Acetyl -malonyl enzyme

Additional complexity occurs at the level of the starter and extender units used for constructing the polyketide scaffold, where the typically 2-4 carbon building blocks such as acetyl-, malonyl-, and propionyl-CoA are selectively used by different PKSs, thus increasing the repertoire of potential products formed. For example, results obtained from work with plant enzymes have shown that larger and more complex starter units such as phenylpropanoid- as well as fatty acyl-CoAs can also serve as efficient substrates (22,23,24). 

As we began this chapter, we saw that photosynthesis traditionally is equated with the process of COg fixation, that is, the net synthesis of carbohydrate from COg. Indeed, the capacity to perform net accumulation of carbohydrate from COg distinguishes the phototrophic (and autotrophic) organisms from het-erotrophs. Although animals possess enzymes capable of linking COg to organic acceptors, they cannot achieve a net accumulation of organic material by these reactions. For example, fatty acid biosynthesis is primed by covalent attachment of COg to acetyl-CoA to form malonyl-CoA (Chapter 25). Nevertheless, this fixed COg is liberated in the very next reaction, so no net COg incorporation occurs. 

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. 

The enzymes that catalyze formation of acetyl-ACP and malonyl-ACP and the subsequent reactions of fatty acid synthesis are organized quite differently in different organisms. We first discuss fatty acid biosynthesis in bacteria and plants, where the various reactions are catalyzed by separate, independent proteins. Then we discuss the animal version of fatty acid biosynthesis, which involves a single multienzyme complex called fatty acid synthase. 

The individual steps in the elongation of the fatty acid chain are quite similar in bacteria, fungi, plants, and animals. The ease of purification of the separate enzymes from bacteria and plants made it possible in the beginning to sort out each step in the pathway, and then by extension to see the pattern of biosynthesis in animals. The reactions are summarized in Figure 25.7. The elongation reactions begin with the formation of acetyl-ACP and malonyl-ACP, which... 

Following the formation of malonyl CoA, another nucleophilic acyl substitution reaction occurs in step 4 to form the more reactive malonyl ACP, thereby binding the malonyl group to an ACP arm of the multienzyme synthase. At this point, both acetyl and malonyl groups are bound to the enzyme, and the stage is set for their condensation. 

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. 

This pathway (the microsomal system ) elongates saturated and unsaturated fatty acyl-CoAs (from Cjg upward) by two carbons, using malonyl-CoA as acetyl donor and NADPH as reductant, and is catalyzed by the microsomal fatty acid elongase system of enzymes (Figure 21-5). Elongation of stearyl-CoA in brain increases rapidly during myehnation in order to provide C22 and C24 fatty acids for sphingoEpids. 

Production of Malonyl-CoA for the Fatty Acid Biosynthesis. Acetyl-CoA serves as a substrate in the production of malonyl-CoA. There are several routes by which acetyl-CoA is supplied to die cytoplasm. One route is the transfer of acetyl residues from the mitochondrial matrix across the mitochondrial membrane into the cyto-plasm. This process resembles a fatty acid transport and is likewise effected with the participation of carnitine and the enzyme acetyl-CoA-camitine transferase. Another route is the production of acetyl-CoA from citrate. Citrate is delivered from the mitochondria and undergoes cleavage in the cytoplasm by the action of the enzyme ATP-citrate lyase ... 

The reaction is catalyzed by the sixth synthetase enzyme-enoylreductase, to form an enzyme-bound butyryl. The butyryl thus synthetized is transferred, through the mediacy of the first synthetase enzyme, acetyltransacylase, onto the SH group (the upper one in the Scheme) initially bound to the acetyl primer. The SH group (the lower one in the Scheme), thus freed, accepts a new malonyl residue ... 

The biosynthesis of polyketides (including chain initiation, elongation, and termination processes) is catalyzed by large multi-enzyme complexes called polyketide synthases (PKSs). The polyketides are synthesized from starter units such as acetyl-CoA, propionyl-CoA, and other acyl-CoA units. Extender units such as malonyl-CoA and methylmalonyl-CoA are repetitively added via a decarboxylative process to a growing carbon chain. Ultimately, the polyketide chain is released from the PKS by cleavage of the thioester, usually accompanied by chain cyclization [49]. 

