Possible copper enzyme homologues of prion protein:
Neurotransmitter Oxygenases

Aponeurocuprein inhibits dopamine beta-monooxygenase

Peptidylglycine alpha-amidating copper enzyme inactivated by sulfite
The multifunctional peptidylglycine alpha-amidating monooxygenase gene

Structure bovine of dopamine-beta-hydroxylase
Mechanism of dopamine beta-hydroxylase
Dopamine beta-hydroxylase: sulfur ligand to copper(I) in reduced enzyme
Role of copper and catalytic mechanism in dopamine beta-hydroxylase
Active site of tetrameric dopamine beta-hydroxylase
Active site labeling of dopamine beta-hydroxylase
CO coordination study of dopamine beta-hydroxylase
Homology of dopamine beta-hydroxylase and peptide alpha-amidating enzyme
bovine dopamine beta-hydroxylase amino acid sequence
Rat dopamine beta-hydroxylase: structure and regulation by reserpine

Aponeurocuprein inhibits dopamine beta-monooxygenase

Markossian KA; Paitian NA; Mikaelyan MV; Nalbandyan RM 
Biochem Biophys Res Commun 138: 1-8 (1986)
[This protein appears exactly once in Medline abstracts, in 1986. Please email if you know any more about it.]

The apoform of neurocuprein, the copper protein from brain and chromaffin granules, was found to be a potent inhibitor of the hydroxylating activity of dopamine beta-monooxygenase, whereas the holoform of neurocuprein has no effect on the activity of the enzyme. The inhibiting capacity of neurocuprein may be due to the property of the apoprotein to chelate copper from the enzyme. A role of neurocuprein as an endogenous protein regulator of dopamine beta-monooxygenase is suggested.


The multifunctional peptidylglycine alpha-amidating monooxygenase gene

Ouafik LH; Stoffers DA; Campbell TA; Johnson RC; Bloomquist BT; Mains RE; Eipper BA 
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205. 

Mol Endocrinol 6: 1571-84 (1992) 
Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction and is dependent on copper, ascorbate, and molecular oxygen. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalyzes the second step of the reaction. Previous studies demonstrated that alternative splicing results in the production of bifunctional PAM proteins that are integral membrane or soluble proteins as well as soluble monofunctional PHM proteins. Rat PAM is encoded by a complex single copy gene that consists of 27 exons and encompasses more than 160 kilobases (kb) of genomic DNA. The 12 exons comprising PHM are distributed over at least 76 kb genomic DNA and range in size from 49-185 base pairs; four of the introns within the PHM domain are over 10 kb in length. Alternative splicing in the PHM region can result in a truncated, inactive PHM protein (rPAM-5), or a soluble, monofunctional PHM protein (rPAM-4) instead of a bifunctional protein. The eight exons comprising PAL are distributed over at least 19 kb genomic DNA. The exons encoding PAL range in size from 54-209 base pairs and have not been found to undergo alternative splicing. The PHM and PAL domains are separated by a single alternatively spliced exon surrounded by lengthy introns; inclusion of this exon results in the production of a form of PAM (rPAM-1) in which endoproteolytic cleavage at a paired basic site can separate the two catalytic domains. The exon following the PAL domain encodes the trans-membrane domain of PAM; alternative splicing at this site produces integral membrane or soluble PAM proteins. The COOH-terminal domain of PAM is comprised of a short exon subject to alternative splicing and a long exon encoding the final 68 amino acids present in all bifunctional PAM proteins along with the entire 3'-untranslated region. Analysis of hybrid cell panels indicates that the human PAM gene is situated on the long arm of chromosome 5.
The reaction product of peptidylglycine alpha-amidating enzyme is a
hydroxyl derivative at alpha-carbon of the carboxyl-terminal glycine. 

Tajima M; Iida T; Yoshida S; Komatsu K; Namba R; Yanagi M; Noguchi M; Okamoto H 
Shiseido Basic Research Laboratories, Kanagawa, Japan. 

