Acetyl-CoA carboxylation

Regulation of enzyme level serves as a coarse control over fatty acid synthesis. In response to changes in physiological state, the levels of the enzymes of fatty acid synthesis fluctuate coordinately. Fatty acid synthesis is also regulated by the direct action of metabolite effectors on key enzymes in the pathway. This means of control is more responsive to sudden alterations in cellular fatty acid requirements. In the case of the committed acetyl-CoA carboxylation step, citrate has been shown to be a positive feed-forward allosteric effector. Since this is the rate-determining step, activation by citrate can effectively adjust the rate of fatty acid synthesis to momentary fluctuations in cellular needs. 

Only within the last few years has it become apparent that the biotin group on enzymes, although covalently bound, is in a dynamic state and shuttles between remote active sites during catalysis. This was proven most directly by work in Roy Vagelos and my laboratories - with the unique E. coli acetyl-CoA carboxylase system. Unlike its counterpart in animal tissues, the carboxylase from E. coli dissociates readily into three protein components all of which are essential foi the overall reaction. Two of these, i.e. biotin carboxylase (BC) and carboxyl transferase (CT), possess catalytic centers for the first [reaction (5)] and second [reaction (6)] half-reactions, respectively, of acetyl-CoA carboxylation. 

The mechanism of chain formation is very complex, but the following scheme provides a general idea of the process. A key player is the mercapto group of an important biological relay compound called coenzyme A (abbreviated HSCoA Figure 19-6). This function binds acetic acid in the form of a thiol ester called acetyl CoA. Carboxylation transforms some... 

When these labeled oxaloacetates enter a second turn of the cycle, both of the carboxyl carbons are lost as CO2, but the methylene and carbonyl carbons survive through the second turn. Thus, the methyl carbon of a labeled acetyl-CoA survives two full turns of the cycle. In the third turn of the cycle, one-half of the carbon from the original methyl group of acetyl-CoA has become one of the carboxyl carbons of oxaloacetate and is thus lost as CO2. In the fourth turn of the cycle, further scrambling results in loss of half of the remaining labeled carbon (one-fourth of the original methyl carbon label of acetyl-CoA), and so on. 

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. 

Eukaryotic cells face a dilemma in providing suitable amounts of substrate for fatty acid synthesis. Sufficient quantities of acetyl-CoA, malonyl-CoA, and NADPH must be generated in the cytosol for fatty acid synthesis. Malonyl-CoA is made by carboxylation of acetyl-CoA, so the problem reduces to generating sufficient acetyl-CoA and NADPH. 

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. 

In animals, acetyl-CoA carboxylase (ACC) is a filamentous polymer (4 to 8 X 10 D) composed of 230-kD protomers. Each of these subunits contains the biotin carboxyl carrier moiety, biotin carboxylase, and transcarboxylase activities, as well as allosteric regulatory sites. Animal ACC is thus a multifunctional protein. The polymeric form is active, but the 230-kD protomers are inactive. The activity of ACC is thus dependent upon the position of the equilibrium between these two forms ... 

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). 

FIGURE 25.3 In the acetyl-CoA carboxylase reaction, the biotin ring, on its flexible tether, acquires carboxyl groups from carbonylphos-phate on the carboxylase subunit and transfers them to acyl-CoA molecules on the transcarboxylase subunits. 

It is also worth noting that the carbon of the carboxyl group that was added to drive this reaction is the one removed by the condensing enzyme. Thus, all the carbons of acetoacetyl-ACP (and of the fatty acids to be made) are derived from acetate units of acetyl-CoA. 

The side-chain carboxylate group of an aspartic acid acts as a base and removes an acidic a proton from acetyl CoA, while the N-H group on the side chain of a histidine acts as an acid and donates a proton to the car bonyl oxygen, giving an enol. 

Assume that acetyl CoA containing a 14C isotopic label in the carboxyl carbon atom is used as starting material for the biosynthesis of mevalonate, as shown in Figure 27.7. At what positions in mevalonate would the isotopic label appear ... 

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]. 

Bicarbonate as a source of CO2 is required in the initial reaction for the carboxylation of acetyl-CoA to mal-onyl-CoA in the presence of ATP and acetyl-CoA carboxylase. Acetyl-CoA carboxylase has a requirement for the vitamin biotin (Figure 21-1). The enzyme is a multienzyme protein containing a variable number of identical subunits, each containing biotin, biotin carboxylase, biotin carboxyl carrier protein, and transcarboxylase, as well as a regulatory allosteric site. The reaction takes place in two steps (1) carboxylation of biotin involving ATP and (2) transfer of the carboxyl to acetyl-CoA to form malonyl-CoA. 

In P-oxidation (Figure 22-2), two carbons at a time are cleaved from acyl-CoA molecules, starting at the carboxyl end. The chain is broken between the 0t(2)- and P(3)-carbon atoms—hence the name P-oxidation. The two-carbon units formed are acetyl-CoA thus, palmi-toyl-CoA forms eight acetyl-CoA molecules. 

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). 

However, if we can design some sophisticated routes to generate carbanion equivalents in the active site of the enzyme, carboxylation reaction might be possible. In fact, acetyl-CoA is carboxylated with the aid of biotin in the biosynthetic pathway of long-chain fatty acids. 

Acetyl-CoA carboxylase (ACCase) carboxylates acetyl-CoA into malo-nyl-CoA and therefore represents the first committed step in fatty acid biosynthesis. ACCase is a multimer essential for cell growth whose components are highly conserved among bacteria, making it a promising broad-spectrum target [8]. 

What p oxidation actually accomplishes is the removal of a C-2 unit as acetyl-CoA from the carboxyl end of the fatty acid. This keeps happening until the fatty acid is completely converted to acetyl-CoA. 

Since neither of the carbons that come in from acetyl-CoA is lost during the first turn of the TCA cycle, it s reasonable to wonder when they are lost. If the label was originally at C-l (the C=0) of acetyl-CoA, it ends up in the two carboxylate groups of oxaloacetate. On the next turn of the TCA cycle (go around again without bringing any more label in from acetyl-CoA) both of these carboxyl groups are lost as C02. So when C-l of acetyl-CoA is labeled, all the label is lost from the TCA-cycle intermediates on the second turn of the cycle. 

It s a lot more complicated when C-2 of the acetyl-CoA is labeled. After the first turn of the cycle, this label ends up on the central carbons of oxaloacetate, and neither of these is lost during the second turn of the cycle. However, on the third turn of the cycle, half the label is lost because half the total label is on the carboxylates of oxaloacetate. On each subsequent turn of the cycle, half the remaining label is lost. The only way to sort this out for yourself is to sit down with the TCA cycle and go round and round. It s a dizzying experience that leaves you a little bit nauseated when it s over. 

Fatty acids are biosynthesized via elongation of C2 units. Here, acetyl-CoA is carboxylated by bicarbonate to form... 

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