaro J.M. The Secretory Granule. Elsevier Biomedical Press, Amsterdam1982Google Scholar, 4Prigge S.T. Mains R.E. Eipper B.A. Amzel L.M. Cell. Mol. Life Sci. 2000; 57: 1236-1259Crossref PubMed Scopus (373) Google Scholar), the majority of mechanistic studies have been completed with the more tractable soluble enzymes. The physiological roles played by soluble and membrane-bound enzymes may be different, but the chemical mechanisms are almost certain to be the same. The in vivo production of PHM is accompanied by the expression of a second activity that leads to the cleavage of the α-hydroxypeptide in Equation 2 to the biologically active, C-terminally carboxamidated peptide and glyoxylate (5Kulathila R. Merkler K.A. Merkler D.J. Nat. Prod. Rep. 1999; 16: 145-154Crossref PubMed Scopus (89) Google Scholar). This second activity is found either in a separate enzyme (peptidylglycine α-amidating lyase) or within a single polypeptide chain that also contains the activity of Equation 2 (peptidylglycine α-amidating monooxygenase). The in vitro study of the hydroxylating activity (Equation 2) is commonly performed with a construct in which the first 41 amino acids of PHM have been deleted, referred to as the PHM catalytic core (PHMcc). Comparison of the primary sequence of PHMcc with the larger DβM indicates a central core of ∼300 amino acids from DβM that is 27% identical and 40% homologous to PHM (6Lamoroux A. Vigny A. Faucon Biguet V. Darmon M.C. Frank R. Henry J.P. Mallet J. EMBO J. 1987; 6: 3931-3937Crossref PubMed Scopus (94) Google Scholar, 7Southan C. Kruse L.I. FEBS Lett. 1989; 255: 116-120Crossref PubMed Scopus (68) Google Scholar, 23Eipper B.A. Quon A.S.W. Mains R.E. Boswell J.S. Blackburn N.J. Biochemistry. 1995; 34: 2857-2865Crossref PubMed Scopus (102) Google Scholar). In addition, DβM contains ∼200 amino acids toward its N terminus and another ∼100 amino acids toward the C terminus that bear no relationship to PHM. Of particular note is the conservation of the ligands to the two coppers per enzyme subunit, designated as CuH and CuM. Using the numbering system of PHM, these are His-107, His-108, and His-172 for the CuH site and His-242, His-244, and Met-314 for the CuM site. Beginning in l997, a series of x-ray structures of different forms of PHM were published. The representation in Fig. 1 illustrates the disposition of the two copper centers per subunit and their respective ligands (8Prigge S.T. Kolhekar A.S. Eipper B.A. Mains R.E. Amzel L.M. Nat. Struct. Biol. 1999; 6: 976-983Crossref PubMed Scopus (159) Google Scholar). Additionally, a substrate analog, N-acetyl, diiodo-Tyr-Gly, is shown to bind close to the CuM site, adjacent to Arg-240, implicating the CM domain as the site of substrate hydroxylation. A recent x-ray structure of PHM with a slowly reacting substrate bound at the CuM site shows the presence of a bound O2 in close proximity to substrate as well (9Prigge S.T. Eipper B.A. Mains R.E. Amzel L.M. Science. 2004; 304: 864-867Crossref PubMed Scopus (358) Google Scholar). The structure of PHM reveals many striking and unexpected features. These include a lack of bridging ligands between the copper sites, consistent with earlier EPR studies that had failed to show any spin coupling between the paramagnetic copper centers (10Klinman J.P. Chem. Rev. 1996; 96: 2541-2561Crossref PubMed Scopus (800) Google Scholar). It is known that the two electrons consumed during substrate hydroxylation are stored in the CuH and CuM sites (11Brenner M. Klinman J.P. Biochemistry. 1