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Fatty acid synthesis- Subjective Questions (Set-2)
Answer-Fatty acids are synthesized by an extramitochondrial system, which is responsible for the complete synthesis of palmitate from acetyl-CoA in the cytosol. Following substances are required for the synthesis of fatty acids-
1) Acetyl co A- As the initial substrate
2) NADPH- As a donor of reducing equivalents
3) Enzymes- Acetyl co A carboxylase and Fatty acid synthase, both are multienzyme complexes.
4) Coenzymes and cofactors- Biotin, Mg++, Mn ++ and NADPH
5) Energy in the form of ATP
Q- Give a brief account of the structural characteristics of fatty acid synthase complex. Highlight the biological advantage of having a multienzyme complex.
Answer- In mammals, the fatty acid synthase complex is a dimer comprising two identical monomers, each containing all seven enzyme activities of fatty acid synthase on one polypeptide chain (Figure-1 and 2)
Each chain is folded into three domains joined by flexible regions (Figure-1).
1) Domain 1, the substrate entry and condensation unit, contains acetyl transferase, malonyl transferase, and β-ketoacyl synthase (condensing enzyme).
2) Domain 2, the reduction unit, contains the acyl carrier protein, β-ketoacyl reductase, dehydratase, and enoyl reductase.
3) Domain 3, the palmitate release unit, contains the thioesterase.
Thus, seven different catalytic sites are present on a single polypeptide chain.
In bacteria and plants, the individual enzymes of the fatty acid synthase system are separate, and the acyl radicals are found in combination with a protein called the acyl carrier protein (ACP). However, in yeast, mammals, and birds, the synthase system is a multienzyme polypeptide complex that incorporates ACP, which takes over the role of CoA. It contains the vitamin pantothenic acid in the form of 4′-phosphopantetheine (Figure-1).
Figure-1 Schematic Representation of Fatty Acid Synthase.-Each of the identical chains in the dimer contains three domains. Domain 1 (blue) contains acetyl transferase (AT), malonyl transferase (MT), and condensing enzyme (CE). Domain 2 (yellow) contains acyl carrier protein (ACP), beta-ketoacyl reductase (KR), dehydratase (DH), and enoyl reductase (ER). Domain 3 (red) contains thioesterase (TE). The flexible phosphopantetheinyl group (green) carries the fatty acyl chain from one catalytic site on a chain to another, as well as between chains in the dimer.
Domain 1 of each chain of this dimer interacts with domains 2 and 3 of the other chain. Thus, each of the two functional units of the synthase consists of domains formed by different chains. Indeed, the arenas of catalytic action are the interfaces between domains on opposite chains.
Biological Advantage of having Multienzyme complex-
1) An advantage of this arrangement is that the synthetic activity of different enzymes is coordinated since it is encoded by a single gene.
2) A multienzyme complex consisting of covalently joined enzymes is more stable than one formed by noncovalent attractions.
3) Furthermore, intermediates can be efficiently handed from one active site to another without leaving the assembly.
Figure-2- Fatty acid synthase multienzyme complex- The —SH of the 4′-phosphopantetheine of one monomer is in close proximity to the —SH of the cysteine residue of the ketoacyl synthase of the other monomer, suggesting a “head-to-tail” arrangement of the two monomers. Though each monomer contains all the partial activities of the reaction sequence, the actual functional unit consists of one-half of one monomer interacting with the complementary half of the other. Thus, two acyl chains are produced simultaneously.
Q- Discuss the steps of de novo synthesis of fatty acids, highlighting the roles of enzymes, coenzymes and the energy consumed during the process of synthesis.
Answer- Fatty acid synthesis takes places in three stages- Initiation, Elongation and Termination,
Formation of Malonyl co A
Fatty acid synthesis starts with the carboxylation of acetyl CoA to malonyl CoA. This irreversible reaction is the committed step in fatty acid synthesis.
The synthesis of malonyl CoA is catalyzed by acetyl CoA carboxylase, which contains a biotin prosthetic group. The carboxyl group of biotin is covalently attached to the € amino group of a lysine residue, as in pyruvate carboxylase and propionyl CoA carboxylase.
As with these other enzymes, a carboxy biotin intermediate is formed at the expense of the hydrolysis of a molecule of ATP. The activated CO2 group in this intermediate is then transferred to acetyl CoA to form malonyl CoA.
Acyl carrier Protein-The intermediates in fatty acid synthesis are linked to an acyl carrier protein. Specifically, they are linked to the sulfhydryl terminus of a phosphopantetheine group, which is, in turn, attached to a serine residue of the acyl carrier protein.
2) Elongation cycle in fatty acid synthesis- Elongation takes place in a cyclic manner, where four processes (Condensation, reduction, dehydration and reduction) are repeated till a fatty acid with a required chain length is synthesized.
