Holocarboxylase synthetase

A single holocarboxylase synthetase (biotin protein ligase, EC 6.3.4.10) acts on the apoenzymes of acetyl CoA, pyruvate, propionyl CoA, and methylcrotonyl CoA carboxylases. Acetyl CoA carboxylase is a cytosolic enzyme, whereas the other three enzymes are mitochondrial. Although holocarboxylase synthetase is found in both the cytosol and mitochondria, it is not clear whether biotin is incorporated into the mitochondrial enzymes before or after they are translocated into the mitochondria. 

Holocarboxylase synthetase from a wide variety of species wUl act on all four apocarboxylases from other species and on a variety of bacterial biotin-dependent apoenzymes. In all the biotin-dependent enzymes investigated to date, the reactive lysine residue is flanked by methionine residues on both sides, and there is a high degree of conservation of the amino sequence around this Met-Lys-Met sequence (Chapman-Smith and Cronan, 1999a, 1999b). 

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Biotin functions to transfer carbon dioxide in a small number of carboxylation reactions. A holocarboxylase synthetase acts on a lysine residue of the apoenzymes of acetyl-CoA carboxylase, pymvate carboxylase, propi-onyl-CoA carboxylase, or methylcrotonyl-CoA carboxylase to react with free biotin to form the biocytin residue of the holoenzyme. The reactive intermediate is 1-7V-carboxybiocytin, formed from bicarbonate in an ATP-dependent reaction. The carboxyl group is then transferred to the substrate for carboxylation (Figure 21—1). 

This enzyme [EC 6.3.4.10], also known as biotin— [propionyl-CoA-carboxylase] ligase and holocarboxylase synthetase, catalyzes the reaction of biotin with ATP and apo-[propanoyl-CoA carbon-dioxide ligase (ADP-forming)] to produce AMP, pyrophosphate, and propa-noyl-CoA carbon-dioxide ligase (ADP-forming). 

OXALOACETATE DECARBOXYASE PROPIONYL-CoA CARBOXYASE PYRUVATE CARBOXYASE TRANSCARBOXYASE BIOTIN HOLOCARBOXYLASE SYNTHETASE BI RAD I CAL CARBENE FREE RADICALS... 

Weiner DL, Grier RE, Wolf (1985) A bioassay for determining biotinidase activity and for discriminating biocytin from biotin using holocarboxylase synthetase-deficient cultured fibroblasts. J Inherit Metab Dis 8 101-102... 

To determine the type of multiple carboxylase deficiency, blood was obtained to determine the biotin holocarboxylase synthetase activity in leukocytes, and serum was sent to determine the biotinidase activity. The results of the serum biotinidase activity returned first and indicated less than 1% of mean normal serum activity, confirming that the child had profound biotinidase deficiency (less than 10% of mean normal serum biotinidase activity). Subsequently, biotin holocarboxylase synthetase activity was found to be normal. Although many states screen for biotinidase deficiency in the newborn period, this child was bom in a state where newborn screening for biotinidase deficiency is not performed. 

Figure 12-2. Metabolic pathways involving the four biotin-dependent carboxylases. The solid rectangular blocks indicate the locations of the enzymes ACC, acetyl-CoA carboxylase PMCC, P-methylcrotonyl-CoA carboxylase PC, pyruvate carboxylase PCC, propionyl-CoA carboxylase. Isolated deficiencies of the first three carboxylases (mitochondrial) have been established (isolated ACC deficiency has not been confirmed). At least the activities of the three mitochondrial carboxylases can be secondarily deficient in the untreated multiple carboxylase deficiencies, biotin holocarboxylase synthetase deficiency and biotinidase deficiency. Lowercase characters indicate metabolites that are frequently found at elevated concentrations in urine of children with both multiple carboxylase deficiencies. The isolated deficiencies have elevations of those metabolites directly related to their respective enzyme deficiency.

Both multiple carboxylase deficiencies are characterized by deficient activities of the three mitochondrial carboxylases in peripheral blood leukocytes prior to biotin treatment. The carboxylase activities increase to near normal or normal after treatment with pharmacological doses of biotin. Patients with biotin holocarboxylase synthetase deficiency have deficient activities of the three mitochondrial carboxylases in fibroblasts incubated in medium with low biotin concentrations (containing only the biotin contributed by fetal calf serum added to the medium for cell growth), whereas patients with biotinidase deficiency have normal carboxylase activities under these conditions. The activities of the carboxylases in biotin holocarboxylase synthetase deficiency become near normal to normal when cultured in medium supplemented with high concentrations of biotin. 

