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Literature summary extracted from

  • Kiema, T.R.; Harijan, R.K.; Strozyk, M.; Fukao, T.; Alexson, S.E.; Wierenga, R.K.
    The crystal structure of human mitochondrial 3-ketoacyl-CoA thiolase (T1): insight into the reaction mechanism of its thiolase and thioesterase activities (2014), Acta Crystallogr. Sect. D, 70, 3212-3225.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.3.1.16 gene ACAA2, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Homo sapiens

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.3.1.16 purified recombinant wild-type and mutant enzymes in apoform and in complex with CoA, hanging drop vapour diffusion method, mixing of 0.002 ml of 4.4 mg/ml protein in 25 mM Tris-HCl pH 8.0, 1 mM DTT, with 0.002 ml of reservoir solution containing 100 mM MES, pH 6.6, or MOPS, pH 7.2, and 14-15% PEG MME 5000, equilibration against 1 ml of reservoir solution, 22°C, method optimization, X-ray diffraction structure determination and analysis at 2.0-3.3 A resolution, modeling Homo sapiens

Protein Variants

EC Number Protein Variants Comment Organism
2.3.1.16 C382A site-directed mutagenesis Homo sapiens
2.3.1.16 C92A site-directed mutagenesis Homo sapiens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.3.1.16 0.0092
-
 acetoacetyl-CoA pH 7.0, 25°C, recombinant wild-type enzyme, degradative reaction Homo sapiens
2.3.1.16 0.25
-
 acetyl-CoA pH 7.0, 25°C, recombinant wild-type enzyme, synthesis reaction Homo sapiens

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
2.3.1.16 mitochondrion
-
 Homo sapiens 5739
-

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.3.1.16 2 acetyl-CoA Homo sapiens reaction of EC 2.3.1.9 CoA + acetoacetyl-CoA
-
 r
2.3.1.16 acyl-CoA + acetyl-CoA Homo sapiens
-
 CoA + 3-oxoacyl-CoA
-
 ?
2.3.1.16 CoA + acetoacetyl-CoA Homo sapiens
-
 2 acetyl-CoA
-
 r
2.3.1.16 additional information Homo sapiens the enzyme catalyzes the thiolytic cleavage of medium-chain to long-chain 3-oxoacyl-CoAs to acetyl-CoA and a fatty acyl-CoA shortened by two C atom, the reaction involves residues C382, C92, and H352, overview. The enzyme can also catalyze the condensation of two acetyl-CoA molecules into acetoacetyl-CoA, EC 2.3.1.9 ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.3.1.16 Homo sapiens P42765 gene ACAA2; gene ACAA2
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.3.1.16 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, cation exchange chromatography, ultrafiltration, and gel filtration, tag cleavage by thrombin Homo sapiens

Reaction

EC Number Reaction Comment Organism Reaction ID
2.3.1.16 acyl-CoA + acetyl-CoA = CoA + 3-oxoacyl-CoA mechanism of the reverse, degradative thiolase reaction, overview. The mode of binding of the acyl moiety favours its transfer to CoA compared with water. Both the thiolytic cleavage and the condensation reaction are catalysed via covalent modification of the nucleophilic cysteine (Cys92) and by stabilization of the negatively charged reaction intermediates by oxyanion holes. The nucleophilic cysteine attacks the beta-carbon of the 3-ketoacyl-CoA and becomes covalently modified. Another cysteine, Cys382, acts first as an acid providing a proton for the leaving acetyl-CoA and next as a base abstracting a proton from the incoming CoA. The activated CoA molecule then attacks the carbonyl C atom of the acylated cysteine and the fatty acyl-CoA is released. The cysteine acting as the nucleophile is activated by a conserved histidine residue (His352). This protonated histidine subsequently forms OAH1 together with a water molecule which is hydrogen-bonded to Asn320. The rate-limiting steps are different in the degradative and biosynthetic thiolases Homo sapiens
a

