AACS is a cytosolic, ATP-dependent, AMP-forming acetoacetate--CoA ligase that activates acetoacetate with CoA to produce acetoacetyl-CoA, AMP, and diphosphate. This reaction consumes a ketone body and supplies cytosolic acetoacetyl-CoA for cholesterol and fatty-acid biosynthesis, providing an anabolic route for ketone-body carbon that bypasses citrate and ATP-citrate lyase. Human AACS transcripts are abundant in kidney, heart, and brain and low in liver, and human pancreatic islets contain abundant acetoacetyl-CoA synthetase in an acetoacetate-to-cytosolic-acyl-CoA pathway. Direct human evidence establishes cytosolic enzyme activity and expression, and a limited recombinant-human study reports kinetic parameters, whereas detailed reaction stoichiometry, substrate breadth, and quantitative lipid-flux evidence derive chiefly from rat and mouse studies.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0030729 acetoacetate-CoA ligase activity | IBA GO_REF:0000033 | ACCEPT | Summary: The phylogenetic annotation assigns the defining acetoacetate-CoA ligase reaction to human AACS. Human AACS is a close ortholog of experimentally characterized rat Aacs. Human islets contain abundant acetoacetyl-CoA synthetase, and a recent peer-reviewed review reports a limited kinetic study of expressed and purified recombinant human AACS. Reason: Acetoacetate-CoA ligase activity is the conserved, defining molecular function of AACS. The IBA evidence chain uses the rat donor, but the human cDNA is 89.3% identical to rat Aacs, human islets contain high levels of the enzyme, and recombinant human AACS has reported acetoacetate and CoA kinetic measurements. These orthogonal data make the phylogenetic transfer sound, while the direct human kinetic report still warrants independent replication. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000644734 · AACS phylogenetic ancestral node SUPPORTS TRANSFER The node propagates the family-defining catalytic activity within the conserved AACS ortholog group. RGD:708522 · rat Aacs (UniProtKB:Q9JMI1) SUPPORTS TRANSFER Rat Aacs carries direct experimental catalytic evidence and is the one-to-one mammalian ortholog used for the human transfer. Supporting Evidence: PMID:12623130 Amino acid sequence of human AACS deduced from the open reading frame showed high homology (89.3%) with that of rat AACS PMID:21454710 Human islets possessed high levels of succinyl-CoA:3-ketoacid-CoA transferase, an enzyme that forms acetoacetate in the mitochondria, and acetoacetyl-CoA synthetase, which uses acetoacetate to form acyl-CoAs in the cytosol. PMID:37356666 Ito et al. (28) measured the Km of the purified rat enzyme to be 8 μM, the Km for AcAc for the expressed and purified human AACS was measured to be 37.6 μM by Aguilo et al. (8), and Bergstrom et al. (3) estimated the Km to be 54 μM in rat liver cytosol. file:human/AACS/AACS-uniprot.txt Reaction=acetoacetate + ATP + CoA = acetoacetyl-CoA + AMP + diphosphate |
| GO:0032024 positive regulation of insulin secretion | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Rat INS-1 832/13 knockdown data support a positive contribution of Aacs to glucose-induced insulin release. Human islets contain abundant acetoacetyl-CoA synthetase and may use the same acetoacetate-to-cytosolic-acyl-CoA route, but human AACS itself was not perturbed in the cited human-islet study. Reason: The IBA transfer is biologically defensible as a beta-cell context-dependent consequence of AACS metabolism, but it is not the enzyme's core evolved function. The causal knockdown evidence is from the rat INS-1 cell line; the human evidence is pathway-level corroboration rather than a direct test that human AACS positively regulates secretion. Propagation Review Root cause: NO FAILURE NON CORE Sources checked: PANTHER:PTN002873124 · AACS insulin-secretion phylogenetic node SUPPORTS TRANSFER The transfer is coherent for mammalian AACS orthologs but represents a beta-cell physiological context rather than the catalytic core function. RGD:708522 · rat Aacs (UniProtKB:Q9JMI1) SUPPORTS TRANSFER The rat donor has IMP evidence from Aacs knockdown in INS-1 832/13 cells; this is direct for rat but not a direct human perturbation. Supporting Evidence: PMID:17724028 However, lowering acetoacetyl-CoA synthetase 80% partially inhibited glucose-induced insulin release indicating formation of SC-CoAs from acetoacetate in the cytosol is important for insulin secretion. PMID:21454710 This information supports previous data that indicate beta cells can use a pathway involving succinyl-CoA:3-ketoacid-CoA transferase and acetoacetyl-CoA synthetase to synthesize and use acetoacetate and suggests human islets may use this pathway more than PC and citrate to form cytosolic acyl-CoAs. |
