Bicarbonate reaction with biotin

Sauers et al. proposed that carboxy phosphate may decompose to give carbon dioxide and inorganic phosphate prior to reaction with biotin (57). These workers suggest that this provides a tamed form of carbon dioxide that is low in entropy in that it is localized in the vicinity of biotin and thus is a much better reactant than bicarbonate. 

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. 

MECHANISM FIGURE 16-16 The role of biotin in the reaction catalyzed by pyruvate carboxylase. Biotin is attached to the enzyme through an amide bond with the e-amino group of a Lys residue, forming biotinyl-enzyme. Biotin-mediated carboxylation reactions occur in two phases, generally catalyzed by separate active sites on the enzyme as exemplified by the pyruvate carboxylase reaction. In the first phase (steps to ), bicarbonate is converted to the more activated C02, and then used to carboxylate biotin. The bicarbonate is first activated by reaction with ATP to form carboxyphosphate (step ), which breaks down to carbon dioxide (step ). In effect, the... 

Claisen reactions involving acetyl-CoA are made even more favourable by first converting acetyl-CoA into malonyl-CoA by a carboxylation reaction with CO2 using ATP and the coenzyme biotin (Figure 2.9). ATP and CO2 (as bicarbonate, HC03-) form the mixed anhydride, which car-boxy lates the coenzyme in a biotin-enzyme complex. Fixation of carbon dioxide by biotin-enzyme complexes is not unique to acetyl-CoA, and another important example occurs in the generation of oxaloacetate from pyruvate in the synthesis of glucose from non-carbohydrate sources... 

The reactive mtermediate is l-AT-carboxy-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. 

Steps 3-4 of Figure 29.5 Carboxytation and Acyl Transfer The third step is a lotul-ing reaction in which acetyl CoA is carboxylated by reaction with HC03 and ATP to yield malonyl CoA plus ADP. This step requires the coenzyme biotin, which is bonded to the lysine residue of acetyl CoA carboxylase and acts as a carrier of CO2. Biotin first reacts with bicarbonate ion to give A carboxybiotin, tvhich then reacts with the enolate ion of acetyl CoA and transfers the CO2 group. Thus, biotin acts as a carrier of CO2, binding it in one step and releasing it in another. 

The formation of A -carboxybiotin from bicarbonate and biotin on an enzyme involves the stoichiometric hydrolysis of ATP to produce ADP and inorganic phosphate (Scheme 17). This is particularly significant because the hydrolysis of ATP is coupled to formation of the carbon-carbon bond (45, 46) but is not explicitly involved in the apparent stoichiometry of the biosynthetic process. Therefore, the function of ATP is cryptic, as it is in most processes in which ATP hydrolysis accompanies a biosynthetic process. Although biotin-dependent reactions are not a general model for other ATP-dependent processes, the patterns that emerge from the study of such a mechanism guides one in thinking about the other processes. In the biotin-dependent case, the reaction with ATP... 

Another possibility within this class of mechanism involves the initial reaction of biotin with ATP, forming ADP and a phosphorylated biotin species. It has been proposed, based on model studies, that such a species would be O-phosphobiotin (52,53). This reacts with bicarbonate to produce iV-carboxybiotin and inorganic phosphate (Scheme 20). The transfer of oxygen from bicarbonate occurs in the second step in this case. Models for the 0-phosphorylation of biotin suggest that such a process can occur readily. 

A third member of the bimolecular then unimolecular reaction class is a variant of the previous mechanism. In this case, the conjugate base of biotin reacts with bicarbonate to produce an addition intermediate that then reacts with ATP (Scheme 23). It is likely that the phosphorus of the terminal group of ATP would preassociate with an oxygen of bicarbonate. In particular, if the anionic center of bicarbonate associates with a cation, the 7r-electron density of bicarbonate would align with the phosphorus of the terminal phosphate of ATP. The addition of the conjugate base of a urea to a carboxylate is an appropriate model for this mechanism. The intermediate should be very reactive toward ATP based on the observation that the conjugate base of a carbonyl hydrate reacts rapidly with an internal phosphate ester (59). 

