Carboxylation biotin-dependent enzymes

FIGURE 18.32 Biotin is covalently linked to a protein via the e-amino group of a lysine residue. The biotin ring is thus tethered to the protein by a 10-atom chain. It functions by carrying carboxyl groups between distant sites on biotin-dependent enzymes. 

Fatty acids with odd numbers of carbon atoms are rare in mammals, but fairly common in plants and marine organisms. Humans and animals whose diets include these food sources metabolize odd-carbon fatty acids via the /3-oxida-tion pathway. The final product of /3-oxidation in this case is the 3-carbon pro-pionyl-CoA instead of acetyl-CoA. Three specialized enzymes then carry out the reactions that convert propionyl-CoA to succinyl-CoA, a TCA cycle intermediate. (Because propionyl-CoA is a degradation product of methionine, valine, and isoleucine, this sequence of reactions is also important in amino acid catabolism, as we shall see in Chapter 26.) The pathway involves an initial carboxylation at the a-carbon of propionyl-CoA to produce D-methylmalonyl-CoA (Figure 24.19). The reaction is catalyzed by a biotin-dependent enzyme, propionyl-CoA carboxylase. The mechanism involves ATP-driven carboxylation of biotin at Nj, followed by nucleophilic attack by the a-carbanion of propi-onyl-CoA in a stereo-specific manner. 

When pyruvate with a chiral methyl group is carboxylated by pyruvate carboxylase the configuration at C-3 is retained. The carboxyl enters from the 2-si side, the same side from which the proton (marked H ) was removed to form the enolate anion (Eq. 14-12). Comparable stereochemistry has been established for other biotin-dependent enzymes.64 65... 

Knowles, J. R., The mechanism of biotin-dependent enzymes. Ann. Rev. Biochem. 58 195, 1989. Review of the chemical mechanism of biotin-dependent carboxylation reactions. 

Biotin is the coenzyme in a small number of carboxylation reactions in mammalian metabolism and some decarboxylation and transcarboxylation reactions in bacteria. Although the biotin-dependent enzymes are cytosolic and mitochondrial, about 25% of tissue biotin is found in the nucleus, much of it bound as thioesters to histones. Biotin has two noncoenzyme functions induction of enzyme synthesis and regulation of the cell cycle. 

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 IdA, thus allowing movement of biotin between the carboxylation and carboxyltransfer sites. 

How is oxaloacetate replenished Mammals lack the enzymes for the net conversion of acetyl CoA into oxaloacetate or any other citric acid cycle intermediate. Rather, oxaloacetate is formed by the carboxylation of pyruvate, in a reaction catalyzed by the biotin-dependent enzyme pyruvate carboxylase. 

Chemical and catalytic mechanisms of carboxyl transfer in biotin-dependent enzymes 02ACR113. 

Acetyl-CoA carboxylase is a biotin-dependent enzyme. It has been purified from microorganisms, yeast, plants, and animals. In animal cells, it exists as an inactive pro-tomer (M.W. 400,000) and as an active polymer (M.W. 4-8 million). The protomer contains the activity of biotin carboxylase, biotin carboxyl carrier protein (BCCP), transcarboxylase, and a regulatory allosteric site. Each protomer contains a biotinyl group bound in amide linkage to the e-amino group of a lysyl residue. 

The answer is b. (Murray, pp 627-661. Scriver, pp 3897-3964. Sack, pp 121-138. Wilson, pp 287-320.) The vitamin biotin is the cofactor required by carboxylating enzymes such as acetyl CoA, pyruvate, and propionyl CoA carboxylases. The fixation of CO2 by these biotin-dependent enzymes occurs in two stages. In the first, bicarbonate ion reacts with adenosine triphosphate (ATP) and the biotin carrier protein moiety of the enzyme in the second, the active CO2 reacts with the substrate—e.g., acetyl CoA. 

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 final examples of CoA-dependent Claisen-like enzymes are the acetyl- and propionyl-CoA carboxylases (EC 6.4.1.2 and EC 6.4.1.3, respectively) in which acyl-CoA-based enolates react with the activated carboxylate bound to the biotin cofactor of these enzymes. Their enzymology is described in the chapter in this series including biotin-dependent enzymes (see Chapters 1.08 and 7.03). 

The first biotin-dependent enzyme to be discovered was acetyl-CoA-car-boxylase, which forms malonic acid and is necessary for the synthesis of fatty acids cf. section 8.5.3). It is assumed that biotin is bonded to a 14-Angstrom-long lysine linker, so that it can swing back and forth between two active centres in the enzyme complex. In the first centre, biotin is carboxylated under the consumption of ATP in the second, the carboxylic acid function is transferred. [115,116]... 

Attwood, P.V., and Wallace, J.C., 2002. Chemical and catalytic mechanisms of carboxyl transfer reactions in biotin-dependent enzymes. Accounts of Chemical Research. 35 113 120. 

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 . 

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). 

Vitamin H (biotin) is present in liver, egg yolk, and other foods it is also synthesized by the intestinal flora. In the body, biotin is covalently attached via a lysine side chain to enzymes that catalyze carboxylation reactions. Biotin-dependent carboxylases include pyruvate carboxylase (see p. 154) and acetyl-CoA carboxylase (see p. 162). CO2 binds, using up ATP, to one of the two N atoms of biotin, from which it is transferred to the acceptor (see p. 108). 