| GO:0005829 cytosol | IEA GO_REF:0000044 | ACCEPT | Summary: The UniProt subcellular-location mapping places AACS in the cytosol, where it activates acetoacetate to acetoacetyl-CoA. The human UniProt statement is by similarity to rat Q9JMI1, and human-islet biochemical data independently describe acetoacetyl-CoA synthetase as using acetoacetate in the cytosol. Reason: Cytosol is the correct compartment for the core AACS reaction and is supported by both conserved mammalian localization and human-islet evidence. The vocabulary mapping is exact rather than a broad cytoplasm default. Propagation Review Root cause: NO FAILURE CORE Sources checked: UniProtKB-SubCell:SL-0091 · cytosol controlled-vocabulary term SUPPORTS TRANSFER The vocabulary mapping lands on the exact compartment. The upstream human UniProt statement is rat-inferred, but human-islet and Reactome evidence independently corroborate cytosolic activity. Supporting Evidence: PMID:21454710 Human islets possessed high levels of succinyl-CoA:3-ketoacid-CoA transferase, an enzyme that forms acetoacetate in the mitochondria, and acetoacetyl-CoA synthetase, which uses acetoacetate to form acyl-CoAs in the cytosol. Reactome:R-HSA-5694494 Acetoacetyl-CoA synthetase (AACS) mediates the activation of acetoacetate (ACA) to the KB acetoacetyl-CoA (ACA-CoA) in the cytosol of cells of lipogenic tissues (Aquilo et al. 2010). |
| GO:0006629 lipid metabolic process | IEA GO_REF:0000002 | MODIFY | Summary: AACS participates in lipid metabolism by supplying cytosolic acetoacetyl-CoA for cholesterol and fatty-acid synthesis. The current parent term loses the experimentally relevant biosynthetic direction of that contribution. Reason: The annotation is related but too broad: AACS channels ketone-derived carbon into lipogenesis rather than participating generically in both lipid synthesis and breakdown. Replace it with GO:0008610 lipid biosynthetic process, which captures the demonstrated pathway direction without overcommitting to one lipid class. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: InterPro:IPR005914 · acetoacetyl-CoA synthetase family SUPPORTS TRANSFER The family assignment supports the broad lipid-metabolism transfer; direct pathway evidence justifies sharpening the target from generic metabolism to the specifically biosynthetic direction of AACS activity. Proposed replacements: lipid biosynthetic process Supporting Evidence: Reactome:R-HSA-5694494 AACS is proposed to provide an alternative supply of acetyl units from that of mitochondrial ketogenesis for de novo lipogenesis and cholesterol synthesis in the brain, based on rat experiments (Endemann et al. 1982, review Cotter et al. 2013). PMID:39471817 AACS LivKO substantially decreased 13C-enrichment into acetyl-CoA and fatty acids (Figures 6E, 6F, S5C, Tables S1, S2, S3). PMID:22985732 We showed that SREBP-2 regulates the expression of AACS and that knockdown of AACS in vivo, by the hydrodynamics method, resulted in the reduction of total blood cholesterol. PMID:6501306 [3-14C]Acetoacetate, injected subcutaneously into rats, was rapidly incorporated into hepatic cholesterol and fatty acids. Injection of radiolabeled acetoacetate, acetate, or glucose resulted in the preferential incorporation of acetoacetate into cholesterol. PMID:7061490 Ketone bodies contribute 19 to 80% of the carbon incorporated into sterols and up to 22% of the carbon incorporated into fatty acids, depending upon the metabolic status of the liver. |
| GO:0030729 acetoacetate-CoA ligase activity | IEA GO_REF:0000120 | ACCEPT | Summary: The combined IEA maps the AACS-specific InterPro family, RHEA:16117 reaction, and EC 6.2.1.16 to acetoacetate-CoA ligase activity. These sources describe the same reaction: ATP- and CoA-dependent conversion of acetoacetate to acetoacetyl-CoA. Reason: This is an appropriately specific, mechanistically exact mapping to the defining AACS reaction. Rhea and EC are reaction-vocabulary corroboration rather than independent experimental evidence; human-islet data and the conserved rat ortholog provide the biological support. Propagation Review Root cause: NO FAILURE CORE Sources checked: InterPro:IPR005914 · acetoacetyl-CoA synthetase family SUPPORTS TRANSFER This is the specific AACS family rather than a generic AMP-binding-enzyme domain mapping. RHEA:16117 · acetoacetate-CoA ligase reaction SUPPORTS TRANSFER The Rhea mapping specifies the exact reaction but is not an independent experimental observation. EC:6.2.1.16 · acetoacetate-CoA ligase SUPPORTS TRANSFER The EC mapping agrees with the specific ligase reaction but is not an independent experimental observation. Supporting Evidence: file:human/AACS/AACS-uniprot.txt Reaction=acetoacetate + ATP + CoA = acetoacetyl-CoA + AMP + diphosphate Reactome:R-HSA-5694494 Acetoacetyl-CoA synthetase (AACS) mediates the activation of acetoacetate (ACA) to the KB acetoacetyl-CoA (ACA-CoA) in the cytosol of cells of lipogenic tissues (Aquilo et al. 2010). |
