PAM (peptidylglycine alpha-amidating monooxygenase) is a bifunctional, copper- and zinc-dependent enzyme of the regulated secretory pathway that converts C-terminally glycine-extended peptides into C-terminally alpha-amidated peptides, a modification many neuropeptides and peptide hormones need for full biological activity. The luminal region contains two catalytic modules that act in sequence. The N-terminal peptidylglycine alpha-hydroxylating monooxygenase (PHM) is a dicopper, ascorbate- and O2-dependent enzyme that hydroxylates the alpha-carbon of the C-terminal glycine. The C-terminal peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) is a zinc- and calcium-containing six-bladed beta-propeller that cleaves the hydroxylated intermediate to release the amidated peptide and glyoxylate. The same chemistry converts N-fatty acylglycines to primary fatty acid amides in vitro (shown for the bovine enzyme). Alternative splicing produces single-pass type I membrane forms, which localise to the trans-Golgi network and secretory granule membranes and carry a cytosolic domain that governs trafficking through the secretory and endosomal pathways, and soluble forms that are secreted with granule contents. PAM is highly expressed in pituitary, islets and heart. In human beta cells it amidates chromogranin A, and coding variants that reduce its activity are associated with type 2 diabetes risk.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0001519 peptide amidation | IDA PMID:12699694 Expression and characterization of human bifunctional peptid... | ACCEPT | Summary: Peptide amidation, the process PAM catalyses, demonstrated with purified recombinant human bifunctional PAM. Reason: Human PAM carries out both steps of C-terminal alpha-amidation of glycine-extended peptides (PHM hydroxylation then PAL N-dealkylation). This is the defining biological process of the enzyme, and the human protein was assayed directly. Supporting Evidence: PMID:12699694 PAM is in charge of the formation of the C-terminal amides of biologically active peptides PMID:12699694 The bifunctional PAM possesses two catalytic domains in a single polypeptide, peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PAL, EC 4.3.2.5) |
| GO:0001519 peptide amidation | IDA PMID:2357221 Human peptidylglycine alpha-amidating monooxygenase: cDNA, c... | ACCEPT | Summary: Peptide amidation by truncated human PAM expressed in COS cells after cDNA cloning. Reason: The cached record is abstract-only, but it reports a functional amidating enzyme from the cloned human cDNA, and the curator saw the full assay. Consistent with later human-protein assays (PMID:12699694, PMID:30054598). Supporting Evidence: PMID:2357221 generated a functional PAM that showed a 20-fold increase of the activity compared to the control PMID:30054598 We subsequently developed a cell-free kinetic assay capable of measuring PAM amidating activity via spectrophotometric detection of converted glyoxylate, a by-product of the amidation reaction |
| GO:0001519 peptide amidation | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA-predicted peptide amidation; matches the experimentally established process. Reason: Redundant with the IDA rows but correct; the electronic prediction agrees with direct human-protein experiments. Supporting Evidence: PMID:12699694 PAM is in charge of the formation of the C-terminal amides of biologically active peptides |
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | TOO GENERAL | Summary: Generic catalytic activity from InterPro PHM/PAL signatures. Reason: True but uninformative: GO:0004504 peptidylglycine monooxygenase activity and GO:0004598 peptidylamidoglycolate lyase activity, both accepted here from human IDA, are descendants of catalytic activity. Supporting Evidence: PMID:12699694 The bifunctional PAM possesses two catalytic domains in a single polypeptide, peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PAL, EC 4.3.2.5) |
| GO:0004497 monooxygenase activity | IEA GO_REF:0000002 | TOO GENERAL | Summary: Monooxygenase activity from the copper type II ascorbate-dependent monooxygenase (PHM) domain signatures. Reason: Correct but entailed by the accepted GO:0004504 peptidylglycine monooxygenase activity (is_a GO:0016715 is_a GO:0004497). Supporting Evidence: PMID:12699694 The bifunctional PAM possesses two catalytic domains in a single polypeptide, peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PAL, EC 4.3.2.5) |
