Affinage

CAT

Glycine N-acyltransferase · UniProt Q6IB77

Audit flag: wrong gene
Length
296 aa
Mass
33.9 kDa
Annotated
2026-06-14
26 papers in source corpus 14 papers cited in narrative 14 extracted findings
Cross-family judge vs UniProt: Affinage preferred

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

GLYAT (acyl-CoA:glycine N-acyltransferase) is a mitochondrial matrix enzyme of liver and kidney that conjugates glycine to acyl-CoA donors, generating N-acylglycine products as a route for detoxification of benzoate and short/medium-chain acyl groups (PMID:7802672, PMID:22475485, PMID:33567294). The purified enzyme is a single-chain monomer of ~30–35 kDa bearing one active site per polypeptide, with a preference for benzoyl-CoA and additional activity toward salicyl-, isovaleryl-, octanoyl-, and fatty acyl-CoA substrates (PMID:457678, PMID:889778, PMID:7802672); by acting on fatty acyl-CoA it feeds a two-enzyme route to fatty acid primary amides via downstream peptidylglycine alpha-amidating enzyme (PMID:8660675). Catalysis proceeds through a general base mechanism in which a catalytic glutamate (Glu226/Glu227) deprotonates the glycine amine to enable nucleophilic attack on the acyl-CoA thioester, a mechanism supported by pH-rescue of an E226Q/E227Q mutant and by the crystal structure of the enzyme bound to benzoyl-CoA revealing a Glu-His catalytic dyad (PMID:22071172, PMID:23237781, PMID:40938199); Arg199 is essential for catalytic turnover, as its loss (R199C) abolishes activity by destabilizing a substrate-binding motif (PMID:23237781, PMID:33803916). Rather than simple Michaelis-Menten or ping-pong behavior, GLYAT displays cooperative (Ferdinand-type) kinetics with sigmoidal glycine activation and benzoyl-CoA substrate inhibition (PMID:28759163, PMID:33803916). Loss-of-function consequences are established in vivo: a homozygous nonsense variant (p.Q108Ter) abolishes activity and causes glycine N-acyltransferase deficiency with developmental delays in humans (PMID:40747359), and GLYAT upregulation in dorsal root ganglion neurons drives a ROS→TRPV1 axis that produces pain hypersensitivity in a mouse osteoarthritis model (PMID:40288509).

Mechanistic history

Synthesis pass · year-by-year structured walk · 11 steps
  1. 1979 High

    Established that glycine conjugation is carried out by discrete acyl-CoA:glycine N-acyltransferase enzymes with a defined ordered kinetic mechanism, settling the basic identity and reaction logic of the activity.

    Evidence Purification to near-homogeneity from bovine liver mitochondria with kinetic mechanism analysis

    PMID:457678

    Open questions at the time
    • Kinetic model later revised to cooperative rather than simple ordered
    • No structural basis for catalysis defined
    • Human enzyme not yet characterized
  2. 1977 High

    Demonstrated the enzyme is a single polypeptide with one acyl-CoA binding active site, defining its architecture and the location of the catalytic site.

    Evidence Photoaffinity labeling with p-azidobenzoyl-CoA plus competitive protection by benzoyl-CoA in beef liver enzyme

    PMID:889778

    Open questions at the time
    • No residue-level identification of catalytic groups
    • No structure
  3. 1986 Medium

    Resolved the subcellular compartment of the activity by showing it is exclusively intramitochondrial, distinguishing it from peroxisomal acyl-CoA metabolism.

    Evidence Subcellular fractionation with organelle-specific activity assays in rat liver

    PMID:3741707

    Open questions at the time
    • Submitochondrial location not pinpointed
    • Human localization not yet shown
  4. 1994 High

    Characterized the human liver enzyme and its substrate preference profile, anchoring later human genetic and mechanistic work to a defined monomeric protein.

    Evidence Purification to homogeneity and kinetic determination of Km for benzoyl-, salicyl-, isovaleryl-, and octanoyl-CoA from human liver

    PMID:7802672

    Open questions at the time
    • Catalytic residues not identified
    • Gene/sequence-level variant effects unaddressed
  5. 1996 Medium

    Extended the enzyme's biological scope by showing it produces N-fatty acylglycines that feed a two-enzyme pathway to fatty acid primary amides.

    Evidence In vitro enzyme assay with N-myristoylglycine and downstream peptidylglycine alpha-amidating enzyme

    PMID:8660675

    Open questions at the time
    • Physiological flux through this route not quantified
    • Single method for the GLYAT step
  6. 2012 High

    Identified the catalytic glutamate (Glu226/Glu227) and showed it acts as a general base, and demonstrated that natural human SNPs including the inactivating R199C alter activity, linking genotype to enzyme function.

