Pyruvate biotin

CO -biotin-enzyme + pyruvate. - biotin-enzyme + oxaloacetate... 

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. 

O Pyruvate undergoes a biotin-dependent carboxylation on the methyl group to give oxaloacetate... 

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-... 

Biotin s biochemical role is becoming clearer. As mentioned, biotin has been implicatfd in C02-fixation. Good examples for this reaction in animals are (a) the combination of pyruvic acid and C02 to form oxaloacetic acid, or aspartic acid (B26, H19) to replace biotin, and (b) the growth stimulation of certain bacteria in the presence of biotin, and bicarbonate (LI). 

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. 

Figure 3.4 Structure of two prosthetic groups (a) biotin (b) lipoate. Biotin functions as a carboxyl group carrier, e.g. in acetyl-CoA carboxylase. Lipoate is presented in its oxidised form (-S-S-). It is a cofactor for pyruvate dehydrogenase and oxoglu-tarate dehydrogenase.

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). 

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. 

This biotin-dependent enzyme [EC 6.4.1.1] catalyzes the reaction of ATP with pyruvate and HCOs to produce ADP, orthophosphate, and oxaloacetate. The enzyme from yeast requires zinc whereas the enzyme isolated from animal tissues needs manganese as well as ace-tyl-CoA. 

This enzyme [EC 2.1.3.1], also known as methylmalonyl-CoA carboxyltransferase, catalyzes the reaction of (5 )-2-methyl-3-oxopropanoyl-CoA with pyruvate to produce propanoyl-CoA and oxaloacetate. The enzyme requires biotin, cobalt, and zinc as cofactors. 

OXALOACETATE DECARBOXYASE PROPIONYL-CoA CARBOXYASE PYRUVATE CARBOXYASE TRANSCARBOXYASE BIOTIN HOLOCARBOXYLASE SYNTHETASE BI RAD I CAL CARBENE FREE RADICALS... 

Biotin (vitamin B ) is widespread in foods and is also synthesized by intestinal bacteria. It is a coenzyme for the carboxylation of pyruvate, acetyl-coenzyme-A (CoA), propionyl CoA, and /1-methyl-crotonyl CoA and is involved in fatty acid formation and in energy release from carbohydrates. In humans deficiencies only occur in patients with an abnormal gut flora and manifests itself as exfoliative dermatitis and alopecia. 

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.)... 

Carboxyl groups are activated in a reaction that splits ATP and joins C02 to enzyme-bound biotin. This activated C02 is then passed to an acceptor (pyruvate in this case) in a carboxylation reaction. 

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... 

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