Substrate Specificities of 3-Oxoacyl-CoA Thiolase A and Sterol Carrier Protein 2/3-Oxoacyl-CoA Thiolase Purified from Normal Rat Liver Peroxisomes

Antonenkov, Vasily D.; Veldhoven, Paul P. Van; Waelkens, Etienne; Mannaerts, Guy P.

doi:10.1074/jbc.272.41.26023

Cited by 140 publications

(109 citation statements)

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“…The final step in peroxisomal ␤-oxidation is the thiolytic cleavage of 3-ketoacyl-CoAs into chain-shortened acyl-CoAs and acetyl-CoA or propionyl-CoA. It can be catalyzed by 3-ketoacyl-CoA thiolase or sterol carrier protein x, which cleaves straight chain ketoacyl-CoAs or both straight and 2-methyl-3-ketoacyl-CoAs, respectively (20). In addition to these enzymes, peroxisomes contain other important proteins that are directly or indirectly involved in the ␤-oxidation.…”

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confidence: 99%

Identification of a Substrate-binding Site in a Peroxisomal β-Oxidation Enzyme by Photoaffinity Labeling with a Novel Palmitoyl Derivative

Kashiwayama

Tomohiro

Narita

et al. 2010

Journal of Biological Chemistry

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Peroxisomes play an essential role in a number of important metabolic pathways including ␤-oxidation of fatty acids and their derivatives. Therefore, peroxisomes possess various ␤-oxidation enzymes and specialized fatty acid transport systems. However, the molecular mechanisms of these proteins, especially in terms of substrate binding, are still unknown. In this study, to identify the substrate-binding sites of these proteins, we synthesized a photoreactive palmitic acid analogue bearing a diazirine moiety as a photophore, and performed photoaffinity labeling of purified rat liver peroxisomes. As a result, an 80-kDa peroxisomal protein was specifically labeled by the photoaffinity ligand, and the labeling efficiency competitively decreased in the presence of palmitoyl-CoA. Mass spectrometric analysis identified the 80-kDa protein as peroxisomal multifunctional enzyme type 2 (MFE2), one of the peroxisomal ␤-oxidation enzymes. Recombinant rat MFE2 was also labeled by the photoaffinity ligand, and mass spectrometric analysis revealed that a fragment of rat MFE2 (residues Trp 249 to Arg 251 ) was labeled by the ligand. MFE2 mutants bearing these residues, MFE2(W249A) and MFE2(R251A), exhibited decreased labeling efficiency. Furthermore, MFE2(W249G), which corresponds to one of the disease-causing mutations in human MFE2, also exhibited a decreased efficiency. Based on the crystal structure of rat MFE2, these residues are located on the top of a hydrophobic cavity leading to an active site of MFE2. These data suggest that MFE2 anchors its substrate around the region from Trp 249 to Arg 251 and positions the substrate along the hydrophobic cavity in the proper direction toward the catalytic center.Peroxisomes are organelles bound by a single membrane, which are present in almost all eukaryotic cells. Peroxisomes are involved in a variety of important metabolic processes including the ␤-oxidation of fatty acids, synthesis of plasmalogen, and bile acids (1). A specialized set of enzymes responsible for the peroxisomal functions are compartmentalized in the organelle, and the deficiency of peroxisomal enzymes causes severe metabolic disease such as acyl-CoA oxidase deficiency and D-bifunctional protein deficiency (2-4). Fibroblasts from patients with these deficiencies exhibit the disturbed peroxisomal ␤-oxidation of very long-chain fatty acids (VLCFAs), 4 and VLCFAs are accumulated in the plasma and tissues of these patients (5). Analyses of these peroxisomal diseases reflect the importance of the peroxisomal ␤-oxidation in organisms along with disclosing the enzymatic organization of the peroxisomal ␤-oxidation system. Peroxisomes are involved in the ␤-oxidation of VLCFAs, branched chain fatty acids such as pristanic acid, and bile acid intermediates such as dihydroxycholestanoic acid and trihydroxycholestanoic acid, which are incompatible with mitochondrial ␤-oxidation (6). Peroxisomal ␤-oxidation of fatty acids proceeds via a four-step pathway as in mitochondria, and multiple enzymes are involved in each step of the...