    Evidence Active-site mutagenesis with pH-rescue kinetics, SNP variant characterization, and molecular modeling of recombinant bovine and human GLYAT

    PMID:22071172 PMID:23237781

    Open questions at the time
    • Catalytic partner (His) of the dyad not yet defined
    • No experimental structure
  7. 2012 Medium

    Confirmed tissue-restricted expression (liver, kidney) and mitochondrial localization of the human protein by direct immunolabeling.

    Evidence Tissue expression analysis and immunolocalization with affinity-purified anti-GLYAT antibody

    PMID:22475485

    Open questions at the time
    • Submitochondrial compartment not resolved by this method
  8. 2017 Medium

    Revised the kinetic model, showing GLYAT follows a cooperative Ferdinand mechanism rather than Michaelis-Menten or ping-pong, reframing how its activity is regulated by substrate.

    Evidence Bi-substrate kinetic analysis by colorimetric and HPLC-ESI-MS/MS methods with allosteric modeling

    PMID:28759163

    Open questions at the time
    • Structural basis of cooperativity unknown
    • Single lab
  9. 2021 Medium

    Confirmed mitochondrial matrix localization in a human cellular context and quantified variant/haplotype effects, establishing Arg199 as critical for catalytic turnover.

    Evidence eGFP-fusion live-cell imaging in HEK293 and kinetic characterization of haplotype variants fitted to two-substrate Hill equations

    PMID:33567294 PMID:33803916

    Open questions at the time
    • Mechanism by which R199 supports turnover inferred from modeling, not structure
    • Single lab
  10. 2025 High

    Provided the first atomic structure and definitive mechanism by capturing the benzoyl-CoA-bound enzyme and showing catalysis via a Glu-His dyad with a potential low-barrier hydrogen bond.

    Evidence X-ray crystallography of apo and benzoyl-CoA-bound bovine GLYAT with active-site mutagenesis

    PMID:40938199

    Open questions at the time
    • Structure of human enzyme not solved
    • Structural basis of cooperative kinetics not directly addressed
  11. 2025 Medium

    Established physiological loss- and gain-of-function consequences: a nonsense variant causes human GLYAT deficiency with developmental delays, and GLYAT upregulation drives ROS→TRPV1 pain signaling in DRG neurons.

    Evidence Human exome sequencing with in vitro activity assay; mouse DRG overexpression/knockout with ROS and TRPV1 readouts and pharmacological rescue

    PMID:40288509 PMID:40747359

    Open questions at the time
    • Single-patient disease evidence
    • Molecular link between GLYAT enzymatic output and ROS generation not defined
    • Pain phenotype shown in mouse only

Open questions

Synthesis pass · forward-looking unresolved questions
  • How GLYAT enzymatic activity mechanistically generates ROS and engages TRPV1, and how its cooperative kinetics are structurally encoded, remain unresolved.
  • No biochemical link between acyl-CoA/glycine conjugation flux and ROS production
  • No structure rationalizing Ferdinand cooperativity
  • Human enzyme structure unsolved

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016740 transferase activity 6
Localization
GO:0005739 mitochondrion 3
Pathway
R-HSA-1430728 Metabolism 2