Biotinidase deficiency and biotin holocarboxylase synthetase deficiency can be definitively diagnosed by direct enzymatic assay. Biotinidase activity in plasma or serum is usually determined by using the artificial substrate, biotinyLp-aminobenz< >ate. If biotinidase activity is present, then biotin is cleaved, releasing jD-aminobenzoatc. The / -aminobenzoate then is reacted with reagents that result in the development of purple color that can be quantitated colorimetrically. In the absence of biotinidase activity,/ -aminobenzoate is not liberated. Biotinidase activity in patients with an isolated carboxylase deficiency or biotin holocarboxylase synthetase deficiency is normal. 

Biotin is attached to the various apocarboxyl-ases by the enzyme biotin holocarboxylase synthetase forming holocarboxylases through the two partial reactions shown below ... 

Figure 12-4. The biotin cycle shows the actions of biotin holocarboxylase synthetase in biotinylating carboxylases and of biotinidase in cleaving biocytin, thereby recycling biotin.

Individuals with untreated biotinidase deficiency develop biotin deficiency because they cannot recycle endogenous biotin. The biotin deficiency subsequently results in the lack of substrate for biotin holocarboxylase synthetase. Without the availability of biotin to be added to the apocarboxylases, multiple carboxylase deficiency occurs, and the abnormal metabolites accumulate. 

MetabolicaUy, biotin is of central importance in lipogenesis, gluconeogen-esis, and the catabolism of branched-chain (and other) amino acids. There are two well-characterized biotin-responsive inborn errors of metabolism, which are fatal if untreated holocarboxylase synthetase deficiency and biotinidase deficiency. In addition, biotin induces a number of enzymes, including glu-cokinase and other key enzymes of glycolysis. Biotinylation of histones may be important in regulation of the cell cycle. 

Figure 11.1. Metabolism of biotin. Holocarboxylase synthetase (biotin protein ligase), EC 6.3.4.10 and biotinidase (biotinamide amidohydrolase), EC 3.5.1.12. Relative molecular mass (Mr) biotin, 244.3 and biocytin, 372.5.

Biotin is bound covalently to enzymes by a peptide fink to the s -amino group of a lysine residue, forrningbiotinyl-s-arnino-lysine orbiocytin (see Figure 11.1). This postsynthetic modification is catalyzed by holocarboxylase synthetase with the intermediate formation of biotinyl-5 -AMP. In bacteria, this intermediate also acts as a potent repressor of all four enzymes of biotin synthesis. 

Holocarboxylase Synthetase Deficiency Genetic deficiency of holocarboxylase synthetase leads to the neonatal form of multiple carboxylase... 

Table 11.1 Abnormal Urinary Organic Acids in Biotin Deficiency and Multiple Carboxylase Deficiency from Lack of Holocarboxylase Synthetase or Biotinidase...

The affected infants have a normal plasma concentration of biotin and excrete normal amounts of biotin in the urine. Skin fibroblasts have extremely low activities of aU four biotin-dependent carboxylases when they are cultured in media containing approximately physiological concentrations of biotin. But, culture with considerably higher concentrations of biotin results in normal activity of aU four carboxylases. The defect is in the affinity of holocarboxylase synthetase for biotin (its is 20- to 70-fold higher than normal). 

Holocarboxylase synthetase deficiency can be diagnosed prenatally by assessing the response of carboxylase activity in cultured amniocytes (obtained by amniocentesis) to the addition of biotin, or by the detection of methylcitric and hydroxyisovaleric acids in the amniotic fluid. Prenatal therapy, by giving the mother 10 mg of biotin per day, results in sufficiently elevated fetal blood concentrations of biotin to prevent the development of organic acidemia at birth. 

Biotinidase Deficiency Genetic lack of biotinidase results in the late-onset variant of multiple carboxylase deficiency. Patients generally present later in life than those with holocarboxylase synthetase deficiency (Section 11.2.2.1) and have a lower than normal blood concentration of biotin. Culture of fibroblasts in media containing low concentrations of biotin results in normal activities of carboxylases, and holocarboxylase synthetase activity is normal. 

Biotin acts to induce glucokinase, phosphofructokinase, and pyruvate kinase (key enzymes of glycolysis), phosphoenolpyruvate carboxykinase (a key enzyme of gluconeogenesis), and holocarboxylase synthetase, acting via a cell-surface receptor linked to formation of cGMP and increased activity of RNA polymerase. The activity of holocarboxylase synthetase (Section 11.2.2) falls in experimental biotin deficiency and increases with a parallel increase in... 

Hyperammonemia occurs in biotin deficiency and the functional deficiency associated with lack of holocarboxylase synthetase (Section 11.2.2.1) and bio-tinidase (Section 11.2.3.1). In deficient rats, the activity of ornithine carbamyl-transferase is two - thirds of that in control animals, as a result of decreased gene expression, although the activities of other urea cycle enzymes are unaffected (Maeda etal., 1996). 

Rodriguez-Melendez R, Cano S, Mendez ST, and Velazquez A (2001) Biotin regulates the genetic expression of holocarboxylase synthetase and mitochondrial carboxylases in mts. Journal of Nutrition 131, 1909-13. 

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