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confidence: 99%

Identification of a Substrate-binding Site in a Peroxisomal β-Oxidation Enzyme by Photoaffinity Labeling with a Novel Palmitoyl Derivative

Kashiwayama

Tomohiro

Narita

et al. 2010

Journal of Biological Chemistry

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“…Percoll, Ultrogel AcA-34, Ampholine PAG plates for isoelectric focusing (pH 3.5±9.5) and pI calibration kits were from Pharmacia. The sources of all other chemicals have been described previously [19]. Medium and long straight chain 3-oxoacyl-CoAs were synthesized enzymatically according to Seubert et al [21] with some modifications [19].…”

Section: Methodsmentioning

confidence: 99%

“…Peroxisomes were prepared by isopycnic centrifugation of the light mitochondrial fraction in a selfgenerating Percoll gradient as described previously [22]. Matrix proteins were extracted from the particles by using three consecutive cycles of sonication and centrifugation [19]. The supernatants obtained after sedimentation of the broken peroxisomes and containing approximately 80% of the total acetoacetyl-CoA thiolase activity found in whole peroxisomes were combined, concentrated by ultrafiltration (Centriprep-10, Amicon) and subjected to conventional chromatography.…”

Section: Preparation Of Peroxisomes and Purification Of Acetoacetyl-cmentioning

confidence: 99%

“…SCP-2/thiolase is active towards medium and long straight chain 3-oxoacyl-CoAs and towards the 3-oxoacyl-CoA derivatives of 2-methylbranched fatty acids (for example, 3-oxopristanoyl-CoA) and of the bile acid intermediates di-and trihydroxycoprostanic acids (24-oxodihydroxycoprostanoyl-CoA and 24-oxotrihydroxycoprostanoyl-CoA). The enzyme which is not or only poorly active towards acetoacetyl-CoA is involved in the peroxisomal b-oxidation of branched fatty acids and in the cleavage of the side chain of cholesterol during bile acid synthesis [19].…”

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“…They catalyse the last step in peroxisomal and mitochondrial b-oxidation. Three different 3-oxoacyl-CoA thiolases have been described in rat liver peroxisomes: 3-oxoacyl-CoA thiolase B (thiolase B, see above), 3-oxoacyl-CoA thiolase A (thiolase A) and sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCP-2/thiolase) [14±16, 18,19]. The constitutively expressed thiolase A and the inducible thiolase B have a virtually identical substrate spectrum [20].…”

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Identification, purification and characterization of an acetoacetyl-CoA thiolase from rat liver peroxisomes

Antonenkov¹,

Croes²,

Waelkens³

et al. 2000

Eur J Biochem

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Acetoacetyl-CoA specific thiolases catalyse the cleavage of acetoacetyl-CoA into two molecules of acetyl-CoA and the synthesis (reverse reaction) of acetoacetyl-CoA. The formation of acetoacetyl-CoA is the first step in cholesterol and ketone body synthesis. In this report we describe the identification of a novel acetoacetyl-CoA thiolase and its purification from isolated rat liver peroxisomes by column chromatography. The enzyme, which is a homotetramer with a subunit molecular mass of 42 kDa, could be distinguished from the cytosolic and mitochondrial acetoacetyl-CoA thiolases by its chromatographic behaviour, kinetic characteristics and partial internal amino-acid sequences. The enzyme did not catalyse the cleavage of medium or long chain 3-oxoacylCoAs. The enzyme cross-reacted with polyclonal antibodies raised against cytosolic acetoacetyl-CoA thiolase. The latter property was exploited to confirm the peroxisomal localization of the novel thiolase in subcellular fractionation experiments.The peroxisomal acetoacetyl-CoA thiolase most probably catalyses the first reaction in peroxisomal cholesterol and dolichol synthesis. In addition, its presence in peroxisomes along with the other enzymes of the ketogenic pathway indicates that the ketogenic potential of peroxisomes needs to be re-evaluated.

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Bifunctional Enzymes of β‐Oxidation (Multifunctional Proteins,MFP1 andMFP2)

Watkins

Mihalik

2002

Wiley Encyclopedia of Molecular Medicine

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Substrate Specificities of 3-Oxoacyl-CoA Thiolase A and Sterol Carrier Protein 2/3-Oxoacyl-CoA Thiolase Purified from Normal Rat Liver Peroxisomes

Cited by 140 publications

References 49 publications

Identification of a Substrate-binding Site in a Peroxisomal β-Oxidation Enzyme by Photoaffinity Labeling with a Novel Palmitoyl Derivative

Identification of a Substrate-binding Site in a Peroxisomal β-Oxidation Enzyme by Photoaffinity Labeling with a Novel Palmitoyl Derivative

Identification, purification and characterization of an acetoacetyl-CoA thiolase from rat liver peroxisomes

Bifunctional Enzymes of β‐Oxidation (Multifunctional Proteins,MFP1 andMFP2)

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