Scheme 23. Reaction of biotin with bicarbonate followed by reaction with ATP to form a phos-phorylated tetrahedral intermediate.

All biotin-requiring enzymes follow the same three steps activation of bicarbonate by ATP, reaction of activated bicarbonate with biotin to form carboxybiotin, and transfer of the carboxyl group from carboxybiotin to the substrate. 

Rittenberg and Bloch showed in the late 1940s that acetate units are the building blocks of fatty acids. Their work, together with the discovery by Salih Wakil that bicarbonate is required for fatty acid biosynthesis, eventually made clear that this pathway involves synthesis of malonyl-CoA. The carboxylation of acetyl-CoA to form malonyl-CoA is essentially irreversible and is the committed step in the synthesis of fatty acids (Figure 25.2). The reaction is catalyzed by acetyl-CoA carboxylase, which contains a biotin prosthetic group. This carboxylase is the only enzyme of fatty acid synthesis in animals that is not part of the multienzyme complex called fatty acid synthase. 

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). 

Step 1 of Figure 29.13 Carboxylation Gluconeogenesis begins with the carboxyl-afion of pyruvate to yield oxaloacetate. The reaction is catalyzed by pyruvate carboxylase and requires ATP, bicarbonate ion, and the coenzyme biotin, which acts as a carrier to transport CO2 to the enzyme active site. The mechanism is analogous to that of step 3 in fatty-acid biosynthesis (Figure 29.6), in which acetyl CoA is carboxylated to yield malonyl CoA. 

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 biotin-dependent enzyme [EC 6.4.1.4] catalyzes the reaction of ATP with 3-methylcrotonyl-CoA and bicarbonate to produce ADP, orthophosphate, and 3-methyl-glutaconyl-CoA. 

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. 

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... 

Reaction buffer 100 mM bicarbonate buffer (pH 8.0-8.3) is preferred for labeling antibody amines (see Note 3). The optimum reaction pH values for labeling antibodies with other reactive biotin derivatives are summarized in Table 3. 

CoA to form malonyl CoA using C02 in the form of bicarbonate HC03 (Fig. 2). This reaction is catalyzed by the enzyme acetyl CoA carboxylase which has biotin as a prosthetic group, a common feature in C02-binding enzymes. One molecule of ATP is hydrolyzed in the reaction, which is irreversible. The elongation steps of fatty acid synthesis all involve intermediates linked to the terminal sulfhydryl group of the phosphopantetheine reactive unit in ACP phosphopantetheine is also the reactive unit in CoA. Therefore, the next steps are the formation of acetyl-ACP and malonyl-ACP by the enzymes acetyl transacylase and malonyl transacylase, respectively (Fig. 2). (For the synthesis of fatty acids with an odd number of carbon atoms the three-carbon propionyl-ACP is the starting point instead of malonyl-ACP.)... 

The reaction takes place in two parts on two different sub-sites on the enzyme. In the first part biotin attacks bicarbonate with a simultaneous attack/hydrolysis by bicarbonate on ATP, resulting in the release of ADP and inorganic phosphate (note the coupling by the enzyme of independent processes in this reaction) ... 

Scheme 24. Parallel reactions of bicarbonate and a carboxylate substrate with the conjugate base of biotin.

Fig. 20.18. Pyruvate carboxylase reaction. Pyruvate carboxylase adds a carboxyl group from bicarbonate (which is in equihbrium with CO2) to pyruvate to form oxaloacetate. Biotin is used to activate and transfer the CO2. The energy to form the covalent biotin-C02 complex is provided by the high-energy phosphate bond of ATP, which is cleaved in the reaction. The enzyme is activated by acetyl CoA.

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