J Biol Chem 265: 9602-5 (1990) 
Abstract The peptidylglycine alpha-amidating enzyme catalyzes a reaction that transforms a carboxyl-terminal glycine-extended precursor into a carboxyl-terminal alpha-amidated peptide. We purified an alpha-amidating enzyme from equine serum by simplified steps including substrate affinity chromatography. With the purified enzyme, we detected an intermediate of the alpha-amidating reaction by high performance liquid chromatography analysis. The production of the intermediate required copper, oxygen, and ascorbate and increased linearly with incubation time. The structure of the intermediate was determined to be a hydroxyl derivative at the carboxyl-terminal glycine by fast atom bombardment mass spectrometry and by proton NMR. The intermediate was readily converted into an alpha-amidated product in alkaline conditions in a nonenzymic fashion. The nonenzymic conversion required no cofactor but was extremely accelerated by the addition of copper ion or at higher temperature. Our data suggest that the direct product of the alpha-amidating reaction is not an alpha-amidated peptide but a hydroxyl derivative at the alpha-carbon of the carboxyl-terminal glycine.
Characterization of peptidylglycine alpha-amidating activities in rat pituitary and brain.
Noguchi M; Takahashi K; Okamoto H 
Department of Biochemistry, Tohoku University School of Medicine, Sendai, Japan. 
Tohoku J Exp Med 156: 191-207 (1988)
Peptidylglycine alpha-amidating activities from rat pituitary, brain and small intestine were compared, utilizing C-terminal analogues of vasoactive intestinal polypeptide (VIP), D-Tyr-Leu-Asn-Gly and D-Tyr-Asn-Gly, and C-terminal analogue of alpha-MSH, D-Tyr-Val-Gly. The three tissues had enzymic activities capable of converting the glycine-extended peptides to the corresponding alpha-amidated ones.. The activities were stimulated in the presence of copper and ascorbate. The alpha-amidating enzymes from these tissues in common have a recognition site for the C-terminal glycine of the glycine-extended precursor regardless of the length and nature of the sequence. No fundamental differences were observed between the catalytic properties of the alpha-amidating activities from these three tissues, raising the possibility that similar enzymes, which may or may not be a single species, are functioning in tissues that produce alpha-amidated polypeptides in vivo.
Bifunctional peptidylglcine alpha-amidating enzyme requires two copper atoms
Kulathila R; Consalvo AP; Fitzpatrick PF; Freeman JC; Snyder LM; Villafranca JJ; Merkler DJ 
Analytical Protein and Organic Chemistry Group, Unigene Laboratories, Inc., Fairfield, New Jersey 07004. 

Arch Biochem Biophys 311: 191-5 (1994) 
The conversion of C-terminal glycine-extended peptides to C-terminal alpha-amidated peptides occurs in two distinct reactions, both of which are catalyzed by bifunctional peptidylglycine alpha-amidating enzyme. The first step is the alpha-hydroxylation of the C-terminal glycine residue and the second step is the dealkylation of the alpha-hydroxyglycine-extended peptide to the alpha-amidated peptide and glyoxylate. We show that the bifunctional enzyme requires 1.9 +/- 0.2 mol of copper/mol of enzyme for maximal dansyl-Tyr-Lys-Gly amidation activity
Peptidylglycine alpha-amidating enzyme is a monooxygenase.
Merkler DJ; Kulathila R; Consalvo AP; Young SD; Ash DE 
Analytical Protein & Organic Chemistry Group, Unigene Laboratories, Inc., Fairfield, New Jersey 07004. 

Biochemistry 31: 7282-8 (1992) 
The biosynthesis of C-terminal alpha-amidated peptides from their corresponding C-terminal glycine-extended precursors is catalyzed by peptidylglycine alpha-amidating enzyme (alpha-AE) in a reaction that requires copper, ascorbate, and molecular oxygen. We have shown that O2 is the source of th e alpha-carbonyl oxygen of pyruvate produced during the amidation. Two one-electron reductions by ascorbate occurred per alpha-AE turnover.
Characterization of peptidylglycine alpha-amidating monooxygenase in bovine hypothalamus.
Chikuma T; Kocha T; Hanaoka K; Kato T; Ishii Y; Tanaka A 
Department of Pharmaceutical Analytical Chemistry, Showa College of Pharmaceutical Sciences, Tokyo, Japan. 