| GO:0004504 peptidylglycine monooxygenase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBD at PTN000073733 (Deuterostomia) for peptidylglycine monooxygenase activity; human PAM (P19021) is one of the descendant experimental evidences, alongside mouse, rat, bovine and zebrafish orthologs. Reason: Human PAM lies inside the deuterostome clade that inherited PHM activity (PANTHER subfamily PTHR10680:SF14), and the activity is directly assayed in human protein. The appearance of P19021 in the WITH/FROM list reflects its own experimental grounding, not circularity. Supporting Evidence: PMID:12699694 The bifunctional PAM possesses two catalytic domains in a single polypeptide, peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PAL, EC 4.3.2.5) PMID:30054598 We subsequently developed a cell-free kinetic assay capable of measuring PAM amidating activity via spectrophotometric detection of converted glyoxylate, a by-product of the amidation reaction |
| GO:0004504 peptidylglycine monooxygenase activity | IDA PMID:12699694 Expression and characterization of human bifunctional peptid... | ACCEPT | Summary: PHM (EC 1.14.17.3) activity of purified recombinant human bifunctional PAM. Reason: Core activity, experimentally established for the human protein; the purified enzyme contains the copper expected for the PHM dicopper site. Supporting Evidence: PMID:12699694 The bifunctional PAM possesses two catalytic domains in a single polypeptide, peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PAL, EC 4.3.2.5) PMID:12699694 Inductively coupled plasma detected 0.62 mol of Zn(2+) and 1.25 mol of Cu(2+) per mol of bifunctional PAM |
| GO:0004504 peptidylglycine monooxygenase activity | IDA PMID:2357221 Human peptidylglycine alpha-amidating monooxygenase: cDNA, c... | ACCEPT | Summary: PHM (EC 1.14.17.3) activity of truncated human PAM expressed in COS cells. Reason: Abstract-only cache, but the abstract reports the cloned enzyme as EC 1.14.17.3 and shows activity of the expressed protein; defer to the curator who read the full text. Supporting Evidence: PMID:2357221 generated a functional PAM that showed a 20-fold increase of the activity compared to the control |
| GO:0004504 peptidylglycine monooxygenase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined electronic annotation (ARBA, Rhea RHEA:21452, EC 1.14.17.3) for PHM activity. Reason: Agrees with the human IDA and the IBA for this activity. Supporting Evidence: PMID:12699694 The bifunctional PAM possesses two catalytic domains in a single polypeptide, peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PAL, EC 4.3.2.5) |
| GO:0004598 peptidylamidoglycolate lyase activity | IDA PMID:12699694 Expression and characterization of human bifunctional peptid... | ACCEPT | Summary: PAL (EC 4.3.2.5) activity of purified human bifunctional PAM; zinc-dependent, inhibited by EDTA and restored by Zn(2+). Reason: Core activity, directly assayed for the human enzyme. Note that PAINT did not propagate GO:0004598 from PTN000073733 (the family contains PHM-only proteins); human PAM nevertheless has direct evidence for PAL activity, so no transfer is needed. Supporting Evidence: PMID:12699694 The bifunctional PAM possesses two catalytic domains in a single polypeptide, peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PAL, EC 4.3.2.5) PMID:12699694 the addition of 1mM EDTA reduced the PAL activity by about 50%, but the decreased activity was recovered by the addition of an excess amount of Zn(2+) |
| GO:0004598 peptidylamidoglycolate lyase activity | IDA PMID:2357221 Human peptidylglycine alpha-amidating monooxygenase: cDNA, c... | ACCEPT | Summary: PAL activity attributed to cloned human PAM expressed in COS cells. Reason: Abstract-only cache: the abstract reports amidating activity but does not separate the PAL step. The full-length enzyme carries a PAL domain and PAL activity is independently established for human PAM (PMID:12699694); accept and defer to the curator. Supporting Evidence: PMID:2357221 generated a functional PAM that showed a 20-fold increase of the activity compared to the control PMID:12699694 The bifunctional PAM possesses two catalytic domains in a single polypeptide, peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PAL, EC 4.3.2.5) |
| GO:0004598 peptidylamidoglycolate lyase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined electronic annotation (ARBA, RHEA:20924, EC 4.3.2.5) for PAL activity. Reason: Agrees with direct human-protein evidence. Supporting Evidence: PMID:12699694 The bifunctional PAM possesses two catalytic domains in a single polypeptide, peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PAL, EC 4.3.2.5) |
| GO:0004598 peptidylamidoglycolate lyase activity | ISS GO_REF:0000024 | ACCEPT | Summary: PAL activity transferred from bovine PAM (P14925), a member of the same PANTHER subfamily. Reason: Consistent with the human IDA; the PAL domain and its zinc-ligand residues are conserved (UniProt Zn binding sites at 582, 687, 783). Supporting Evidence: PMID:12699694 The bifunctional PAM possesses two catalytic domains in a single polypeptide, peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PAL, EC 4.3.2.5) PMID:19604476 The active site is formed by a Zn(II) ion coordinated by three histidine residues; the substrate binds to this site with its alpha-hydroxyl group coordinated to the Zn(II) ion. |