The first formation of a carbon-carbon bond occurs between malonyl and acetyl units bound to fatty acid synthase. After reduction, dehydration, and further reduction, the acyl enzyme is condensed with more malonyl-CoA and the cycle is repeated until the acyl chain grows to C16. When the growing fatty acid reaches a chain length of 16 carbons, the acyl group is hydrolyzed to give the free fatty acid. 

Members of the CHS/STS family of condensing enzymes are relatively modest-sized proteins of 40-47 kDa that function as homodimers. Each enzyme typically reacts with a cinnamoyl-CoA starter unit and catalyzes three successive chain extensions with reactive acetyl groups derived from enzyme catalyzed decarboxylation of malonyl-CoA.11 Release of the resultant tetraketide together with or prior to polyketide chain cyclization and/or decarboxylation yields chalcone or resveratrol (a stilbene). Notably, CHS and STS catalyze identical reactions up to the formation of the intermediate tetraketide. Divergence occurs during the termination step of the biosynthetic cascade as each tetraketide intermediate undergoes a distinct cyclization reaction (Fig. 12.2). 

CHS orchestrates the condensation, cyclization, and aromatization of one p-coumaroyl-CoA and three malonyl-CoA molecules to produce chalcone (Fig. 12.2).22 Transfer of the p-coumaroyl moiety from the CoA-linked starter molecule to Cys 164 within the active site initiates the reaction sequence. Next, the sequential condensation of three acetate units, derived from malonyl-CoA, with the enzyme-bound coumaroyl moiety forms a tetraketide intermediate. Inherent in the condensation reaction is decarboxylation of malonyl-CoA to an acetyl-CoA carbanion that serves as a nucleophile during the successive chain elongation... 

Figure 12.8 A. 2-PS reaction. B. Surface representations of the CHS (left) and 2-PS (right) active site cavities are shown. The catalytic cysteines (red), the three positions that convert CHS into 2-PS (green), and the substitution that does not affect product formation (blue) are highlighted. C. TLC analysis of CHS, 2-PS, and CHS mutant enzymes. The radiogram shows the radiolabeled products produced by incubation of each protein with [14C]malonyl-CoA and either p-coumaroyl-CoA (C) or acetyl-CoA (A). Numbering of mutants corresponds to CHS with 2-PS numbering in parenthesis. Positions of reaction products and their identities are indicated.

By 1960 it was clear that acetyl CoA provided its two carbon atoms to the to and co—1 positions of palmitate. All the other carbon atoms entered via malonyl CoA (Wakil and Ganguly, 1959 Brady et al. 1960). It was also known that 3H-NADPH donated tritium to palmitate. It had been shown too that fatty acid synthesis was very susceptible to inhibition by p-hydroxy mercuribenzoate, TV-ethyl maleimide, and other thiol reagents. If the system was pre-incubated with acetyl CoA, considerable protection was afforded against the mercuribenzoate. In 1961 Lynen and Tada suggested tightly bound acyl-S-enzyme complexes were intermediates in fatty acid synthesis in the yeast system. The malonyl-S-enzyme complex condensed with acyl CoA and the B-keto-product reduced by NADPH, dehydrated, and reduced again to yield the (acyl+2C)-S-enzyme complex. Lynen and Tada thought the reactions were catalyzed by a multifunctional enzyme system. 

Triglyceride and fatty acid synthesis are promoted by insulin stimulation of liver and adipose tissues by causing the phosphorylation of the first and controlling enzyme in the pathway acetyl-CoA carboxylase (see Section 6.3.2). This enzyme catalyses the formation of malonyl-CoA and requires both allosteric activation by citrate and covalent modification for full activity. 

The fatty acid synthesis pathway can be seen to occur in two parts. An initial priming stage in which acetyl-CoA is converted to malonyl-CoA by a carboxylation reaction (Figure 6.9) is followed by a series of reactions which occur on a multi-enzyme complex (MEC), which achieves chain elongation forming C16 palmitoyl-CoA. The whole process occurs in the cytosol. 

Figure 7.15 Inhibition of acetyl-CoA carboxylase by cyclic AMP dependent protein kinase and AMP dependent protein kinase the dual effect of glucagon. Phosphorylation of acetyl-CoA carboxylase by either or both enzymes inactivates the enzyme which leads to a decrease in concentration of malonyl-CoA, and hence an increase in activity of carnitine palmitoyltransferase-I and hence an increase in fatty acid oxidation. Insulin decreases the cyclic AMP concentration maintaining an active carboxylase and a high level of malonyl-CoA to inhibit fatty acid oxidation.

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