Pyruvate carboxylase biotin

Pyruvate carboxylase is the most important of the anaplerotie reactions. It exists in the mitochondria of animal cells but not in plants, and it provides a direct link between glycolysis and the TCA cycle. The enzyme is tetrameric and contains covalently bound biotin and an Mg site on each subunit. (It is examined in greater detail in our discussion of gluconeogenesis in Chapter 23.) Pyruvate carboxylase has an absolute allosteric requirement for acetyl-CoA. Thus, when acetyl-CoA levels exceed the oxaloacetate supply, allosteric activation of pyruvate carboxylase by acetyl-CoA raises oxaloacetate levels, so that the excess acetyl-CoA can enter the TCA cycle. 

Two particularly interesting aspects of the pyruvate carboxylase reaction are (a) allosteric activation of the enzyme by acyl-coenzyme A derivatives and (b) compartmentation of the reaction in the mitochondrial matrix. The carboxy-lation of biotin requires the presence (at an allosteric site) of acetyl-coenzyme A or other acylated coenzyme A derivatives. The second half of the carboxylase reaction—the attack by pyruvate to form oxaloacetate—is not affected by CoA derivatives. 

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 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-... 

Pyruvate carboxylase is a mitochondrial enzyme and like other carboxylase or decarboxylase enzymes requires biotin as coenzyme. The biotin is firmly attached to the enzyme protein (i.e. a prosthetic group) via a lysine residue. The role of biotin is to hold the C02 in the correct orientation to allow its incorporation into the pyruvate. 

Pyruvate carboxylase is a mitochondrial enzyme requiring biotin. It is activated by acetyl CoA (fiom p oxidation). The product oxaloacetate (OAA), a citric add cyde intermediate, cannot leave the mitochondria but is reduced to malate that can leave via the malate shuttle. In the cytoplasm, malate is reoxidized to OAA. 

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). 

Pyruvate is first transported from the cytosol into mitochondria or is generated from alanine within mitochondria by transamination, in which the a-amino group is removed from alanine (leaving pyruvate) and added to an a-keto carboxylic acid (transamination reactions are discussed in detail in Chapter 18). Then pyruvate carboxylase, a mitochondrial enzyme that requires the coenzyme biotin, converts the pyruvate to oxaloacetate (Fig. 14-17) ... 

The reaction involves biotin as a carrier of activated HCO3 (Fig. 14-18). The reaction mechanism is shown in Figure 16-16. Pyruvate carboxylase is the first regulatory enzyme in the gluconeogenic pathway, requiring acetyl-CoA as a positive effector. (Acetyl-CoA is produced by fatty acid oxidation (Chapter 17), and its accumulation signals the availability of fatty acids as fuel.) As we shall see in Chapter 16 (see Fig. 16-15), the pyruvate carboxylase reaction can replenish intermediates in another central metabolic pathway, the citric acid cycle. 

FIGURE 14-18 Role of biotin in the pyruvate carboxylase reaction. 

The pyruvate carboxylase reaction requires the vitamin biotin (Fig. 16-16), which is the prosthetic group of the enzyme. Biotin plays a key role in many carboxyla-tion reactions. It is a specialized carrier of one-carbon groups in their most oxidized form C02. (The transfer of one-carbon groups in more reduced forms is mediated by other cofactors, notably tetrahydrofolate and 5-adenosylmethionine, as described in Chapter 18.)... 

Pyruvate carboxylase has four identical subunits, each containing a molecule of biotin covalently attached through an amide linkage to the -amino group of a specific Lys residue in the enzyme active site. Carboxylation of pyruvate proceeds in two steps (Fig. 16-16) first, a carboxyl group derived from HCO3 is attached to biotin,... 

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... 

When intermediates are shunted from the citric acid cycle to other pathways, they are replenished by several anaplerotic reactions, which produce four-carbon intermediates by carboxylation of three-carbon compounds these reactions are catalyzed by pyruvate carboxylase, PEP carboxykinase, PEP carboxylase, and malic enzyme. Enzymes that catalyze carboxylations commonly employ biotin to activate C02 and... 

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. 

Carboxylation of pyruvate to oxaloacetate (OAA) by pyruvate carboxylase is a biotin-dependent reaction (see Figure 8.24). This reaction is important because it replenishes the citric acid cycle intermediates, and provides substrate for gluconeogenesis (see p. 116). 

Pyruvate is converted to phosphoenolpyruvate (PEP) by pyruvate carboxylase and PEP carboxykinase. The carboxylase requires biotin and ATP, and is allosterically activated by acetyl CoA. PEP carboxykinase, which requires GTP, is the rate-limiting step in gluconeo genesis. The transcription of its mRNA is increased by glucagon and decreased by insulin. 

PEP carboxylase is lacking from animal tissues and fungi. In these creatures PEP is converted to pyruvate, which is then carboxylated to oxaloacetate with coupled cleavage of ATP by the action of pyruvate carboxylase (Eq. 14-3), an enzyme that not only utilizes bicarbonate ion but also contains biotin. However, there are mechanistic similarities between its action and that of PEP carboxylase. 

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... 

A bound divalent metal ion, usually Mn2+, is required in the transcarboxylation step. A possible function is to assist in enolization of the carboxyl acceptor. However, measurement of the effect of the bound Mn2+ on 13C relaxation times in the substrate for pyruvate carboxylase indicated a distance of 0.7 ran between the carbonyl carbon and the Mn2+, too great for direct coordination of the metal to the carbonyl oxygen.68 Another possibility is that the metal binds to the carbonyl of biotin as indicated in Eq. 14-11. Pyruvate carboxylase utilizes two divalent metal ions and at least one monovalent cation.683... 

Eight enzyme-catalyzed reactions are involved in the conversion of acetyl-CoA into fatty acids. The first reaction is catalyzed by acetyl-CoA carboxylase and requires ATP. This is the reaction that supplies the energy that drives the biosynthesis of fatty acids. The properties of acetyl-CoA carboxylase are similar to those of pyruvate carboxylase, which is important in the gluconeogenesis pathway (see chapter 12). Both enzymes contain the coenzyme biotin covalently linked to a lysine residue of the protein via its e-amino group. In the last section of this chapter we show that the activity of acetyl-CoA carboxylase plays an important role in the control of fatty acid biosynthesis in animals. Regulation of the first enzyme in a biosynthetic pathway is a strategy widely used in metabolism. 

Biotin Cofactor for some carboxylases acetyl CoA carboxylase, pyruvate carboxylase, [1-methylcrotonyl... 

Thus pyruvate carboxylase generates oxaloacetate for gluconeogenesis but also must maintain oxaloacetate levels for citric acid cycle function. For the latter reason, the activity of pyruvate carboxylase depends absolutely on the presence of acetyl CoA the biotin prosthetic group of the enzyme cannot be carboxy-lated unless acetyl CoA is bound to the enzyme. This allosteric activation by acetyl CoA ensures that more oxaloacetate is made when excess acetyl CoA is present. In this role of maintaining the level of citric acid cycle intermediates, the pyruvate carboxylase reaction is said to be anaplerotic, that is filling up. ... 

Acetyl-CoA is a positive effector of pyruvate carboxylase, a biotin enzyme. The correct answer is 1,3-diphosphoglyceric acid. It is produced by the oxidation of glyceraldehyde-3-phosphate in the presence of P,. 

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