| GO:0005507 copper ion binding | IEA GO_REF:0000002 | ACCEPT | Summary: Copper ion binding from the copper type II ascorbate-dependent monooxygenase domain signatures. Reason: PHM is a dicopper enzyme; purified human PAM contains about 1.25 mol Cu per mol, matching two copper sites with partial occupancy. The copper ions are the catalytic cofactor of the monooxygenase step. Supporting Evidence: PMID:12699694 Inductively coupled plasma detected 0.62 mol of Zn(2+) and 1.25 mol of Cu(2+) per mol of bifunctional PAM PMID:19604476 PHM is a dicopper, ascorbate-dependent monooxygenase that catalyzes the stereospecific Ξ±-hydroxylation of glycine-extended precursor peptides by molecular oxygen (O2). |
| GO:0005507 copper ion binding | ISS GO_REF:0000024 | ACCEPT | Summary: Copper ion binding transferred from bovine PAM. Reason: Supported by human metal analysis and conserved copper ligands (UniProt binding sites 102, 103, 167, 237, 239, 309). Supporting Evidence: PMID:12699694 Inductively coupled plasma detected 0.62 mol of Zn(2+) and 1.25 mol of Cu(2+) per mol of bifunctional PAM file:human/PAM/PAM-uniprot.txt Note=Binds 2 Cu(2+) ions per subunit. {ECO:0000250|UniProtKB:P14925}; |
| GO:0005509 calcium ion binding | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Calcium ion binding transferred from bovine PAM; the PAL beta-propeller binds a structural Ca(2+). Reason: The calcium site is structural (stabilising the PAL propeller), shown in the rat PAL crystal structure; human PAM conserves the Ca-binding residues (517, 584, 784). True, but not part of the catalytic function. Supporting Evidence: PMID:19604476 The calcium binds to three Ξ²-strands in a fashion that is consistent with its important structural role in enhancing the stability of the protein |
| GO:0005515 protein binding | IPI PMID:9837933 P-CIP1, a novel protein that interacts with the cytosolic do... | REMOVE | Summary: Interaction of the PAM cytosolic domain with P-CIP1 (RASSF9) in vitro. Reason: Generic protein binding is uninformative. The reported interaction (with the rat PAM-1 cytosolic domain, relevant to PAM trafficking) is not disputed, but no more specific molecular function of PAM is established by it; removal under the protein-binding policy does not mean the interaction is false. Supporting Evidence: PMID:9837933 In vitro, P-CIP1 interacts with the cytosolic domain of wild type PAM-1 |
| GO:0005576 extracellular region | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT IBD at PTN000840899 placing family members in the extracellular region (seeded by Drosophila and rat). Reason: Soluble PAM forms (alternative splicing isoforms 3/4 and luminal domains released by cleavage) are secreted from granules; human recombinant luminal PAM is constitutively released, and amidating activity is measurable in human plasma. Peptide amidation itself happens mainly in the TGN/secretory granule lumen, so the extracellular location is real but peripheral. Supporting Evidence: PMID:30054598 In line with previous observations, PAM was constitutively released into supernatant PMID:39137152 In addition, a reduced level of PAM-amidating activity (PAM-AMA) in plasma was observed in subjects with diabetes |
| GO:0005576 extracellular region | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Extracellular region from UniProt subcellular location (Secreted, isoforms 3 and 4). Reason: As for the IBA row: secreted soluble PAM is real, but not the main site of action. Supporting Evidence: file:human/PAM/PAM-uniprot.txt SUBCELLULAR LOCATION: [Isoform 3]: Secreted. Note=Secreted from PMID:39137152 In addition, a reduced level of PAM-amidating activity (PAM-AMA) in plasma was observed in subjects with diabetes |
| GO:0006518 peptide metabolic process | IEA GO_REF:0000002 | TOO GENERAL | Summary: Peptide metabolic process from the PHM/PAL InterPro entry. Reason: Entailed by the accepted GO:0001519 peptide amidation (is_a peptide modification is_a peptide metabolic process). Supporting Evidence: PMID:12699694 PAM is in charge of the formation of the C-terminal amides of biologically active peptides |
