M-CSA Mechanism and Catalytic Site Atlas

Peptidylglycine monooxygenase

peptidylglycine monooxygenase is a copper protein that acts on the terminal glycine residue of peptidylglycines. Peptidylglycines with a neutral amino acid residue in the penultimate position are the best substrates for the enzyme. In Rattus norvegicus this is functionality is part of a bifunctional enzyme. The monooxygenase part produces an unstable peptidyl(2-hydroxyglycine) as its product, which is then transported and dismutated to glyoxylate and the corresponding desglycine peptide amide by the lyase part. The C-terminal amidation of peptides such as neuropeptides is essential for full biological activity and involved in the final step of biosynthesis of alpha-melanotropin and related biologically active peptides.

Reference Protein and Structure

Sequence: P14925 (1.14.17.3, 4.3.2.5) (Sequence Homologues) (PDB Homologues)
Biological species: Rattus norvegicus (Norway rat)
PDB: 1sdw - Reduced (Cu+) peptidylglycine alpha-hydroxylating monooxygenase with bound peptide and dioxygen (1.85 Å)
Catalytic CATH Domains: 2.60.120.230 2.60.120.310 (see all for 1sdw)
Cofactors: Copper(2+) (2), L-ascorbate (1), Water (3) Metal MACiE

Click To Show Structure

Enzyme Reaction (EC:1.14.17.3)

dioxygen

CHEBI:15379

L-ascorbate

CHEBI:38290

glycino(1-) group

CHEBI:83148

→

water

CHEBI:15377

L-dehydroascorbate

CHEBI:58539

alpha-hydroxyglycino(1-) group

CHEBI:85224

Enzyme reaction links: IntEnz ENZYME ExplorEnz

Alternative enzyme names: PAM, PAM-A, PAM-B, Peptide alpha-amidating enzyme, Peptide alpha-amide synthase, Peptide-alpha-amide synthetase, Peptidyl alpha-amidating enzyme, Peptidylglycine 2-hydroxylase, Peptidylglycine alpha-amidating monooxygenase, Peptidylglycine alpha-hydroxylase, Synthase, peptide alpha-amide,

Summary
Step 1
Step 2
Step 3
Step 4
Step 5
Step 6
Step 7
Products
All Steps

Introduction

Ascorbate donates a single electron to one of the Cu(II) centres, which causes the hydroxide ligand to dissociate from the Cu(II) centre. A second ascorbate donates a single electron to the other Cu(II) centre, which causes the water ligand to dissociate from the Cu(II) centre. The two semidehydroascorbates disproportionate to yield ascorbate and dehydroascorbate (it is not clear at what stage this reaction occurs. For clarity it is shown at the end of the reaction cycle as occurring outside the enzyme's active site). One of the Cu(I) centres donates a single electron to a dioxygen molecule, which causes it to bind in a bidentate manner and displace the water ligand. The bound superoxide abstracts a hydrogen from the peptide substrate, and water displaces one of the bonds between the superoxide and the Cu(II) centre. In a homolytic substitution, the O-O bond is broken resulting in the hydroxylation of the peptide intermediate and the Cu(II) bound peroxo. Water coordinates to the Cu(I) centre, initiating a single electron relay through His108, Gln170, water and the peptide product to the Cu(II) centre, which then donates the electron to the bound oxo group, which deprotonates a water molecule to regenerate the active site.

Catalytic Residues Roles

UniProt	PDB* (1sdw)
His108	His108(66)A	Part if the Copper A binding site (also known as the CuM site), and part of a single electon relay chain that links the two copper sites.	single electron relay, hydrogen bond donor, metal ligand, single electron acceptor, single electron donor
Gln170	Gln170(128)A	Part of the single electron relay chain that links the two copper sites.	single electron relay, single electron donor, single electron acceptor, hydrogen bond acceptor
His107, His172	His107(65)A, His172(130)A	Forms part of the Copper A binding site (also known as the CuM site).	metal ligand
His244, Met314, His242	His244(202)A, Met314(272)A, His242(200)A	Forms part of the Copper B binding site (also known as the CuH site).	metal ligand

*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

redox reaction, radical formation, elimination (not covered by the Ingold mechanisms), overall reactant used, decoordination from a metal ion, intermediate formation, cofactor used, bimolecular homolytic addition, bimolecular nucleophilic substitution, coordination to a metal ion, hydrogen transfer, radical propagation, bimolecular homolytic substitution, overall product formed, proton transfer, electron transfer, radical termination, coordination, intermediate terminated, native state of enzyme regenerated, electron relay, reaction occurs outside the enzyme, native state of cofactor regenerated