| GO:0005515 protein binding | IPI PMID:25416956 A proteome-scale map of the human interactome network. | MARK AS OVER ANNOTATED | Summary: This IPI annotation represents an AACS-CMTM5 binary interaction reported by a proteome-scale human interactome screen. The interaction may be reproducible, but the generic protein-binding term neither defines the interaction mechanism nor describes AACS catalysis. Reason: GO:0005515 is an uninformative umbrella term and should not be treated as a core molecular function. The AACS-CMTM5 edge is better retained in the interaction database until a specific biochemical role or complex context is established. Supporting Evidence: PMID:25416956 Here, we describe a systematic map of ?14,000 high-quality human binary protein-protein interactions. file:human/AACS/AACS-uniprot.txt Q86V21; Q96DZ9: CMTM5; NbExp=3; IntAct=EBI-10259474, EBI-2548702; |
| GO:0046951 ketone body biosynthetic process | TAS Reactome:R-HSA-77111 | MODIFY | Summary: Reactome:R-HSA-77111 is a mitochondrial hepatic ketone-body-synthesis pathway that contains the AACS reaction, but propagating the parent pathway term to AACS reverses the reaction's local direction: AACS consumes the ketone body acetoacetate in the cytosol to form acetoacetyl-CoA for downstream metabolism. Reason: This is a pathway-polarity error, not merely an overly broad term. AACS is a ketone-body-utilizing enzyme and does not synthesize ketone bodies. Replace GO:0046951 with GO:0046952 ketone body catabolic process to reflect consumption of acetoacetate. Propagation Review Root cause: PROPAGATION BAD Failure modes: ROLE CONFLATION COMPARTMENT OR COMPLEX MISMATCH Sources checked: Reactome:R-HSA-77111 · Synthesis of Ketone Bodies SUPPORTS SOURCE BUT NOT TARGET The pathway correctly describes mitochondrial ketogenesis, but assigning its biosynthetic process term to cytosolic AACS reverses the role of the AACS reaction, which consumes acetoacetate. Proposed replacements: ketone body catabolic process Supporting Evidence: Reactome:R-HSA-77111 Ketone body synthesis proceeds via the synthesis of ccetoacetic acid in three steps from acetyl CoA, followed by the reduction of acetoacetic acid to beta-hydroxybutyrate. In the body, these reactions occur in the mitochondria of liver cells (Sass 2011). Reactome:R-HSA-5694494 Acetoacetyl-CoA synthetase (AACS) mediates the activation of acetoacetate (ACA) to the KB acetoacetyl-CoA (ACA-CoA) in the cytosol of cells of lipogenic tissues (Aquilo et al. 2010). |
| GO:0030729 acetoacetate-CoA ligase activity | TAS Reactome:R-HSA-5694494 | ACCEPT | Summary: The human Reactome event states that AACS ligates CoA to acetoacetate to form acetoacetyl-CoA in the cytosol, which is precisely GO:0030729. Reason: The event is reaction-specific and matches the defining AACS molecular function. Reactome explicitly notes that its physiological proposal is based substantially on rat experiments, while human cDNA, human-islet enzyme evidence, and a limited recombinant-human kinetic report corroborate the human assignment. Supporting Evidence: Reactome:R-HSA-5694494 Acetoacetyl-CoA synthetase (AACS) mediates the activation of acetoacetate (ACA) to the KB acetoacetyl-CoA (ACA-CoA) in the cytosol of cells of lipogenic tissues (Aquilo et al. 2010). PMID:21454710 Human islets possessed high levels of succinyl-CoA:3-ketoacid-CoA transferase, an enzyme that forms acetoacetate in the mitochondria, and acetoacetyl-CoA synthetase, which uses acetoacetate to form acyl-CoAs in the cytosol. PMID:37356666 The Km for CoASH was measured to be 10 μM (28) for the enzyme purified from rat liver and 2.3 μM for the recombinant human AACS (8). |
