Publications

• Crystallographic and kinetic studies of human mitochondrial acetoacetyl-CoA thiolase: the importance of potassium and chloride ions for its structure and function.

  Haapalainen A.M., Merilaeinen G., Pirilae P.L., Kondo N., Fukao T., Wierenga R.K.

  Thiolases are CoA-dependent enzymes which catalyze the formation of a carbon-carbon bond in a Claisen condensation step and its reverse reaction via a thiolytic degradation mechanism. Mitochondrial acetoacetyl-coenzyme A (CoA) thiolase (T2) is important in the pathways for the synthesis and degrad ... >> More

  Thiolases are CoA-dependent enzymes which catalyze the formation of a carbon-carbon bond in a Claisen condensation step and its reverse reaction via a thiolytic degradation mechanism. Mitochondrial acetoacetyl-coenzyme A (CoA) thiolase (T2) is important in the pathways for the synthesis and degradation of ketone bodies as well as for the degradation of 2-methylacetoacetyl-CoA. Human T2 deficiency has been identified in more than 60 patients. A unique property of T2 is its activation by potassium ions. High-resolution human T2 crystal structures are reported for the apo form and the CoA complex, with and without a bound potassium ion. The potassium ion is bound near the CoA binding site and the catalytic site. Binding of the potassium ion at this low-affinity binding site causes the rigidification of a CoA binding loop and an active site loop. Unexpectedly, a high-affinity binding site for a chloride ion has also been identified. The chloride ion is copurified, and its binding site is at the dimer interface, near two catalytic loops. A unique property of T2 is its ability to use 2-methyl-branched acetoacetyl-CoA as a substrate, whereas the other structurally characterized thiolases cannot utilize the 2-methylated compounds. The kinetic measurements show that T2 can degrade acetoacetyl-CoA and 2-methylacetoacetyl-CoA with similar catalytic efficiencies. For both substrates, the turnover numbers increase approximately 3-fold when the potassium ion concentration is increased from 0 to 40 mM KCl. The structural analysis of the active site of T2 indicates that the Phe325-Pro326 dipeptide near the catalytic cavity is responsible for the exclusive 2-methyl-branched substrate specificity. << Less

  Biochemistry 46:4305-4321(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Molecular cloning and nucleotide sequence of complementary DNA for human hepatic cytosolic acetoacetyl-coenzyme A thiolase.

  Song X.-Q., Fukao T., Yamaguchi S., Miyazawa S., Hashimoto T., Orii T.

  Complementary DNA for human cytosolic acetoacetyl-CoA thiolase (CT) was cloned with the use of anti-[human CT] antibody. The human CT cDNA clone (HCT10) has a 1479-bp insert and a 1191-base open reading frame encoding 397 amino acid residues. Partial polypeptide sequences from purified human CT we ... >> More

  Complementary DNA for human cytosolic acetoacetyl-CoA thiolase (CT) was cloned with the use of anti-[human CT] antibody. The human CT cDNA clone (HCT10) has a 1479-bp insert and a 1191-base open reading frame encoding 397 amino acid residues. Partial polypeptide sequences from purified human CT were present in the deduced sequence. In vivo expression analysis showed that HCT10 encoded potassium-ion non-activated acetoacetyl-CoA thiolase with no 3-ketooctanoyl-CoA thiolase activity, which is characteristic for CT. The deduced amino acid sequence has a 34-57% homology with 4 other human thiolases and 4 acetoacetyl-CoA thiolases of microorganisms. << Less

  Biochem. Biophys. Res. Commun. 201:478-485(1994) [PubMed] [EuropePMC]

• Comparison of the stability and substrate specificity of purified peroxisomal 3-oxoacyl-CoA thiolases A and B from rat liver.

  Antonenkov V.D., Van Veldhoven P.P., Waelkens E., Mannaerts G.P.