Initially, a priming molecule of acetyl-CoA combines with a cysteine —SH group catalyzed by acetyl transacylase (Figure-3). Malonyl-CoA combines with the adjacent —SH on the 4′-phosphopantetheine of ACP of the other monomer, catalyzed by malonyl transacylase (reaction-1), to form acetyl (acyl)-malonyl enzyme.
Malonyl transacylase is highly specific, whereas acetyl transacylase can transfer acyl groups other than the acetyl unit, though at a much slower rate. Fatty acids with an odd number of carbon atoms are synthesized starting with propionyl ACP, which is formed from propionyl CoA by acetyl transacylase.
a) Condensation- The acetyl group attacks the methylene group of the malonyl residue, catalyzed by 3-ketoacyl synthase, forming 3-ketoacyl enzyme (acetoacetyl enzyme) (reaction 2), freeing the cysteine —SH group.
In the condensation reaction, a four-carbon unit is formed from a two carbon unit and a three-carbon unit, and CO2 is released.
Why is the four-carbon unit not formed from 2 two-carbon units? In other words, why are the reactants acetyl ACP and malonyl ACP rather than two molecules of acetyl ACP?
The answer is that the equilibrium for the synthesis of acetoacetyl ACP from two molecules of acetyl ACP is highly unfavorable. In contrast, the equilibrium is favorable if malonyl ACP is a reactant because its decarboxylation contributes a substantial decrease in free energy. In effect, ATP drives the condensation reaction, though ATP does not directly participate in the condensation reaction. Rather, ATP is used to carboxylate acetyl CoA to malonyl CoA. The free energy thus stored in malonyl CoA is released in the decarboxylation accompanying the formation of acetoacetyl ACP.
Although HCO3 – is required for fatty acid synthesis, its carbon atom does not appear in the product. Rather, all the carbon atoms of fatty acids containing an even number of carbon atoms are derived from acetyl CoA.
The acetoacetyl group is then delivered to three active sites in domain 2 of the opposite chain to reduce it to a butyryl unit.
b) Reduction-The next three steps in fatty acid synthesis reduce the keto group at C-3 to a methylene group . First, acetoacetyl ACP is reduced to d-3-hydroxybutyryl ACP. This reaction differs from the corresponding one in fatty acid degradation in two respects: (1) the d rather than the l isomer is formed; and (2) NADPH is the reducing agent, whereas NAD+ is the oxidizing agent in β oxidation. This difference exemplifies the general principle that NADPH is consumed in biosynthetic reactions, whereas NADH is generated in energy-yielding reactions.
c) Dehydration-d-3-hydroxybutyryl ACP is dehydrated to form trans-Δ 2-enoyl ACP (α-β unsaturated acyl ACP)
d) Reduction-The final step in the cycle reduces α-β unsaturated acyl ACP to butyryl ACP.
NADPH is again the reductant, whereas FAD is the oxidant in the corresponding reaction in β-oxidation.
These last three reactions a reduction, a dehydration, and a second reduction convert acetoacetyl ACP into butyryl ACP, which completes the first elongation cycle. This saturated C4 unit then migrates from the phosphopantetheinyl sulfur atom on ACP to the cysteine sulfur atom on the condensing enzyme. The synthase is now ready for another round of elongation.
Second round of Elongation
In the second round of fatty acid synthesis, butyryl ACP condenses with malonyl ACP to form a C6-β-ketoacyl ACP. This reaction is like the one in the first round, in which acetyl ACP condenses with malonyl ACP to form a C4-β- ketoacyl ACP. Reduction, dehydration, and a second reduction convert the C6-β-ketoacyl ACP into a C6-acyl ACP, which is ready for a third round of elongation.
Figure-3 – Biosynthesis of long-chain fatty acids. Details of how addition of a malonyl residue causes the acyl chain to grow by two carbon atoms. (Cys, cysteine residue; Pan, 4′-phosphopantetheine.)
3) Termination of fatty acid synthesis
The elongation cycles continue until C16-acyl ACP is formed.
Five more rounds of condensation and reduction produce a palmitoyl (C16) chain on the condensing enzyme, which is hydrolyzed to palmitate by the thioesterase on domain 3 of the opposite chain. This intermediate is a good substrate for a thioesterase that hydrolyzes C16-acyl ACP to yield palmitate and ACP. The thioesterase acts as a ruler to determine fatty acid chain length. In mammary gland, there is a separate thioesterase specific for acyl residues of C8, C10, or C12, which are subsequently found in milk lipids.The free palmitate must be activated to acyl-Co A before it can proceed via any other metabolic pathway. Its usual fate is esterification into acylglycerols, chain elongation or desaturation, or esterification to cholesteryl ester.
The equation for the overall synthesis of palmitate from acetyl-Co A and malonyl-Co A is