Source Tissue

EC Number Source Tissue Comment Organism Textmining
2.3.1.16 adrenal gland
-
 Homo sapiens
-
2.3.1.16 heart
-
 Homo sapiens
-
2.3.1.16 kidney
-
 Homo sapiens
-
2.3.1.16 liver
-
 Homo sapiens
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.3.1.16 2 acetyl-CoA reaction of EC 2.3.1.9 Homo sapiens CoA + acetoacetyl-CoA
-
 r
2.3.1.16 3-oxodecanoyl-CoA + CoA degradation of 3-oxodecanoyl-CoA into acetyl-CoA and octanoyl-CoA by human mitochondrial 3-ketoacyl-CoA thiolase, substrate binding mode and reaction mechanism, overview Homo sapiens acetyl-CoA + octanoyl-CoA
-
 ?
2.3.1.16 acyl-CoA + acetyl-CoA
-
 Homo sapiens CoA + 3-oxoacyl-CoA
-
 ?
2.3.1.16 CoA + acetoacetyl-CoA
-
 Homo sapiens 2 acetyl-CoA
-
 r
2.3.1.16 additional information the enzyme catalyzes the thiolytic cleavage of medium-chain to long-chain 3-oxoacyl-CoAs to acetyl-CoA and a fatty acyl-CoA shortened by two C atom, the reaction involves residues C382, C92, and H352, overview. The enzyme can also catalyze the condensation of two acetyl-CoA molecules into acetoacetyl-CoA, EC 2.3.1.9 Homo sapiens ?
-
 ?
2.3.1.16 additional information active site structure and CoA binding structure, and enzyme substrate specificity, detailed overview Homo sapiens ?
-
 ?

Subunits

EC Number Subunits Comment Organism
2.3.1.16 homotetramer thiolases share a structurally conserved thiolase core domain composed of topologically similar N-terminal and C-terminal subdomains and a more variable loop domain with structural features involved in the tetramerization and substrate specificity Homo sapiens

Synonyms

EC Number Synonyms Comment Organism
2.3.1.16 mitochondrial 3-ketoacyl-CoA thiolase
-
 Homo sapiens

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.3.1.16 25
-
 assay at Homo sapiens

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.3.1.16 1.4
-
 acetyl-CoA pH 7.0, 25°C, recombinant wild-type enzyme, synthesis reaction Homo sapiens
2.3.1.16 14.8
-
 acetoacetyl-CoA pH 7.0, 25°C, recombinant wild-type enzyme, degradative reaction Homo sapiens

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.3.1.16 8
-
 thiolase reaction Homo sapiens

General Information

EC Number General Information Comment Organism
2.3.1.16 evolution the rate-limiting steps are different in the degradative and biosynthetic thiolases. Thiolases share a structurally conserved thiolase core domain composed of topologically similar N-terminal and C-terminal subdomains and a more variable loop domain with structural features involved in the tetramerization and substrate specificity Homo sapiens
2.3.1.16 additional information the nucleophilic cysteine attacks the beta-carbon of the 3-ketoacyl-CoA and becomes covalently modified. Another cysteine, Cys382, acts first as an acid providing a proton for the leaving acetyl-CoA and next as a base abstracting a proton from the incoming CoA. The activated CoA molecule then attacks the carbonyl C atom of the acylated cysteine and the fatty acyl-CoA is released. The cysteine acting as the nucleophile is activated by a conserved histidine residue (His352) Homo sapiens
2.3.1.16 physiological function thiolases are enzymes which are involved in lipid metabolism, catalysing the biological Claisen condensation and thiolytic cleavage reactions using a cysteine thiol in the active site. The enzyme catalyzes the last step of the mitochondrial beta-oxidation pathway and is also involved in the synthesis of acetoacetyl-CoA for the generation of ketone bodies, cf. EC 2.3.1.9 Homo sapiens