Biotin, carboxylations with function

Neither catalytic component contains a trace of bound biotin. The biotin prosthetic group is covalently linked to the third component, carboxyl carrier protein (CCP—biotin). As with other biotin enzymes, the bicyclic ring of the prosthetic group resides at the distal end of a flexible 14 A side chain which allows it to act as a mobile carboxyl carrier between the two catalytic centers as illustrated below. A large number of biotin-dependent enzymes which carry out diverse reaction types are now known. All of these reactions proceed through a carboxylated intermediate with the carboxybiotinyl prosthetic group functioning as a mobile carboxyl carrier . 

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). 

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. 

Biotin acts as a carboxyl group carrier in a series of carboxylation reactions, a function originally suggested by the fact that aspartate partially replaces biotin in promoting the growth of the yeast Torula cremonis. Aspartate was known to arise by transamination from oxaloacetate, which in turn could be formed by carboxylation of pyruvate. Subsequent studies showed that biotin was needed for an enzymatic ATP-dependent reaction of pyruvate with bicarbonate ion to form oxaloacetate (Eq. 14-3). This is a (3 carboxylation coupled to the hydrolysis of ATP. 

Amine-reactive biotinylation reagents contain functional groups off biotin s valeric acid side chain that are able to form covalent bonds with primary amines in proteins and other molecules. Two basic types are commonly available NHS esters and car-boxylates. NHS esters spontaneously react with amines to form amide linkages (Chapter 2, Section 1.4). Carboxylate-containing biotin compounds can be coupled to amines via a carbodiimide-mediated reaction using EDC (Chapter 3, Section 1.1). 

All compounds of the arachidonic acid cascade as well as many peptides and biotin contain the carboxyl function. After seeing the value of the Barton-McCombie reaction, it was logical to consider if similar chemistry could be carried out with the carboxyl function.1 In particular, decarboxylation to the corresponding radical seemed a promising way to replace -C02H by -H. 

The important function of biotin is its role as coenzyme for carboxylase, which catalyses carbon dioxide fixation or carboxylation reaction. The epsilon amino group of lysine in carboxylase enzymes combines with the carboxyl group of biotin to form covalently linked biotinyl carboxyl carrier protein (BCCP or biocytin) (Figure 6.8). This serves as an intermediate carrier of carbon dioxide. The carboxylation of acetyl CoA to malonyl CoA in presence of acetyl CoA carboxylase requires biotin as coenzyme. Propionyl carboxylase and pyruvate carboxylase are also associated with biotin. 

Biotin is ds-tetrahydro-2-oxothieno[3,4-d]-imidazoiine-4 valeric acid (Figure 30-18). The vitamin in most organisms occurs mauily bound to protein. The -amino group of the lysyi side chain of protein is linked via an amide function, involving the carboxyl group of the valeryl side chain of biotin. In addition, some biotin is linked noncovalently as a complex with avidin, a protein in egg white. 

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