Evidence

Reading pass · 14 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1979 Two distinct acyl-CoA:glycine N-acyltransferases were purified to near-homogeneity from bovine liver mitochondria, each consisting of a single polypeptide chain (~33 kDa). Preliminary kinetic studies were consistent with a sequential (ordered) reaction mechanism in which acyl-CoA binds first, glycine adds before CoA leaves, and the peptide product dissociates last. Enzyme purification, substrate specificity assays, stoichiometric cleavage assays, mass spectrometry of products, kinetic analysis The Journal of biological chemistry High 457678
1977 Acyl-CoA:glycine N-acyltransferase from beef liver is composed of a single polypeptide (~35 kDa) with one active site per molecule, as demonstrated by photoaffinity labeling with p-azidobenzoyl-CoA. Benzoyl-CoA protects the enzyme from photoinactivation, confirming that p-azidobenzoyl-CoA targets the acyl-CoA binding active site. Photoaffinity labeling, UV irradiation, competitive protection assay with benzoyl-CoA Biochemistry High 889778
1986 Acyl-CoA:glycine N-acyltransferase is exclusively an intramitochondrial enzyme in rat liver; no activity was detected in peroxisomes despite their capacity to produce acyl-CoAs. Subcellular fractionation, organelle-specific enzyme activity assays Biochemical medicine and metabolic biology Medium 3741707
1994 Human mitochondrial acyl-CoA:glycine N-acyltransferase (ACGNAT) was purified to homogeneity from adult liver; it is a monomer of 30 kDa (pI 6.8). Km values for benzoyl-CoA, salicyl-CoA, isovaleryl-CoA, and octanoyl-CoA were determined, establishing the enzyme's substrate preference profile. Protein purification to homogeneity, SDS-PAGE, isoelectric focusing, enzyme kinetics Biochemical and biophysical research communications High 7802672
1996 N-fatty acylglycines are enzymatically produced from fatty acyl-CoA and glycine by acyl-CoA:glycine N-acyltransferase, and N-myristoylglycine is a substrate for peptidylglycine alpha-amidating enzyme, establishing a two-enzyme biosynthetic route to fatty acid primary amides. In vitro enzyme assay with N-myristoylglycine substrate, measurement of (V/K)app Archives of biochemistry and biophysics Medium 8660675
2011 The catalytic glutamate residue (Glu226 in bovine GLYAT) was identified as essential for activity: an E226Q mutant showed decreased activity at pH 8.0 that could be compensated by increasing pH, indicating Glu226 deprotonates glycine to facilitate nucleophilic attack on the acyl-CoA. Recombinant expression, E226Q active-site mutagenesis, enzyme kinetics at varying pH, protein modeling Drug metabolism and disposition High 22071172
2012 Single nucleotide polymorphisms in human GLYAT alter enzyme activity: N156S variant is more active, F168L is less active, and R199C is completely inactive. An E227Q active-site mutant is also inactive, confirming Glu227 as the catalytic residue in human GLYAT. The R199C inactivity is attributed to destabilization of an α-loop-α motif important for substrate binding in the GNAT superfamily. Site-directed mutagenesis of recombinant human GLYAT, expression, purification, enzymatic characterization, molecular modeling with CoA Gene High 23237781
2012 Human GLYAT is expressed specifically in liver and kidney, and the protein localizes to mitochondria, as confirmed by immunolabeling. Tissue-specific expression analysis, cellular immunolocalization with affinity-purified anti-GLYAT antibody Biochemical and biophysical research communications Medium 22475485
2017 Human GLYAT exhibits mechanistic kinetic cooperativity (sigmoidal substrate activation with glycine variable and substrate inhibition with benzoyl-CoA variable) consistent with a Ferdinand enzyme mechanism, rather than the previously assumed Michaelis-Menten or ping-pong mechanism. Bi-substrate kinetic analysis by colorimetric and HPLC-ESI-MS/MS methods, allosteric sigmoidal enzyme kinetic modeling Journal of biochemical and molecular toxicology Medium 28759163
2021 Human GLYAT wild-type protein localizes to the mitochondrial matrix when expressed as an eGFP fusion in HEK293 cells, confirming its intramitochondrial localization in a human cellular context. The p.(Gln61Leu) variant shows decreased specific activity and the p.(Asn156Ser) variant shows somewhat increased activity compared to wild-type. Recombinant expression in E. coli and HEK293 cells, eGFP fusion localization imaging, enzyme activity assay after purification Biochimie Medium 33567294
2021 GLYAT exhibits cooperative substrate binding (two-substrate Hill kinetics) and the rare 156Asn>Ser,199Arg>Cys haplotype reduces kcat to ~9.8% of the most abundant haplotype while increasing affinity for benzoyl-CoA, demonstrating that Arg199 is critical for catalytic turnover. Recombinant expression of haplotype variants, kinetic characterization fitted to two-substrate Hill equation International journal of molecular sciences Medium 33803916
2025 The first crystal structure of bovine GLYAT was solved in apo form and bound to benzoyl-CoA. Structural analysis and mutagenesis of key residues revealed that catalysis proceeds via a general base mechanism driven by a potential low-barrier hydrogen bond (LBHB) formed between a catalytic Glu-His dyad, with Glu deprotonating the glycine amine for nucleophilic attack on the acyl-CoA thioester. X-ray crystallography (apo and benzoyl-CoA-bound structures), active-site mutagenesis, functional validation Biochemistry High 40938199
2025 GLYAT is upregulated in dorsal root ganglion (DRG) neurons in a mouse osteoarthritis pain model. GLYAT overexpression increases reactive oxygen species (ROS) levels in DRG neurons and upregulates TRPV1, causing mechanical and thermal hyperalgesia; GLYAT loss-of-function alleviates pain-like behaviors. ROS clearance or TRPV1 blockade in GLYAT-overexpressing mice rescues hyperalgesia, placing GLYAT upstream of ROS→TRPV1 in pain signaling. Transcriptomic sequencing of DRG, viral overexpression and genetic knockout in mice, ROS measurement, TRPV1 immunolabeling, pharmacological rescue with ROS scavenger and TRPV1 blocker, behavioral nociception assays The journal of pain Medium 40288509
2025 A homozygous nonsense variant (p.Q108Ter) in human GLYAT abolishes GLYAT enzymatic activity in vitro, causing glycine N-acyltransferase deficiency associated with developmental delays, confirming that GLYAT enzymatic activity is required for normal glycine conjugation metabolism in humans. Whole exome sequencing, Sanger confirmation, in vitro activity assay of the nonsense variant JIMD reports Medium 40747359

