N-carboxybiotin

FIGURE 25.2 (a) The acetyl-CoA carboxylase reaction produces malonyl-CoA for fatty acid synthesis, (b) A mechanism for the acetyl-CoA carboxylase reaction. Bicarbonate is activated for carboxylation reactions by formation of N-carboxybiotin. ATP drives the reaction forward, with transient formation of a carbonylphosphate intermediate (Step 1). In a typical biotin-dependent reaction, nncleophilic attack by the acetyl-CoA carbanion on the carboxyl carbon of N-carboxybiotin—a transcarboxylation—yields the carboxylated product (Step 2). 

Biotin (5) is the coenzyme of the carboxylases. Like pyridoxal phosphate, it has an amide-type bond via the carboxyl group with a lysine residue of the carboxylase. This bond is catalyzed by a specific enzyme. Using ATP, biotin reacts with hydrogen carbonate (HCOa ) to form N-carboxybiotin. From this activated form, carbon dioxide (CO2) is then transferred to other molecules, into which a carboxyl group is introduced in this way. Examples of biotindependent reactions of this type include the formation of oxaloacetic acid from pyruvate (see p. 154) and the synthesis of malonyl-CoA from acetyl-CoA (see p. 162). 

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. 

Structures of biotinyl enzyme and N -carboxybiotin. The reactive portions of the coenzyme and the active intermediate are shown in red. In carboxylase enzymes, biotin is covalently bonded to the proteins by an amide linkage between its carboxyl group and a lysyl- -NH2 group in the polypeptide chain. 

The coenzymatic function of biotin appears to be to mediate the carboxylation of substrates by accepting the ATP-activated carboxyl group and transferring it to the carboxyl acceptor substrate. There is good reason to believe that the enzymatic sites of ATP-dependent carboxylation of biotin are physically separated from the sites at which N -carboxybiotin transfers the carboxyl group to acceptor substrates, that is, the transcarboxylase sites. In fact, in the case of the acetyl-CoA carboxylase from E. coli (see chapter 18), these two sites reside on two different subunits, while the biotinyl group is bonded to a third, a small subunit designated biotin carboxyl carrier protein. 

Biotin (D 10) is the prosthetic group of carboxylating and transcarboxylating enzymes. It is reversibly transformed to N -carboxybiotin ... 

Carboxylases bind free carbon dioxide in an ATP-dependent reaction, form N -carboxybiotin, and transfer the carboxy group of this compound to the acceptor molecule ... 

Carboxyl transferases catalyze the transfer of a carboxy group from a donor via N -carboxybiotin to an acceptor ... 

Only recently was it possible to demonstrate that free V-N carboxybiotin derivatives are enzymatically active carboxyl donors. 

Kinetics and reaction mechanism for CO2 exchange in 2-imidazolidinone-l-carboxyhc acid lithium salt (Li 20) were investigated by Lihs and Caudle [134]. N-Carboxyimidazolidone anion, 20, was probed as an analogue for N -carboxybiotin and synthesized as the lithium salt by deprotonation of 2-imidazolidone 21 with phenyllithium and further reaction of the resulting lithium amide with carbon dioxide. The study was addressed to ascertain the viability of unimolecular CO2 elimination from... 

Lihs FJ, Caudle MT (2002) Kinetics and mechanism for CO2 scrambling in a N-carboxyi-midazolidone analogue for N -carboxybiotin. J Am Chem Soc 124 11334-11341... 

The isoimide intermediate (0-acyl urea) reacts with bicarbonate and then decomposes to give I -N-carboxybiotin and phosphate ion. Kluger synthesized an interesting model compound to prove the existence of his intermediate (see page 470). 

The mechanism of the CO2 transfer reaction with acetyl CoA to give malonyl CoA is thought to involve CO2 as the reactive species. One proposal is that loss of CO2 is favored by hydrogen-bond formation between the N-carboxybiotin carbonyl group and a nearby acidic site in the enz3mie. Simultaneous deprotonation of acetyl CoA by a basic site in the enzyme gives a thioester enolate ion that can react with CO2 as it is formed (Figure 29.6). 