  The specific activities and substrate specificities of 3-oxoacyl-CoA thiolase A (thiolase A) purified from normal rat liver peroxisomes and 3-oxoacyl-CoA thiolase B (thiolase B) isolated from livers of rats treated with the peroxisome proliferator clofibrate were virtually identical. The enzymes c ... >> More

  The specific activities and substrate specificities of 3-oxoacyl-CoA thiolase A (thiolase A) purified from normal rat liver peroxisomes and 3-oxoacyl-CoA thiolase B (thiolase B) isolated from livers of rats treated with the peroxisome proliferator clofibrate were virtually identical. The enzymes could be distinguished by their N-terminal amino acid sequences, their isoelectric points and their stability, the latter being higher for thiolase A. Contrary to thiolase B, which showed a marked cold lability in the presence of KCl by dissociating into monomers with poor activity, thiolase A retained its full activity and its homodimeric structure under these conditions. << Less

  Biochim. Biophys. Acta 1437:136-141(1999) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• The crystal structure of human mitochondrial 3-ketoacyl-CoA thiolase (T1): insight into the reaction mechanism of its thiolase and thioesterase activities.

  Kiema T.R., Harijan R.K., Strozyk M., Fukao T., Alexson S.E., Wierenga R.K.

  Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 Å resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active s ... >> More

  Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 Å resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of the Zoogloea ramigera biosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2-Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance. << Less

  Acta Crystallogr. D 70:3212-3225(2014) [PubMed] [EuropePMC]

• Substrate specificities of 3-oxoacyl-CoA thiolase A and sterol carrier protein 2/3-oxoacyl-CoA thiolase purified from normal rat liver peroxisomes. Sterol carrier protein 2/3-oxoacyl-CoA thiolase is involved in the metabolism of 2-methyl-branched fatty acids and bile acid intermediates.

  Antonenkov V.D., Van Veldhoven P.P., Waelkens E., Mannaerts G.P.

  The two main thiolase activities present in isolated peroxisomes from normal rat liver were purified to near homogeneity. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the first enzyme preparation displayed a single band of 41 kDa that was identified as 3-oxoacyl-CoA thiolase A (th ... >> More

  The two main thiolase activities present in isolated peroxisomes from normal rat liver were purified to near homogeneity. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the first enzyme preparation displayed a single band of 41 kDa that was identified as 3-oxoacyl-CoA thiolase A (thiolase A) by N-terminal amino acid sequencing. The second enzyme preparation consisted of a 58- and a 46-kDa band. The 58-kDa polypeptide reacted with antibodies raised against either sterol carrier protein 2 or the thiolase domain of sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCP-2/thiolase), formerly also called sterol carrier protein X, whereas the 46-kDa polypeptide reacted only with the antibodies raised against the thiolase domain. Internal peptide sequencing confirmed that the 58-kDa polypeptide is SCP-2/thiolase and that the 46-kDa polypeptide is the thiolase domain of SCP-2/thiolase. Thiolase A catalyzed the cleavage of short, medium, and long straight chain 3-oxoacyl-CoAs, medium chain 3-oxoacyl-CoAs being the best substrates. The enzyme was inactive with the 2-methyl-branched 3-oxo-2-methylpalmitoyl-CoA and with the bile acid intermediate 24-oxo-trihydroxycoprostanoyl-CoA. SCP-2/thiolase was active with medium and long straight chain 3-oxoacyl-CoAs but also with the 2-methyl-branched 3-oxoacyl-CoA and the bile acid intermediate. In peroxisomal extracts, more than 90% of the thiolase activity toward straight chain 3-oxoacyl-CoAs was associated with thiolase A. Kinetic parameters (Km and Vmax) were determined for each enzyme with the different substrates. Our results indicate the following: 1) the two (main) thiolases present in peroxisomes from normal rat liver are thiolase A and SCP-2/thiolase; 2) thiolase A is responsible for the thiolytic cleavage of straight chain 3-oxoacyl-CoAs; and 3) SCP-2/thiolase is responsible for the thiolytic cleavage of the 3-oxoacyl-CoA derivatives of 2-methyl-branched fatty acids and the side chain of cholesterol. << Less

  J. Biol. Chem. 272:26023-26031(1997) [PubMed] [EuropePMC]

  This publication is cited by 9 other entries.