Source papers

Stage 0 corpus · 26 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
1979 Benzoyl-coenzyme A:glycine N-acyltransferase and phenylacetyl-coenzyme A:glycine N-acyltransferase from bovine liver mitochondria. Purification and characterization. The Journal of biological chemistry 71 457678
2013 Glycine conjugation: importance in metabolism, the role of glycine N-acyltransferase, and factors that influence interindividual variation. Expert opinion on drug metabolism & toxicology 60 23650932
1996 Fatty acid amide biosynthesis: a possible new role for peptidylglycine alpha-amidating enzyme and acyl-coenzyme A: glycine N-acyltransferase. Archives of biochemistry and biophysics 59 8660675
2010 Identification of glycine N-acyltransferase-like 2 (GLYATL2) as a transferase that produces N-acyl glycines in humans. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 47 20305126
2012 Designation of enzyme activity of glycine-N-acyltransferase family genes and depression of glycine-N-acyltransferase in human hepatocellular carcinoma. Biochemical and biophysical research communications 41 22475485
1994 Purification to homogeneity of mitochondrial acyl coa:glycine n-acyltransferase from human liver. Biochemical and biophysical research communications 34 7802672
1986 Acyl-CoA:glycine N-acyltransferase: organelle localization and affinity toward straight- and branched-chained acyl-CoA esters in rat liver. Biochemical medicine and metabolic biology 32 3741707
1978 A radiochemical assay for glycine N-acyltransferase activity. Some properties of the enzyme in rat and rabbit. The Biochemical journal 32 666745
1977 Photoaffinity labeling of acyl-coenzyme A:glycine N-acyltransferase with p-azidobenzoyl-coenzyme A. Biochemistry 31 889778
2016 Glycine N-acyltransferase-like 3 is responsible for long-chain N-acylglycine formation in N18TG2 cells. Journal of lipid research 23 27016726
2015 Conservation of the coding regions of the glycine N-acyltransferase gene further suggests that glycine conjugation is an essential detoxification pathway. Gene 23 26149650
2012 Characterisation of the influence of genetic variations on the enzyme activity of a recombinant human glycine N-acyltransferase. Gene 21 23237781
2012 Reversible lysine acetylation regulates activity of human glycine N-acyltransferase-like 2 (hGLYATL2): implications for production of glycine-conjugated signaling molecules. The Journal of biological chemistry 16 22408254
2011 Enzymatic characterization and elucidation of the catalytic mechanism of a recombinant bovine glycine N-acyltransferase. Drug metabolism and disposition: the biological fate of chemicals 15 22071172
1997 Developmental profile of mitochondrial glycine N-acyltransferase in human liver. The Journal of pediatrics 15 9202629
2010 Genetic polymorphisms of glycine N-acyltransferase (GLYAT) in a French Caucasian population. Xenobiotica; the fate of foreign compounds in biological systems 14 20925583
2019 Characterization of glycine-N-acyltransferase like 1 (GLYATL1) in prostate cancer. The Prostate 13 31376196
2009 Genetic polymorphisms of glycine N-acyltransferase in Japanese individuals. Drug metabolism and pharmacokinetics 13 19252341
2014 Expression, purification, and characterization of mouse glycine N-acyltransferase in Escherichia coli. Protein expression and purification 12 24576660
2021 Functional Characterisation of Three Glycine N-Acyltransferase Variants and the Effect on Glycine Conjugation to Benzoyl-CoA. International journal of molecular sciences 10 33803916
2021 Frequent sequence variants of human glycine N-acyltransferase (GLYAT) and inborn errors of metabolism. Biochimie 9 33567294
2017 New insights into the catalytic mechanism of human glycine N-acyltransferase. Journal of biochemical and molecular toxicology 7 28759163
2023 Downregulation of Glycine N-Acyltransferase in Kidney Renal Clear Cell Carcinoma: A Bioinformatic-Based Screening. Diagnostics (Basel, Switzerland) 5 38066746
2025 Glycine N-acyltransferase deficiency in sensory neurons suppresses osteoarthritis pain. The journal of pain 4 40288509
2025 Glycine N-Acyltransferase Deficiency due to a Homozygous Nonsense Variant in the GLYAT: A Novel Inborn Error of Metabolism. JIMD reports 1 40747359
2025 Structure of Bovine Glycine N-Acyltransferase Clarifies Its Catalytic Mechanism. Biochemistry 0 40938199

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