Carboxybiotin. The structure of biotin suggested that bicarbonate might be incorporated reversibly into its position 2. However, this proved not to be true and it remained for F. Lynen and associates to obtain a clue from a "model reaction." They showed that purified P-methylcrotonyl-CoA carboxylase promoted the carboxylation of free biotin with bicarbonate (H14C03 ) and ATP. While the carboxylated biotin was labile, treatment with diazomethane (Eq. 14-6) gave a stable dimethyl ester of N-l -carboxybiotin.53 54 The covalently bound biotin at active sites of enzymes was also successfully labeled with 14C02 Treatment of the labeled enzymes with diazomethane followed by hydrolysis with trypsin and pepsin gave authentic N-l -carboxybiocytin. It was now clear that the cleavage of ATP is required to couple the C02 from HCOs to the biotin to form carboxybiotin. The enzyme must... 

Recall that, in aqueous solutions, CO2 exists as HCO3 with the aid of carbonic anhydrase (Section 9.2). The HCO3 is activated to carboxyphosphate. This activated CO2 is subsequently bonded to the N-1 atom of the biotin ring to form the carboxybiotin-enzyme intermediate (see Figure 16.27). The CO2 attached to the biotin is quite activated. The A G° for its cleavage... 

While A-carboxybiotin is responsible for transfer of the carboxyl group, biotin derivatives are not involved in the transfer of more reduced one-carbon units. The next lowest oxidation state at carbon involves the transfer of an aldehyde carboxyl —HC=0. This is equivalent to substitution for the hydroxyl group of formic acid. The leaving group in this case is a derivative of tetrahydrofolate, which contains the carbon as a formamide derivative. Amide resonance is a powerful factor in maintaining the C-N bond. Rotation of the carboxyl out of the plane of the amine will weaken the bond by disruption of resonance. 

Biotin (see Scheme 40) is the prosthetic group associated with the biotin-dependent carboxylases and transcarboxylases. The role of biotin is as a carboxyl group carrier. At the first active site, biotin is carboxylated at the N(l) position to form A-carboxybiotin, which acts as CO2 transport. At the second subsite, the carboxyl group is transferred from A-carboxybiotin to substrate. The mechanism of neither half-reaction has been fully defined (for recent discussions see Blonski et al., 1987 Thatcher et ai, 1986). 

At the same period, X-ray work on the active (-l-)-form revealed that the two rings are cis fused, and the hydrogens of all three asymmetric carbons are in a cis relationship. The structure shows that the N-3 of the ureido function is hindered from reaction by the five carbon side chain of valeric acid. The distance between N-3 and C-6 is only 0.28 nm. Only then did the mechanism of action of biotin become clear a C02-transfer reaction is achieved through the reversible formation of T-Af-carboxybiotin. 

Now that we have examined objectively three mechanisms that agree with the experimental results, we should examine the real problem in biotin chemistry. Indeed, in recent years a controversy has arisen regarding the exact site on biotin to which the carboxyl group is attached. During isolation and characterization by Lynen of the relatively unstable free carboxybiotin, particularly at acidic pH, the product was converted to the more stable dimethyl ester with diazomethane (343, 344). This derivative was subsequently identified as r-N-methoxycarbonyl-( + )-biotin methyl ester. The same product was also obtained by enzymatic degradation of enzyme-bound biotin. Figure 7.16 shows some of these transformations. 

Decarboxylation studies of N-carboxy-2-imidazolidone revealed the poor leaving ability of the imidazolidone anion. Metal ions such as Cu(II) or Mn(II) prevent decarboxylation. The sensitivity of carboxyimidazolidone to specific acid catalysis is probably the most remarkable feature of this compound and could account for the fact that carboxybiotin-enzyme intermediates are unstable. The neutral form however can decarboxylate in a unimolecular pathway via a six-membered transition state. 

These convincing data showed that the I -N-ureido position of biotin serves as the site for carboxyl transfer with biotin enzymes. Lane also correctly pointed out that N O carboxyl migration might have preceded the participation of carboxybiotin in the enzymatic process. However, the well-established thermodynamic and kinetic stabilities of iV-acyl and JV-carboxy-2-imidazolidone derivatives render this possibility unlikely. Moreover, the urea carboxylase component of ATP-amidolyase, also a biotin-dependent enzyme, reversibly carboxylates urea to form iV-carboxyurea, a known example of carboxylation at the N-ureido position (333). 

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