3- methylcrotonyl-coenzyme

Coenzymes Coenzyme A Benzoyl-coenzyme A reductase, acetyl-coenzyme A carboxylase, 3 -methylcrotonyl-coenzyme-A carboxylase... 

Gibson KM, Bennett MJ, Naylor EW, Morton DH (1998) 3-Methylcrotonyl-coenzyme a carboxylase deficiency in amish/mennonite adults identified by detection of increased acylcarnitines in blood spots of their children. J Pediatr 132 519-523... 

The method described is suitable for the assay of four biotin-containing carboxylases pyruvate carboxylase, acetyl-coenzyme A carboxylase, propionyl-coenzyme A carboxylase, and 3-methylcrotonyl-coenzyme A carboxylase. The assays do not require radioisotopes and are suitable for use in clinical laboratories. 

Substrates and products are separated by reversed-phase chromatography at 45°C on a Nucleosil Qs column (4.6 mm X 250 mm). For assay of acetyl-coenzyme A carboxylase, propionyl-coenzyme A carboxylase, and 3-methylcrotonyl-coenzyme A carboxylase, a linear gradient from solvent A (0.1 M sodium phosphate buffer, pH 2.1) to solvent B (methanol-solvent A, 80 20, v/v) was applied in 15 minutes at a flow rate of 1.5 mL/min. Quantitation was based on the absorbance of the product (malonyl-CoA, methylmalonyl-CoA, and 3-methylglutaconyl-CoA, respectively) at 260 nm. For assay of pyruvate carboxylase, pyruvate was separated by isocratic elution using 0.1 M sodium phosphate buffer (pH 2.1) containing 0.1 M sodium sulfate. Quantitation was based on the disappearance of pyruvate as followed at 210 nm. 

Diez, T.A., Wurtele, E.S., and Nikolau, B.J. (1994) Purification and characterization of 3-methylcrotonyl-Coenzyme A carboxylase from leaves of Zea mays. Arch. Biochem. Biophys. 310, 64-75. 

Baldet P, Alban C, Axiotis S, Douce R. Characterization of biotin and 3-methylcrotonyl-coenzyme A carboxylase in plants. Plant Physiol, 1992 99 450-455. 

The chemistry of a fourth coenzyme was at least partially elucidated in the period under discussion. F. Lynen and coworkers treated P-methylcrotonyl coenzyme A (CoA) carboxylase with bicarbonate labelled with 14C, and discovered that one atom of radiocarbon was incorporated per molecule of enzyme. They postulated that an intermediate was formed between the enzyme and C02, in which the biotin of the enzyme had become car-boxylated. The carboxylated enzyme could transfer its radiolabelled carbon dioxide to methylcrotonyl CoA more interestingly, they found that the enzyme-COz compound would also transfer radiolabelled carbon dioxide to free biotin. The resulting compound, carboxybiotin [4], was quite unstable, but could be stabilized by treatment with diazomethane to yield the methyl ester of N-carboxymethylbiotin (7) (Lynen et al., 1959). The identification of this radiolabelled compound demonstrated that the unstable material is N-carboxybiotin itself, which readily decarboxylates esterification prevents this reaction, and allows the isolation and identification of the product. Lynen et al. then postulated that the structure of the enzyme-C02 compound was essentially the same as that of the product they had isolated from the reaction with free biotin, but where the carbon dioxide was inserted into the bound biotin of the enzyme (Lynen et al., 1961). Although these discoveries still leave significant questions to be answered as to the detailed mechanism of the carboxylation reactions in which biotin participates as coenzyme, they provide a start toward elucidating the way in which the coenzyme functions. 

Eall RR, ML Hector (1977) Acyl-coenzyme A carboxylases. Homologous 3-methylcrotonyl-CoA and geranyl-CoA carboxylases from Pseudomonas citronellis. Biochemistry 16 4000-4005. 

