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Lipid Biosynthesis Study Guide: What Pathways, Mechanisms, And Enzymes Are Involved In Fatty Acid Synthesis?
Introduction: Fatty Acid Synthesis Differentiated From Lipid Degradation Pathways
Like in other biochemical pathways, the lipid biosynthesis pathway is separate and differently strategized than the degradation pathway. There are four key differences:
- 1. Fatty acid synthesis is CYTOSOLIC
- 2. Fatty acid synthesis is performed by ONE multi-function enzyme in animals: fatty acid synthase. (Plants and bacteria do have separate enzymes.)
- 3. Fatty acid synthesis uses NADP+/NADPH.
- 4. Fatty acid synthesis intermediates are covalently bound to the –SH groups of ACPs (acyl carrier proteins).
Fatty acid biosynthesis uses the following mechanism.
- · Chain built by addition of two-carbon units (acetyl-CoA)
- · Acetyl-CoAs are activated by conversion to malonyl-CoA
- · Malonyl-CoA is decarboxylated and this is what drives chain growth
- · Chain continues elongation to sixteen carbons (palmitic acid)
- · Palmitic acid can then be lengthened and have double bonds added by other processes
- · Net reaction: Acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 13 H+ + H2O è palmitate- + 7 HCO3- + 8 CoASH + 14 NADP+
- · Palmitate is rapidly converted to CoA esters and subsequently used to form phospholipids and triglycerides
Cells need cytosolic acetyl-CoA for the manufacture of fatty acids. Acetyl-CoA is produced via three pathways: amino acid degradation (cytosol), fatty acid oxidation (mitochondria), and glycolysis (cytosolic pyruvate goes to mitochondrial acetyl-CoA). Even though AA degradation acetyl-CoA is already in the cytosol, it isn’t enough for lipid synthesis. Glycolytic and FA degradation acetyl Co-A is trapped in the mitochondria, so the only way to access it is to use a shuttle. Acetyl-CoA is linked with OAA (oxaloacetate) to form citrate, which is transported from the mito matrix into the cytosol. Here, it is liberated from the OAA by the ATP-citrate lyase enzyme. The OAA is shuttled back to the mitochondria in the form of pyruvate or malate, so it is “recycled” and can bring more acetyl-CoA units.
When acetate units (acetyl-CoA) are carboxylated to malonyl-CoA, it becomes committed to fatty acid synthesis – that is, the acetyl-to-malonyl-CoA conversion is the biochemically committed step in the fatty acid synthesis process. The conversion is catalyzed by acetyl-CoA carboxylase, which contains a prosthetic biotin group. In animals, this carboxylase is the ONLY fatty acid synthesis enzyme that is not part of the multienzyme fatty acid synthase complex.
The biotin prosthetic group is covalently attached to an active lysine. In E. coli, the enzyme consists of three subunits: the biotin carboxylase, which adds CO2 to the prosthetic group, carboxylase, which transfers the activated CO2 to acetyl-CoA, and the actual biotin carboxyl carrier protein.
ACC in animals is a polymer-type protein with multiple subunits called “protomers.” Each protomer is composed of the sites for each activity described above, as well as allosteric regulatory sites. The ACC enzyme is regulated by conversion between the active polymer configuration and the inactive protomer configuration. Regulation occurs by feedback inhibition and feedforward activation: palmitoyl-CoA, the final product, pushes equilibrium in the direction of the inactive conformation in a classic example of biochemical feedback inhibition. Conversely, citrate (which signals availability of NADPH and acetyl-CoA – more on that later) nudges equilibrium towards the active polymer conform by an allosteric activation mechanism.
The regulation of acetyl-CoA carboxylase has another layer. The enzyme has eight to ten sites on each subunit that can be phosphorylated, some of which have a regulatory function. If all of these sites are phosphorylated, the carboxylase enzyme has low affinity for citrate and thus is only active at high citrate concentrations. If it’s unphosphorylated, on the other hand, it has a higher affinity for citrate and thus can operate functionally at low citrate molarities. (Remember that citrate binding is an important allosteric activator, and consequently, it’s important for the enzyme to be able to function with low levels of citrate). Palmitoyl-CoA inhibition works using a similar mechanism in the opposite direction: the fully phosphorylated enzyme is easily inhibited by low concentrations of palmitoyl-CoA, so with even a little product, the enzyme shuts down. However, when dephosphorylated, it is only inhibited by sky high levels of palmitoyl-CoA. Consequently, the dephosphorylated enzyme is more conducive to pushing fatty acid synthesis forward towards completion, because it is more easily activated and less easily inhibited.
14 NADPH are needed to produce palmitate. Along with acetyl-CoA availability, this is one of the major limiting factors. NADPH is produced by two primary pathways: the pentose phosphate pathway and the malic enzyme pathway. Malate dehydrogenase and malic enzyme can convert NADH from glycolysis into NADPH. For every citrate that enters the cytosol, one acetyl-CoA and one malate are produced. Subsequent malate oxidation results in production of one NADPH and one acetyl-CoA (the remaining compound is OAA, discussed earlier). Since 8 acetyl-CoAs are used to produce palmitate, this process can provide 8 of the NADPHs needed as well. This means that the other 6 NADPH must be derived from the pentose phosphate pathway.
Moving Towards Synthesis: Acyl Carrier Proteins and Fatty Acid Synthase
Acetyl and malonyl groups are not transferred straight from coenzyme A to the synthesizing chain. First, they’re passed to a phosphopantetheine group on an Acyl Carrier Protein (an ACP). The specialized phosphopantheine group is structurally similar to CoA, but larger. In animals, this enzyme and all subsequent FA reactions are catalyzed by the homodimeric fatty acyl synthase 1 enzyme. (In lower organisms, the FAS 2 collection is separated).
The fatty acid chain elongation starts with a transfer of the acyl group of acetyl-Coa to the acyl carrier protein by malonyl-CoA-acetyl-CoA-ACP transacylase. (MAT for short).
Fatty Acid Synthesis: Decarboxylative Condensation, Reduction of B-Carbonyl Group,
The B-ketoacyl-ACP synthase (KS) condenses the acyl group with malonyl-ACP to produce a B-ketoacyl-ACP intermediate (hence the enzyme name). In the first go around, this intermediate is acetoacetyl-ACP. After acetyl group transfer to MAT, the ACP thiol sulfur attacks to form an acetyl-ACP complex. The acetyl is transferred to a sulfur on the KS cysteine group. The thiol on ACP can then acquire the malonyl. Subsequently, the malonyl group is decarboxylated, creating a highly reactive carbanion intermediate that attacks the acetate group. In this process, two carbons are added to the growing chain. (Malonyl is used instead of acetyl because the reaction is thermodynamically unfavorable, but the decarboxylation drives it forward).
After this, the process follows steps that are not unlike those in B-oxidation of fatty acids, just in reverse. However, there are some key differences. The first step is reduction of the B-carbonyl group by a reductase (KR) which forms a B-alcohol. The second step is dehydration by a dehydrogenase, and the final step is a second reduction by a trans-enoyl ACP reductase, which causes the chain to be saturated. The differences between these steps and B-oxidation are as follows: the alcohol has the D configuration, and NADPH is used (as opposed to L configuration and NAD/FAD in oxidation).
Overall, the first “turn” of the cycle results in the production of a butyryl (four carbon) group. Each subsequent turn adds two carbons. The KS is the limiting factor – it can’t handle substrates bigger than 16 Cs, so other steps must be taken to convert the palmitic acid product into longer or unsaturated fatty acids.
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