Certain enzymes catalyze their reactions by way of a multisite mechanism in which the covalently linked intermediate is attached to a long arm that swings from one subsite to another subsite within the enzyme. In some cases, the covalently tethered intermediate can actually be transferred between subunits that form the active site. An example is Propionibacterium shermanii transcarboxylase an enzyme that catalyzes the biotin-dependent conversion of methylmalonyl-CoA and pyruvate to propionyl-CoA and oxaloacetate. Carboxylated biotin allows the two catalytic subsites to operate on the same reaction intermediate. 

In this cycle, one molecule of acetyl-CoA is formed from two molecules of bicarbonate (Figure 3.5). The key carboxylating enzyme is the bifunctional biotin-dependent acetyl-CoA/propionyl-CoA carboxylase. In Bacteria and Eukarya, acetyl-CoA carboxylase catalyzes the first step of fatty acid biosynthesis. However, Archaea do not contain fatty acids in their lipids, and acetyl-CoA carboxylase cannot serve as the key enzyme of fatty acid synthesis rather, it is responsible for autotrophy. 

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. 

Apart from the true Claisen condensations discussed in the previous section in which the electrophilic reaction partner is another thioester, a number of enzymes also catalyze related Claisen-like condensations in which an acyl-CoA-based nucleophile reacts with other electrophilic carbonyl groups such as ketones, aldehydes, and the carboxylate group of carboxy-biotin. The most important examples of such enzymes are hydroxymethylglutaryl-CoA (HMG-CoA) synthase, citrate and homocitrate synthase (HCS), malate and ct-isopropylmalate synthase (ct-IPMS), and the biotin-dependent acetyl- and propionyl-CoA carboxylases. 

After dehydrogenation to 234, X = SCoA, the catabolism of leucine 205 (Scheme 62c) differs from that of the other branched-chain amino acids. A biotin-dependent carboxylation leads to the acid 236, X = SCoA, which is hydrated to HMG-CoA 237, a compound involved in isoprenoid biosynthesis. Feeding stereospecifically labeled samples of leucine in studies of terpenoid biosynthesis indicated that the ( )-methyl group was carboxylated without isomerization of the double bond (181, 182). Messner, Cornforth et al. (215) investigated the hydration 236 = 237 catalyzed by the enzyme 3-methyl-glutaconyl-CoA hydratase (EC 4. 2. 1. 18) and showed that the reversible reaction had syn stereospecificity. 

PEPC catalyses carboxylation of phosphoenol pyruvate (PEP), employing bicarbonate as carboxyl donor (Cooper and Wood, 1971). As in all such enzymes employing bicarbonate in place of CO2, the enzyme must activate bicarbonate towards carboxyl transfer. In this respect, as in others, PEPC resembles biotin-dependent carboxylases. Two distinct mechanisms are postulated for PEPC one involving the intermediacy of carboxyphosphate, the other a cyclic transition state and pseudorotation at phosphorus. 

Biotin enzymes carboxylases that use biotin as a cofactor. The biotin is bound via an amide bond to the E-amino group of a specific lysine residue in the enzyme protein, i.e. B.e. contain a biotinyllysyl residue. Free (+)-E-V-biotinyl-L-lysine actually occurs in yeast extract, and is known as biocytin. During catalysis, N-atom 1 of the biotin residue is carboxylated in an ATP-dependent reaction ATP + HCOj" + bioti-nyl-enzyme (I) -> ADP + Pj + carboxybiotinyl-en-zyme (II). The carboxyl group is then transferred from (II) to the carboxylase substrate (II) + substrate —> (I) + carboxylated substrate. 

Thus, Lynen proposed that—like free biotin—the biotinyl prosthetic group of the enzyme undergoes carboxylation on its I -nitrogen after which carboxyl transfer to the acyl-CoA substrate occurs. This and the results of isotopic exchange experiments carried out in several laboratories, including our own, with various biotin-dependent carboxylases led to the well-known 2-step reaction sequence. 

Proof that a carboxylated en2yme intermediate (enzyme-COf) actually participates in biotin-dependent carboxylations was provided by Yoshito Kaziro in Severn Ochoa s laboratory at New York University. They were able to isolate enzyme-C02 and show an exact stoichiometry between bound biotin and the active carboxy group. Importantly, the isolated enzyme-COr transferred its labile carboxy group to propionyl-CoA yielding methylmalonyl-CoA [reaction (3)] or underwent quantitative decarboxylation upon exposure to ADP, P and Mg + [reverse of reaction (2)]. 

These biochemical transformations occur on a multienzyme complex composed of at least three dissimilar proteins biotin carrier protein (MW = 22,000), biotin carboxylase (MW = 100,000) and biotin transferase (MW = 90,000). Each partial reaction is specifically catalyzed at a separate subsite and the biotin is covalently attached to the carrier protein through an amide linkage to a lysyl a-amino group of the carrier protein (338, 339). In 1971, J. Moss and M. D. Lane, from Johns Hopkins University proposed a model for acetyl-CoA carboxylase of E, coli where the essential role of biotin in catalysis is to transfer the fixed CO2, or carboxyl, back and forth between two subsites. Consequently, reactions catalyzed by a biotin-dependent carboxylase proceed though a carboxylated enzyme complex intermediate in which the covalently bound biotinyl prosthetic poup acts as a mobile carboxyl carrier between remote catalytic sites (Fig. 7.13). 

---