Neurochem Int 25: 349-54 (1994) 
In many peptide hormones and neuropeptides, the carboxyl-terminal alpha-amide structure is essential in eliciting their biological activity. In the present study, an enzymatic activity capable of converting 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-Gly(Dabsyl-Gly-Phe -Gly) to 4-dimethylaminoazo-benzene-4'-sulfonyl-Gly-L-Phe-NH2(Dabsyl- Gly-Phe-NH2) was investigated in bovine hypothalamus. Two molecular forms of amidating activity were identified by size-exclusion chromatography and the molecular weight of the two enzymes were estimated to be 49 kDa and 69 kDa.
Peptidyl-alpha-hydroxyglycine alpha-amidating lyase. Purification, characterization, and expression.
Eipper BA; Perkins SN; Husten EJ; Johnson RC; Keutmann HT; Mains RE 
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205. 

J Biol Chem 266: 7827-33 (1991) 
The production of alpha-amidated peptides from their glycine-extended precursors is a two-step process involving the sequential action of two catalytic domains encoded by the bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) precursor. The NH2-terminal third of the PAM precursor contains the first enzyme, peptidylglycine alpha-hydroxylating monooxygenase (PHM), a copper, molecular oxygen, and ascorbate-dependent enzyme. The middle third of the PAM precursor contains the second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). The COOH-terminal third of the PAM precursor encodes a transmembrane domain and a hydrophilic domain that may form a cytoplasmic tail.
Proteolytic processing of prohormone
Bundgaard JR; Cowland JB; Vuust J; Rehfeld JF 
Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Denmark. 

DNA Cell Biol 15: 147-57 (1996) 
A novel system for heterologous expression of prohormones based on transient transfection of the HIT beta-cell line was established using human progastrin as a model. Progastrin was expressed at high levels compared to other gene transfer systems in endocrine cells, and the processing pattern was similar to that of normal antral gastrin cells. Thus, gastrin was partially tyrosine O-sulfated and carboxyamidated. . Glucose increased both the level of gastrin gene expression and maturation to carboxyamidated peptides, indicating that glucose influences the activity of the amidation enzyme complex, peptidylglycine alpha-amidating mono-oxygenase (PAM), in insulin cells. The mutant peptides displayed sulfation-dependent processing, supporting our recent suggestion that tyrosine sulfation increases the proteolytic processing of prohormones.

Peptidylglycine alpha-amidating copper enzyme inactivated by sulfite

Merkler DJ; Kulathila R; Francisco WA; Ash DE; Bell J 
Analytical Protein and Organic Chemistry Group, Unigene Laboratories, Inc., Fairfield, NJ 07004, USA. 

FEBS Lett 366: 165-9 (1995) 
Peptidylglycine alpha-amidating enzyme (alpha-AE) and dopamine beta-monooxygenase (D beta M), two copper-dependent monooxygenases that have catalytic and structural similarities, are irreversibly inactivated by sodium sulfite. . Sulfite inactivation of alpha-AE is specific for the monooxygenase reaction of this bifunctional enzyme and amidated peptides provide protection against the inactivation. Consequently, the sulfite-mediated inactivation of alpha-AE and D beta M most likely results from the transition metal-catalyzed oxidation of sulfite to the sulfite radical, SO3-.

Structure bovine of dopamine-beta-hydroxylase

Wu HJ; Parmer RJ; Koop AH; Rozansky DJ; O'Connor DT 
Department of Medicine, University of California, San Diego. 

J Neurochem 55: 97-105 (1990) 
Dopamine-beta-hydroxylase (DBH), the enzyme that catalyzes the conversion of dopamine to norepinephrine, remains the topic of many unanswered questions. We isolated DBH cDNA clones from a bovine adrenal medulla. The longest cDNA had an open reading frame encoding an entire mature DBH 578 amino acid (64,808 dalton) polypeptide chain, though lacking a portion of the signal peptide. Additional 5' clones, obtained by the polymerase chain reaction, established the sequence of a 19 amino acid signal peptide. The mature protein sequence was 84% homologous to that of human pheochromocytoma DBH, including preservation of four potential copper ligand sites (HH or HXH) and substrate binding domains. There were no hydrophobic (putative membrane spanning) domains, other than the signal peptide. All available DBH peptide and protein sequence data can be accounted for by the cDNA-deduced 578 amino acid mature protein primary structure. Prokaryotic DBH expression yielded a 65-kilodalton DBH-immunoreactive peptide that differed from eukaryotic adrenal DBH only in N-linked, endoglycosidase F-sensitive glycosylation in the latter. Comparison of the DBH primary structure with other reported sequences did not indicate that DBH is a member of any known gene family. The results suggest that a single DBH gene encodes a message specifying a single DBH polypeptide chain.