Biotin, an essential water-soluble B-complex vitamin, is the coenzyme for four human carboxylases (Fig. 12-2) These include the three mitochondrial enzymes pyruvate carboxylase, which converts pyruvate to oxaloacetate and is the initial step of gluconeogenesis propionyl-CoA carboxylase, which catabolizes several branched-chain amino acids and odd-chain fatty acids and 3-methylcrotonyl-CoA carboxylase, which is involved in the catabolism of leucine and the principally cytosolic enzyme, acetyl-CoA carboxylase, which is responsible for the... 

Biotin is a coenzyme for the carbon dioxide fixation reactions catalyzed by acetyl-CoA carboxylase (Chapter 19), propionyl-CoA carboxylase, pyruvate carboxylase, and S-methylcrotonyl-CoA carboxylase. Car-boxylation reactions that do not require biotin are the addition of Ce to the purine ring (Chapter 27), the formation of carbamoyl phosphate (Chapter 17), and the y-carboxylation of glutamyl residues of several of the clotting factors, which requires vitamin K (Chapter 36). 

Some of the pioneering studies of biotin s mechanism of action in carboxylation reactions were done in Lynen s laboratory on -methylcrotonyl CoA carboxylase [74]. Lynen s early findings on that coenzyme have since, either in his own or in other laboratories, been extended to many other carboxylases. 

Biotin serves as a covalently bound coenzyme for acetyl-CoA carboxylases (ACC) 1 and 2, pyruvate carboxylase (PC), propionyl-CoA carboxylase (PCQ and 3-methylcrotonyl-CoA carboxylase (MCQ in mammals and other metazoans (Zempleni et al. 2009). Additional carboxylases exist in microbes (Knowles 1989). The attachment of biotin to the s-amino group of a spedlic lysine residue in holocarboxylases is catalysed by holocarboxylase synthetase (HLCS) or microbial orthologs such as BirA. Biotinylation of carboxylases requires ATP to produce the energy-rich intermediate biotinyl-5 -AMP (Zempleni et al. 2009). 

Biotin is a covalently bound coenzyme for acetyl-CoA carboxylases 1 and 2, 3-methylcrotonyl-CoA carboxylase, propionyl-CoA carboxylase and pyruvate carboxylase, which play essential roles in macronutrient metabolism. 

The inborn errors of L-leucine catabolism present biochemically with branched-chain amino and/or organic aciduria [1]. These disorders include maple syrup disease (MSD branched-chain a-ketoacid dehydrogenase (BCKD) deficiency), isovaleric acidemia (isovaleryl-coenzyme A (CoA) dehydrogenase deficiency), isolated 3-methylcrotonyl-CoA carboxylase deficiency, the 3-methylglutaconic acidurias (3-methylglutaconyl-CoA hydratase deficiency, Barth syndrome, and other disorders in which the primary defect has not been demonstrated), and 3-hydroxy-3-methylglutaric aciduria (3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase deficiency). 

Fig. 6.1. The L-leucine degradative pathway. Reactions for which inherited metabolic disorders have not been conclusively identified include A, leucine-isoleucine aminotransferase and the majority of the 3-methylglutaconic acidurias (6.6-6.7). 6.1, Branched-chain a-ketoacid dehydrogenase (BCKD) complex, a reaction also occurring in the initial steps of L-isoleucine and L-valine degradation 6.2, isovaleryl-CoA dehydrogenase 6.3, 3-methylcrotonyl-CoA carboxylase 6.4, 3-methylglutaconyl-CoA hydra-tase 6.8, HMG-CoA lyase. Pathologic urinary metabolites used as specific markers in the differential diagnosis are presented in squares. Abbreviation Co A, coenzyme A...

Tietz and Ochoa could detect no biotin in their enzyme preparation although in biotin deficiency carboxylation of propionyl CoA is greatly diminyied. After it was established that biotin is the coenzyme of the carboxylation enzyme of jS-methylcrotonyl CoA lOJf) it was soon shown that this is also true for the propionyl CoA carboxylase 105a, lOSb). 

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