| GO:0030729 acetoacetate-CoA ligase activity | ISS GO_REF:0000024 | ACCEPT | Summary: This ISS annotation transfers acetoacetate-CoA ligase activity from the experimentally characterized rat ortholog Q9JMI1 to human AACS. Reason: Rat Q9JMI1 has direct catalytic and cytosolic-localization evidence, and the transfer is strengthened by 89.3% human-rat sequence identity, membership in the same specific AACS subfamily, detection of acetoacetyl-CoA synthetase in human islets, and a limited recombinant-human kinetic report. The GOA evidence path remains an ISS transfer even though direct human catalysis provides external corroboration. Propagation Review Root cause: NO FAILURE CORE Sources checked: UniProtKB:Q9JMI1 · rat Aacs SUPPORTS TRANSFER The one-to-one rat ortholog supplies the ISS evidence path for the exact reaction; the limited recombinant-human kinetic report independently corroborates the transferred activity. Supporting Evidence: PMID:12623130 Amino acid sequence of human AACS deduced from the open reading frame showed high homology (89.3%) with that of rat AACS file:human/AACS/AACS-uniprot.txt Evidence={ECO:0000250|UniProtKB:Q9JMI1}; PMID:37356666 Ito et al. (28) measured the Km of the purified rat enzyme to be 8 μM, the Km for AcAc for the expressed and purified human AACS was measured to be 37.6 μM by Aguilo et al. (8), and Bergstrom et al. (3) estimated the Km to be 54 μM in rat liver cytosol. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-5694494 | ACCEPT | Summary: The Reactome reaction is explicitly cytosolic, consistent with AACS activation of acetoacetate outside mitochondria and with human-islet biochemical evidence. Reason: Cytosol is the correct location for the core AACS reaction. Reactome's detailed mechanistic model relies substantially on rat work rather than direct human localization microscopy, but the compartment is independently described in the human-islet study and is conserved across mammalian AACS. Supporting Evidence: Reactome:R-HSA-5694494 Acetoacetyl-CoA synthetase (AACS) mediates the activation of acetoacetate (ACA) to the KB acetoacetyl-CoA (ACA-CoA) in the cytosol of cells of lipogenic tissues (Aquilo et al. 2010). PMID:21454710 Human islets possessed high levels of succinyl-CoA:3-ketoacid-CoA transferase, an enzyme that forms acetoacetate in the mitochondria, and acetoacetyl-CoA synthetase, which uses acetoacetate to form acyl-CoAs in the cytosol. |
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Download this section (compressed HTML)Q: Can the published recombinant-human AACS kinetic values, including the reported ATP Km, be independently reproduced and extended to define substrate range, cation requirements, reaction stoichiometry, and the catalytic competence of each annotated splice isoform?
Q: In which native human tissues and nutritional states does AACS make a quantitatively important contribution to cholesterol or fatty-acid synthesis relative to the citrate--ATP-citrate-lyase route?
Q: Does endogenous AACS causally support glucose-stimulated insulin secretion in human beta cells, or is the phenotype seen in rat INS-1 cells species- or model-specific?
Experiment: Purify the canonical Q86V21 product, each annotated alternative isoform, and an active-site mutant; measure AMP, diphosphate, and acetoacetyl-CoA formation across defined acetoacetate, CoA, ATP, and cation concentrations, and confirm products by targeted mass spectrometry.
Hypothesis: Full-length human AACS reproduces the reported acetoacetate-CoA ligase activity and kinetics, whereas one or more truncated splice isoforms have reduced activity.
Type: recombinant enzymology and isoform comparison
Experiment: Perform CRISPR knockout and wild-type versus catalytic-dead rescue in human neural and adipocyte models with confirmed endogenous AACS, using hepatocytes as a low-expression comparator. Trace labeled acetoacetate into acetoacetyl-CoA, HMG-CoA, cholesterol, and fatty acids, and compare flux with and without ATP-citrate-lyase inhibition.
Hypothesis: AACS supplies a measurable fraction of cytosolic acetoacetyl-CoA and downstream sterol and fatty-acid carbon in lipogenic human cells.
Type: genetic rescue and stable-isotope metabolomics
Experiment: Perturb AACS in primary human islets or stem-cell-derived beta cells using CRISPRi, rescue with wild-type or catalytic-dead Q86V21, and quantify acetoacetyl-CoA pools, stimulus-coupled insulin secretion, viability, and mitochondrial versus cytosolic carbon flux.
Hypothesis: Human beta-cell AACS contributes directly to glucose-stimulated insulin secretion by supplying cytosolic short-chain acyl-CoAs.
Type: human beta-cell perturbation and metabolic flux analysis
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