References

Chen P et al. (2004), J Am Chem Soc, 126, 4991-5000. Oxygen Activation by the Noncoupled Binuclear Copper Site in Peptidylglycine α-Hydroxylating Monooxygenase. Reaction Mechanism and Role of the Noncoupled Nature of the Active Site. DOI:10.1021/ja031564g. PMID:15080705.
Abad E et al. (2014), J Biol Chem, 289, 13726-13738. Reaction mechanism of the bicopper enzyme peptidylglycine α-hydroxylating monooxygenase. DOI:10.1074/jbc.M114.558494. PMID:24668808.
Chauhan S et al. (2014), Biochemistry, 53, 1069-1080. Binding of copper and silver to single-site variants of peptidylglycine monooxygenase reveals the structure and chemistry of the individual metal centers. DOI:10.1021/bi4015264. PMID:24471980.
Rudzka K et al. (2013), J Biol Inorg Chem, 18, 223-232. Coordination of peroxide to the Cu(M) center of peptidylglycine α-hydroxylating monooxygenase (PHM): structural and computational study. DOI:10.1007/s00775-012-0967-z. PMID:23247335.
Meliá C et al. (2013), Chemistry, 19, 17328-17337. Investigation of the hydroxylation mechanism of noncoupled copper oxygenases by ab initio molecular dynamics simulations. DOI:10.1002/chem.201301000. PMID:24259416.
Kline CD et al. (2013), Biochemistry, 52, 2586-2596. HHM motif at the CuH-site of peptidylglycine monooxygenase is a pH-dependent conformational switch. DOI:10.1021/bi4002248. PMID:23530865.
Bauman AT et al. (2011), Biochemistry, 50, 10819-10828. A copper-methionine interaction controls the pH-dependent activation of peptidylglycine monooxygenase. DOI:10.1021/bi201193j. PMID:22080626.
Chufán EE et al. (2010), J Am Chem Soc, 132, 15565-15572. Differential reactivity between two copper sites in peptidylglycine α-hydroxylating monooxygenase. DOI:10.1021/ja103117r. PMID:20958070.
Woertink JS et al. (2010), Inorg Chem, 49, 9450-9459. Spectroscopic and computational studies of an end-on bound superoxo-Cu(II) complex: geometric and electronic factors that determine the ground state. DOI:10.1021/ic101138u. PMID:20857998.
McIntyre NR et al. (2009), J Am Chem Soc, 131, 10308-10319. Imino-oxy acetic acid dealkylation as evidence for an inner-sphere alcohol intermediate in the reaction catalyzed by peptidylglycine alpha-hydroxylating monooxygenase. DOI:10.1021/ja902716d. PMID:19569683.
Bauman AT et al. (2006), J Biol Chem, 281, 4190-4198. The hydrogen peroxide reactivity of peptidylglycine monooxygenase supports a Cu(II)-superoxo catalytic intermediate. DOI:10.1074/jbc.M511199200. PMID:16330540.
Siebert X et al. (2005), Biophys J, 89, 3312-3319. The catalytic copper of peptidylglycine alpha-hydroxylating monooxygenase also plays a critical structural role. DOI:10.1529/biophysj.105.066100. PMID:16100265.
Prigge ST et al. (2004), Science, 304, 864-867. Dioxygen Binds End-On to Mononuclear Copper in a Precatalytic Enzyme Complex. DOI:10.1126/science.1094583. PMID:15131304.
Prigge ST et al. (2000), Cell Mol Life Sci, 57, 1236-1259. New insights into copper monooxygenases and peptide amidation: structure, mechanism and function. DOI:10.1007/pl00000763. PMID:11028916.
Prigge ST et al. (1999), Nat Struct Biol, 6, 976-983. Substrate-mediated electron transfer in peptidylglycine alpha-hydroxylating monooxygenase. DOI:10.1038/13351. PMID:10504734.
Prigge ST et al. (1997), Science, 278, 1300-1305. Amidation of bioactive peptides: the structure of peptidylglycine alpha-hydroxylating monooxygenase. PMID:9360928.

Step 1. Ascorbate donates a single electron to one of the Cu(II) centres, which causes the hydroxide ligand to dissociate from the Cu(II) centre.

Residue	Roles
His108(66)A	hydrogen bond donor
Gln170(128)A	hydrogen bond acceptor
His107(65)A	metal ligand
His108(66)A	metal ligand
His172(130)A	metal ligand
His242(200)A	metal ligand
His244(202)A	metal ligand
Met314(272)A	metal ligand