Biotin 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 is involved in carboxylation and decarboxylation reactions. It is covalently bound to its enzyme. In the carboxylase reaction, C02 is first attached to biotin at the ureido nitrogen, opposite the side chain in an ATP-dependent reaction. The activated C02 is then transferred from carboxybiotin to the substrate. The four enzymes of the intermediary metabolism requiring biotin as a prosthetic group are pyruvate carboxylase (pyruvate oxaloacetate), propionyl-CoA-carboxylase (propionyl-CoA methylmalonyl-CoA), 3-methylcroto-nyl-CoA-carboxylase (metabolism of leucine), and actyl-CoA-carboxylase (acetyl-CoA malonyl-CoA) [1]. 

Mitochondrial pyruvate carboxylase catalyzes the cat-boxylation of pymvate to oxaloacetate, an ATP-tequit-ing reaction in which the vitamin biotin is the coenzyme. Biotin binds CO2 from bicatbonate as carboxybiotin ptiot to the addition of the COj to pym-... 

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. 

Propionyl-CoA is first carboxylated to form the d stereoisomer of methylmalonyl-CoA (Pig. 17—11) by propionyl-CoA carboxylase, which contains the cofactor biotin. In this enzymatic reaction, as in the pyruvate carboxylase reaction (see Pig. 16-16), C02 (or its hydrated ion, HCO ) is activated by attachment to biotin before its transfer to the substrate, in this case the propionate moiety. Formation of the carboxybiotin intermediate requires energy, which is provided by the cleavage of ATP to ADP and Pi- The D-methylmalonyl-CoA thus formed is enzymatically epimerized to its l stereoisomer by methylmalonyl-CoA epimerase (Pig. 17-11). The L-methylmal onyl -CoA then undergoes an intramolecular rearrangement to form succinyl-CoA, which can enter the citric acid cycle. This rearrangement is catalyzed by methylmalonyl-CoA mutase, which requires as its coenzyme 5 -deoxyadenosyl-cobalamin, or coenzyme Bi2, which is derived from vitamin B12 (cobalamin). Box 17—2 describes the role of coenzyme B12 in this remarkable exchange reaction. 

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... 

Biotin-dependent decarboxylases act as sodium ion pumps in Klebsiella74 and in various anaerobes.22 75 For example, oxaloacetate is converted to pyruvate and bound carboxybiotin.74 743 The latter is decarboxylated... 

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. 

Structure of A -carboxybiotin linked to biotin carboxyl carrier protein (BCCP). BCCP is one of the components of acetyl-CoA carboxylase isolated from E. coli. 

Figure 12-3- The structure of d-biotin and /V-carboxybiotin covalently attached to an -amino group of a lysyl residue of a carboxylase. Biocytin is the biotin similarly attached to the e-amino group of a lysine. The dotted line indicates the amide bond linkage.

One of the important points at which CO2 enters as a reagent carried by biotin is in fatty acid biosynthesis where C02 is transferred to the enol of acetyl CoA. A magnesium(II) ion is also required and we may imagine the reaction as a nucleophilic attack of the enol on the magnesium salt of carboxybiotin. Most of the C02 transfers we have met take place by mechanisms of this sort nucleophilic attack on a bound molecule of C02, usually involving a metal ion. 

Avidin is a tetrameric protein and binds 4 mol of biotin per tetramer it also binds iV-carboxybiotin with a somewhat lower affinity. The unit of avidin activity is that amount which will bind 1 /rg (4.09 nmol) of biotin commercially available avidin has an activity of 10 to 15 units per mg of protein. 

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. 

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... 

The activated carboxyl group is then transferred from carboxybiotin to pyruvate to form oxaloacetate. The long, flexible link between biotin and the enzyme enables this prosthetic group to rotate from one active site of the enzyme (the ATP-bicarbonate site) to the other (the pyruvate site). 

The first partial reaction of pyruvate carboxylase, the formation of carboxybiotin, depends on the presence of acetyl CoA. Biotin is not carboxylated unless acetyl CoA is bound to the enzyme. Acetyl CoA has no effect on the second partial reaction. The allosteric activation of pyruvate carboxylase by acetyl CoA is an important physiological control mechanism that will be discussed in Section 17.3.1. 

The synthesis of malonyl CoA is catalyzed by acetyl CoA carboxylase, which contains a biotin prosthetic group. The carboxyl group of biotin is covalently attached to the e amino group of a lysine residue, as in pyruvate carboxylase (Section 16.3.2) and propionyl CoA carboxylase (Section 22.3.3). As with these other enzymes, a carboxybiotin intermediate is formed at the expense of the hydrolysis a molecule of ATP. The activated CO2 group in this intermediate is then transferred to acetyl CoA to form malonyl CoA. 

Biotin performs vital metabolic functiun.s in important carboxyliition processes in the form of carboxybiotin. which is in combination with a carboxylase, as represented below. 

The answer is d. (Murray, pp 627-661. Scrivcr, pp 3897-3964. Sack, pp 121—138. Wilson, pp 287-320.) The key enzymatic step of fatty acid synthesis is the carboxylation of acetyl CoA to form malonyl CoA. The carboxyl of biotin is covalently attached to an -amino acid group of a lysine residue of acetyl CoA carboxylase. The reaction occurs in two stages. In the first step, a carboxybiotin is formed ... 

The reaction begins with the ATP-dependent carboxylation of the biotin cofactor of the enzyme. The carboxylase abstracts a proton from the a-carbon of the acetyl-CoA to generate a reactive carbanion. The carbanion attacks the carbon of carboxybiotin to yield mal-onyl-CoA and biotinate. The biotinate is protonated by the enzyme to regenerate its biotin form. 

After completion of the first sequence, carboxybiotin moves to the second site, where the carboxyl group is transferred from biotin to pyruvate, forming oxal-oacetate. In essence, a proton and a carboxyl group trade places in this step. Isotope effects indicate that proton removal from pyruvate is not concerted with carboxylation (60, 61). The lack of positional isotope exchange (62) during this process presumably is because the active complex is isolated from solvent, rather than because of a lack of exchangeable sites. 

The formation of a bond between the carboxylate group derived from bicarbonate and a carbon adjacent to a carbonyl group is indicative of a reaction catalyzed by an enzyme that utilizes biotin as a cofactor (Scheme 16). The recent review by Knowles covers many recent discoveries relating to the role of enzymes in these reactions (43). (Editor s note For additional aspects of biotin-dependent carboxylation, see Chapter 6 by O Leary.) Most biotin-dependent enzymes promote a two-step process in which Ai-carboxybiotin serves as an intermediate in a process involving the exchange of the caiix)xylate group derived from bicarbonate for a proton at the a-carbon of the carbonyl compound (44). 

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... 

A more subtle exchange, which is indicative of internal return in the formation of an intermediate, can be detected in some enzymes through the use of what Middelfort and Rose call positional isotope exchange (32). However, this class of experiments has also given no evidence of an intermediate in that no positional isotope exchange is observed (43). Together, the exchange results show that all the reaction components, biotin, ATP, bicarbonate, and substrate, are necessary in order for an enzyme to produce A-carboxybiotin. 

A -carboxybiotin. The mechanism involves trapping of the addition product between bicarbonate and the conjugate base of biotin by the terminal group of ATP. This mechanism might give positional isotope exchange if the groups were bound loosely. 

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. 

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