| GO:0008270 zinc ion binding | IDA PMID:12699694 Expression and characterization of human bifunctional peptid... | ACCEPT | Summary: Zinc content of purified human PAM and Zn-restoration of EDTA-inhibited PAL activity. Reason: Zinc is the catalytic metal of the PAL active site (three-His site in the PAL structure); human PAM contains Zn and needs it for PAL activity. Supporting Evidence: PMID:12699694 Inductively coupled plasma detected 0.62 mol of Zn(2+) and 1.25 mol of Cu(2+) per mol of bifunctional PAM PMID:12699694 the addition of 1mM EDTA reduced the PAL activity by about 50%, but the decreased activity was recovered by the addition of an excess amount of Zn(2+) PMID:19604476 The active site is formed by a Zn(II) ion coordinated by three histidine residues; the substrate binds to this site with its alpha-hydroxyl group coordinated to the Zn(II) ion. |
| GO:0010043 response to zinc ion | IDA PMID:12699694 Expression and characterization of human bifunctional peptid... | REMOVE | Summary: Response to zinc ion, based on restoration of EDTA-reduced PAL activity of purified PAM by excess Zn(2+). Reason: The experiment is in vitro cofactor depletion and reconstitution of a purified enzyme, which shows that PAL needs zinc as a cofactor. That is already captured by zinc ion binding (GO:0008270, accepted). GO:0010043 describes a change in cell or organism state in response to zinc; nothing in the study measures such a response. Supporting Evidence: PMID:12699694 the addition of 1mM EDTA reduced the PAL activity by about 50%, but the decreased activity was recovered by the addition of an excess amount of Zn(2+) |
| GO:0016020 membrane | HDA PMID:19946888 Defining the membrane proteome of NK cells. | MODIFY | Summary: Membrane, from a proteomic survey of NK-cell membranes. Reason: PAM isoforms 1 and 2 are single-pass type I membrane proteins, so membrane is correct but too general. The specific membrane is the secretory granule membrane (also annotated here by ISS and supported by human islet data). Proposed replacements: secretory granule membrane Supporting Evidence: file:human/PAM/PAM-uniprot.txt TRANSMEM 864..887 PMID:30054598 revealed co-localization of endogenous PAM with both insulin and glucagon vesicles |
| GO:0016020 membrane | IEA GO_REF:0000120 | MODIFY | Summary: Membrane, from InterPro and UniProt subcellular location. Reason: Correct but too general; refine to secretory granule membrane, which the gene already carries. Proposed replacements: secretory granule membrane Supporting Evidence: file:human/PAM/PAM-uniprot.txt TRANSMEM 864..887 PMID:34089560 an ascorbate- and copper-dependent membrane enzyme that enters secretory granules along with its soluble substrates |
| GO:0016715 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced ascorbate as one donor, and incorporation of one atom of oxygen | IEA GO_REF:0000002 | TOO GENERAL | Summary: Ascorbate-dependent monooxygenase activity from PHM domain signatures. Reason: Accurate description of the PHM reaction class (ascorbate as electron donor), but entailed by the accepted GO:0004504 peptidylglycine monooxygenase activity, which is_a GO:0016715. Supporting Evidence: PMID:35380039 ascorbate binds to the enzyme to accomplish reduction |
| GO:0030658 transport vesicle membrane | IEA GO_REF:0000044 | MODIFY | Summary: Transport vesicle membrane, mapped from UniProt 'secretory vesicle membrane'. Reason: PAM resides in the membrane of regulated secretory (dense-core) granules and the TGN. In GO, secretory granule membrane is not under transport vesicle membrane, so the SubCell mapping lands on a sibling branch. Replace with secretory granule membrane (lateral correction). Proposed replacements: secretory granule membrane Supporting Evidence: PMID:30054598 revealed co-localization of endogenous PAM with both insulin and glucagon vesicles PMID:34089560 an ascorbate- and copper-dependent membrane enzyme that enters secretory granules along with its soluble substrates |
| GO:0030667 secretory granule membrane | ISS GO_REF:0000024 | ACCEPT | Summary: Secretory granule membrane, transferred from rat PAM (P10731). Reason: Integral PAM enters secretory granules with its soluble substrates. Human islet immuno-EM places endogenous PAM on insulin and glucagon vesicles, which supports the transfer. Supporting Evidence: PMID:34089560 an ascorbate- and copper-dependent membrane enzyme that enters secretory granules along with its soluble substrates PMID:30054598 revealed co-localization of endogenous PAM with both insulin and glucagon vesicles |