Rat dopamine beta-hydroxylase: structure and regulation by reserpine

McMahon A; Geertman R; Sabban EL 
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla 10595. 

J Neurosci Res 25: 395-404 (1990) 
The rat 2445 nucleotide sequence revealed a single open reading frame of 1860 nucleotides and a 3' untranslated region containing two polyadenylation addition signals. The cDNA coded for a 620 amino acid protein of 69,883 daltons. Six potential glycosylation sites and one potential phosphorylation site were identified. Amino acid residues likely to be involved in the active site of DBH and in copper ligand binding were identified. The N-terminal 42 amino acids appeared to constitute a typical but unusually long signal sequence. Hydropathy analysis indicated that this N-terminal region contained the only extensive hydrophobic domain and thus constituted the only obvious potential membrane attachment site. DBH mRNA levels were induced in vivo in rat adrenals upon treatment with reserpine.
CO coordination study of dopamine beta-hydroxylase
Pettingill TM; Strange RW; Blackburn NJ 
Department of Chemical and Biological Sciences, Oregon Graduate Institute of Science and Technology, Beaverton 97006-1999. 

J Biol Chem 266: 16996-7003 (1991) 
The carbon monoxide complex of ascorbate-reduced dopamine beta-hydroxylase has been prepared identifying the CO-binding site as the O2-binding site. Analysis of extended x-ray absorption spectroscopy data is most consistent with an average coordination per Cu of 2-3 histidines, 0.5 CO, and 0.5 S atoms as ligands, and absorption edge comparisons indicates pseudo-4 coordination as the most likely geometry at each Cu(I)center. The results can be interpreted by a model involving inequivalent 4-coordination at each Cu(I) center in the CO adduct with CuA His3S...CuB His2 CO-X as the coordination most consistent with all of the data.

Active site labeling of dopamine beta-hydroxylase

Farrington GK; Kumar A; Villafranca JJ 
Department of Chemistry, Pennsylvania State University, University Park 16802. 

J Biol Chem 265: 1036-40 (1990)
Each inhibitor labeled a unique tyrosine in the enzyme corresponding to Tyr477 in the primary sequence of the bovine enzyme and a unique histidine (His249 and an arginine at position 503.

Active site of tetrameric dopamine beta-hydroxylase

Blackburn NJ; Concannon M; Shahiyan SK; Mabbs FE; Collison D 
Department of Chemistry, University of Manchester Institute of Science and Technology, United Kingdom. 

Biochemistry 27: 6001-8 (1988) 
Results suggest a lower limit of ca. 7 A for the separation between the two copper ions per subunit and thus rule out a type 3 site in the oxidized enzyme. The data are most consistent with Cu(II) sites consisting of two or three N (imidazole) and one or two O donor ligands in the coordination sphere. The similarity in EPR spectra and power saturation of 8- and 4-Cu derivatives suggests that the difference in Cu-binding constants may be due either to differences in the identity of an axial ligand or to solvation effects in the active site.

Role of copper and catalytic mechanism in dopamine beta-hydroxylase

Klinman JP; Brenner M 
Department of Chemistry, University of California, Berkeley 94720. 

Prog Clin Biol Res 274: 227-48 (1988) 
Although classically characterized as a Type II copper protein, recent work has shown that D beta H requires two coppers per subunit for optimal activity. A major challenge has been to elucidate the relationship of these copper sites to one another, specifically whether there exists a single binuclear copper site or distinct metal sites catalyzing separate functions. The data support separate binding sites for reductants and product/substrate and hence, separate functions for each copper per subunit. A detailed catalytic mechanism for D beta H is discussed.

