The Role of Biotin in Carboxylation Reactions

The reactive intermediate is 1-ALcarboxy-biotin (see Figure 11.1) bound to a lysine residue of the enzyme as biocytin, which is formed from enzyme-bound biocytin by reaction with bicarbonate.

The biotin-dependent carboxylases catalyze a two-step reaction:

1. enzyme-biotin + ATP + HCO3- ^ enzyme-biotin-COOH + ADP + Pi

2. enzyme-biotin- COOH + acceptor ^ enzyme-biotin + acceptor-COOH.

In the bacterial biotin-dependent decarboxylases, reaction 2 proceeds from right to left, followed by decomposition of the carboxy-biotin to biotin and CO2.

The role of ATP in the carboxylation of biotin is unclear. It is possible that biotin is O-phosphorylated during the carboxylation reaction. However, evidence suggests that the immediate reactive species that carboxylates biotin is carboxyphosphate, as in the (biotin-independent) reaction of carbamyl phosphate synthetase in urea and pyrimidine synthesis.

Steady-state kinetic analysis shows that biotin-dependent reactions proceed by way of a two-site ping-pong mechanism; the two-part reactions are catalyzed at distinct sites in the enzyme. These sites may be on the same or different polypeptide chains in different biotin-dependent enzymes. The e-amino linkage of lysine to the side chain of biotin in biocytin allows considerable movement of the coenzyme - the distance from C-2 of lysine to C-5 of biotin is 14A, thus allowing movement of biotin between the carboxylation and carboxyltransfer sites.

In mammals and birds, there are four biotin-dependent carboxylases: acetyl CoA carboxylase, pyruvate carboxylase, propionyl CoA carboxylase, and methylcrotonyl CoA carboxylase. Congenital deficiency of three of the four human biotin-dependent carboxylases has been reported.

11.2.1.1 Acetyl CoA Carboxylase Acetyl CoA carboxylase catalyzes the first and rate-limiting step of fatty acid synthesis: carboxylation of acetyl CoA to malonyl CoA. The mammalian enzyme is activated allosterically by citrate and isocitrate, and inhibited by long-chain fatty acyl CoA derivatives. It is also activated in response to insulin and inactivated in response to glucagon.

Tissues that oxidize fatty acids, but do not synthesize them, such as muscle, also have acetyl CoA carboxylase and form malonyl CoA to regulate the activity of carnitine palmitoyltransferase, and thus control the uptake of fatty acids into the mitochondria for f-oxidation.

There are no unequivocal reports of acetyl CoA carboxylase deficiency; presumably impairment of this key enzyme in lipogenesis would not be compatible with intrauterine development.

11.2.1.2 Pyruvate Carboxylase Pyruvate carboxylase catalyzes the car-boxylation of pyruvate to oxaloacetate - both the first committed step of gluconeogenesis from pyruvate and also an important anaplerotic reaction, permitting repletion of tricarboxylic acid cycle intermediates and hence fatty acid synthesis. The mammalian enzyme is activated allosterically by acetyl CoA, which accumulates when there is a need for increased activity of pyruvate carboxylase to synthesize oxaloacetate to permit increased citric acid cycle activity or for gluconeogenesis (Attwood, 1995; Jitrapakdee and Wallace, 1999).

Pyruvate carboxylase is also important in lipogenesis. Citrate is transported out of mitochondria and cleaved in the cytosol to provide acetyl CoA for fatty acid synthesis; the resultant oxaloacetate is reduced to malate, which undergoes oxidative decarboxylation to pyruvate, a reaction that provides at least half of the NADPH required for fatty acid synthesis. Pyruvate reenters the mitochondria and is carboxylated to oxaloacetate to maintain the process.

Mammalian pyruvate carboxylase has four identical subunits, and the isolated monomer will catalyze the complete reaction. By contrast, three distinct subunits can be isolated from acetyl CoA carboxylase of Escherichia coli and spinach chloroplasts: a biotinyl carrier protein, biotin carboxylase, and car-boxyl transferase.

Genetic deficiency of pyruvate carboxylase does not cause the expected hypoglycemia. Rather, it seems that depletion of tissue pools of oxaloacetate results in impaired activity of citrate synthase, and a slowing of citric acid cycle activity, leading to accumulation of lactate, pyruvate, and alanine, and also increased accumulation of acetyl CoA, resulting in ketosis. Affected infants have serious neurological problems and rarely survive. A less severe variant of the disease is associated with low residual activity of pyruvate carboxylase.

11.2.1.3 Propionyl CoA Carboxylase Propionyl CoA carboxylase catalyzes the carboxylation of propionyl CoA to methylmalonyl CoA, which undergoes a vitamin B12-dependent isomerization to succinyl CoA (see Figure 10.13). This reaction provides a pathway for the oxidation, through the tricarboxylic acid cycle, of propionyl CoA arising from the catabolism of isoleucine, valine, odd-carbon fatty acids, and the side chain of cholesterol.

Propionic acidemia caused by propionyl CoA carboxylase deficiency causes severe ketosis and acidosis, resulting in failure to thrive and mental retardation, and is generally fatal in infancy. Some reports of ketotic hyperglycinemia may also, with hindsight, be attributed to propionyl CoA carboxylase deficiency.

11.2.1.4 Methylcrotonyl CoA Carboxylase Methylcrotonyl CoA carboxylase catalyzes the conversion of methylcrotonyl CoA, arising from the cata-bolism of leucine, to methylglutaconyl CoA. This in turn undergoes hydroxy-lation catalyzed by crotonase, yielding hydroxymethyl-glutaryl CoA, which is cleaved to acetyl CoA and acetoacetate.

Methylcrotonyl CoA carboxylase deficiency is the least severe of the carboxylase deficiencies. Maintenance on a low-protein diet, to minimize the burden of leucine that must be catabolized, prevents the development of metabolic acidosis. At higher intakes of protein, the affected infants become hypoglycemic and comatose.

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Responses

  • arvid
    How does biotin facilitate carboxalate reaction?
    6 months ago
  • christina
    How does biotin facilitate carboxylase reaction?
    5 months ago

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