| GO:0034599 cellular response to oxidative stress | IC PMID:39880951 C-terminal amides mark proteins for degradation via SCF-FBXO... | REMOVE | Summary: Cellular response to oxidative stress, inferred by curator from the FBXO31 C-terminal amide degron paper. Reason: I read the full text (PMC11821526). The paper shows that C-terminal amide-bearing proteins (CTAPs) form after oxidative stress by non-enzymatic oxidative backbone cleavage, which it reconstitutes in vitro without any enzyme, and that SCF-FBXO31 recognises and degrades them. PAM appears only as the enzyme that amidates secreted peptide hormones. The authors suggest CTAPs might also come from 'a to date unknown PAM-like lyase', which implies PAM itself was not shown to take part. No experiment in the paper perturbs or measures PAM, and PAM acts in the lumen of the secretory pathway, not the cytosol where CTAPs are cleared. The inference is not supported. Supporting Evidence: PMID:39880951 This mechanism facilitates binding and turnover of endogenous CTAPs that are formed after oxidative stress. |
| GO:0051604 protein maturation | IDA PMID:2357221 Human peptidylglycine alpha-amidating monooxygenase: cDNA, c... | KEEP AS NON CORE | Summary: Protein maturation, reflecting PAM's role in the final processing step of glycine-extended peptide precursors. Reason: Amidation is the final maturation step for many peptide hormones (for example, PAM amidates chromogranin A in human beta cells). The specific process is captured by GO:0001519 peptide amidation, which is not a descendant of protein maturation in GO, so this row is not a candidate for MODIFY-by-refinement. It is correct but broader than the core description. Supporting Evidence: PMID:2357221 generated a functional PAM that showed a 20-fold increase of the activity compared to the control PMID:30054598 We also show that PAM amidates the granule packaging factor Chromogranin A (CgA) |
| GO:0062112 fatty acid primary amide biosynthetic process | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Fatty acid primary amide biosynthesis, transferred from bovine PAM (P14925), which converts N-fatty acylglycines to primary fatty acid amides plus glyoxylate. Reason: Same two-step PHM/PAL chemistry on a different glycine-extended substrate, transferred within PANTHER subfamily PTHR10680:SF14 from bovine PAM. It is not assayed for the human enzyme and its physiological weight relative to peptide amidation is uncertain, so it is kept as non-core. Supporting Evidence: file:human/PAM/PAM-uniprot.txt step conversion of an N-fatty acylglycine to a primary fatty acid amide |
| GO:0070062 extracellular exosome | HDA PMID:19199708 Proteomic analysis of human parotid gland exosomes by multid... | KEEP AS NON CORE | Summary: Detected in proteomic analysis of human parotid-gland exosomes. Reason: High-throughput detection only. Plausible, because integral PAM cycles through endosomes and soluble PAM is secreted, but extracellular vesicles are not where PAM acts. |
| GO:0070062 extracellular exosome | HDA PMID:23533145 In-depth proteomic analyses of exosomes isolated from expres... | KEEP AS NON CORE | Summary: Detected in proteomic analysis of urinary exosomes from expressed prostatic secretions. Reason: As for the parotid exosome row: high-throughput detection, peripheral to function. |
| GO:0005802 trans-Golgi network | IDA PMID:30054598 Type 2 diabetes risk alleles in PAM impact insulin release f... | NEW | Summary: Integral-membrane human PAM localises to the trans-Golgi network in the human beta-cell line EndoC-betaH1, where secretory granules form and where peptide amidation starts. Reason: Direct human-cell localisation data. TGN is a well-established site of PAM in neuroendocrine cells and complements the granule membrane location. This is a location annotation, not a process claim. Supporting Evidence: PMID:30054598 Integral membrane WT-PAM localized to the trans-Golgi network (TGN), where secretory granules originate, whereas luminal WT-PAM displayed a punctate staining pattern and co-localized with endogenous insulin |
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Download this section (compressed HTML)Q: Does human PAM contribute at all to cytosolic C-terminal amide-bearing protein fragments formed under oxidative stress, or are these entirely non-enzymatic as PMID:39880951 suggests? Should UniProt's FUNCTION statement on oxidative-stress amidation be revised?
Q: Is fatty acid primary amide (e.g. oleamide) biosynthesis by PAM physiologically significant in human tissues, given that it has only been shown in vitro with the bovine enzyme?
Q: Should GO represent ascorbate as the electron donor of PHM (L-ascorbic acid binding) for PAM, given kinetic evidence for ascorbate binding in the rat PHM domain?
Experiment: Knock out or chemically inhibit PAM (4-phenyl-3-butenoic acid) in human cells, apply oxidative stress, and quantify C-terminally amidated cytosolic fragments by proteomics. This would test whether PAM contributes to CTAP formation.
Experiment: Run targeted lipidomics of primary fatty acid amides in PAM-deficient human cells or in carriers of the PAM loss-of-function variants Ser539Trp and Asp563Gly.
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