Dopamine beta-hydroxylase: sulfur ligand to copper(I) in reduced enzyme

Blackburn NJ; Hasnain SS; Pettingill TM; Strange RW 
Department of Chemical and Biological Sciences, Oregon Graduate Institute 0f Science and Technology, Beaverton 97006-1999. 

J Biol Chem 266: 23120-7 (1991) 
The structure of the copper sites in oxidized and reduced dopamine beta-hydroxylase has been studied by extended x-ray absorption fine structure spectroscopy. An histidine-rich active site has been found to be present with an average histidine coordination of between two and three histidine ligands per copper. In the oxidized protein, the data support four-coordination, involving two to three imidazole groups at 1.99 A with additional ligands derived from water or exogenous O-donor groups at an average distance of 1.94 A. A hypothesis for the structure of the copper sites has been proposed involving inequivalent CuA(His)3(H2O)...CuB-(His)2X(H2O) coordination in the oxidized enzyme, which upon reduction loses coordinated water and coordinates a sulfur probably from a methionine.

Sequence similarity between dopamine beta-hydroxylase and peptide alpha-amidating enzyme: evidence for a conserved catalytic domain.

Southan C; Kruse LI 
Department of Medicinal Chemistry, Smith Kline & French Research Limited, Welwyn, England. 

FEBS Lett 255: 116-20 (1989) 
A comparison of human dopamine beta-hydroxylase (EC 1.14.17.1) with bovine peptide C-terminal alpha-amidating enzyme (EC 1.14.17.3), revealed a 28% identity extending throughout a common catalytic domain of approximately 270 residues. The shared biochemical properties of these two enzymes from neurosecretory granules suggests that the sequence similarity reflects a genuine homology and provides a structural basis for a new family of copper type II ascorbate-dependent monooxygenases.

Mechanism-based inhibitors of dopamine beta-hydroxylase.


The copper-containing monooxygenase dopamine beta-hydroxylase catalyzes the hydroxylation of dopamine at the benzylic position to form norepinephrine.
Mechanism-based inhibitors for dopamine beta-hydroxylase have been used as probes of the mechanism of catalysis. The variety of such inhibitors that have
been developed for this enzyme can be divided into three groups: (i) those in which the inactivating species is formed by abstraction of a hydrogen atom to form a
radical intermediate; (ii) those in which the inactivating species is formed by abstraction of an electron to form an epoxide-like intermediate; and (iii) those in
which the product is the inactivating species. A mechanism consistent with inactivation by all three groups of inhibitors which proposes that hydroxylation of
dopamine by dopamine beta-hydroxylase involves formation of a benzylic radical has been developed. The benzylic radical is formed by abstraction of a hydrogen atom from the substrate by a high-potential copper-oxygen species. 


bovine dopamine beta-hydroxylase amino acid sequence

MQA H LS H QPCWSSLPSPSVREAASMYGTAVAIFLVILVAALRGS EPPESPFPY H IPLDPEGILELSWNVSYVQEII H FQLQVQGLRAGVLFGMSDRGEMENA DLIMLWSDGDRAYFADAWSDRKGQI H LDSQQDYQLLQAQRTRDGLSLLFKRPFVTCDP KDYVIEDDTV H LVYGILEEPFQSLEAINTSGL H TGLLRVQLLKSEVPTPSMPEDVQTM DIRAPDILIPDNEQTYWCYITELPPRFPR H H IIMYEAIVTEGNEALV H H MEVFQCAAE SEDFPQFNGPCDSKMKPDRLNYCR H VLAAWALGAKAFYYPKEAGVPFGGPGSSPFLRL EV H Y H NPRKIQGRQDSSGIRLPYTATLRRYDAGIMELGLVYTPLMAIPPQETAFVLTG YCTDKCTQMALQDSGI H IFASQL H T H LTGRKVVTVLARDGQERKEVNRDN H YSP H FRE IRMLKKVVTVYPGDVLITSCTYNTENKTLATVGGFGILEEMCVNYV H YYPQTELELCK SAVDDGFLQKYF H MVNRFSSEEVCTCPQASVPQQFSSVPWNSFNRNMLKALYDYAPIS M H CNKTSAVRFPGEWNLQPLPKITSTLEEPTPRCPIRQTQSPANPTVPITTGGRC