1. Fatty Acid Biosynthesis
Can occur in the cytoplasm of most animal cells, but the liver is the major site for this process
Fatty acids are synthesized when the diet is low in fat or high in carbohydrate or protein (most from glucose via pyruvate)
A large quantity of NADPH is needed for this process and is provided by the pentose phosphate pathway and oxidation of malate to form pyruvate by malic enzyme
Oxidation is the business of taking organized structures and breaking them down, getting the energy out so the energy can be used (catabolic). In biosynthesis is taking pieces that are separated and bringing those together to synthesis a complex hydrocarbon or fatty acid (anabolic).
They are energetically the opposite even though they a lot of structural similarities. One of the major differences si that the synthesis of fatty acids along with most other biosyntheses occurs in the cytoplasm where as oxidation occurs in the mitochondria so we need to get everything together in the cytoplasm to have this successful biosynthesis. The other thing to keep in mind is the logic (physiological logic) of biosynthesis (fatty acid biosynthesis in particular) is going to occur when times are good, when nutrients are abundant, when cellular energy charges are high and the cell can afford to store away smaller structures (in particular acetyl-CoA) as larger molecules (fatty acid) need to be stored to be used another day. So things have to be abundant. If you are in a situation where you are demanding a lot of energy biosynthesis of fatty acids is not going to occur. Another thing you need to keep in mind is NADPH drives this reductive biosynthesis. NADPH is the nicotine adenine dinucleotide with a phosphate group on it, that tags those electrons when it is in the form of NADPH for biosynthetic reactions. We have seen one example already in the reduction of glutathione for the maintenance of reducing potential. Not exactly a macromolecular biosynthesis but a synthesis nonetheless of reduced glutathione. NADPH comes from the pentose phosphate pathway, so the pentose phosphate pathway will be functioning in cells that are actively synthesizing, taking glucose and instead of oxidizing it for energy it will convert it (oxidize it) through the glucose-6-phosphate dehydrogenase (first step of the pentose phosphate pathway) and off you go with a bunch of NADPH. There is another way you can get NADPH is that is the oxidation of malate by an enzyme called the malic enzyme. Malate is oxidized to pyruvate and malic enzyme takes on NAD+ and reduces it to NADPH. So there is a second source of reducing potential for the biosynthesis.

2. Acetyl-CoA which is the main precursor driving biosynthesis of fatty acids is produced in the mitochondrion usually from glycolysis and the production (activity) of pyruvate dehydrogenase. If the mitochondrion has done its job and delivered enough ATP (no longer a need for ATP) then the cycle will slow down and oxaloacetate will accumulate because the cycle is essentially locked there and instead of condensing acetyl-CoA to form citrate for oxidation, that citrate will build up in the mitochondrion because it is not being being consumed by oxidation. So the abundant citrate will be transported to the cytoplasm (again under conditions when things are good and there is no need for generating ATP, so its basically a by-product of mitochondrial pyruvate dehydrogenase making more acetyl-CoA that is needed by the matrix) citrate goes to the cytoplasm and is converted back to oxaloacetate and acetyl-CoA, so the net effect here is to move acetyl-CoA across the inner mitochondrial membrane and into the cytoplasm. Then the oxaloacetate is reduced by malate dehydrogenase (same enzyme that is in the TCA cycle but just running in the reverse, instead of oxaloacetate it is making malate and consuming NADH and of course again since the energy charge in the cell is high there will be a strong reducing potential, NADH is abundant. If the cell was depleted in NADH this reaction wouldn’t happen but instead NAD+ would be going to stimulate oxidative pathways, so you can regenerate NADH.
Here is the malic enzyme, it takes malate and oxidizes it to pyruvate, it uses NAD+ as a coenzyme and reduces NADPH as a result of getting reduced as the malate gets oxidized. And of course pyruvate is good stuff, its for the TCA cycle where it can be shipped back to the mitochondrial matrix where it can be converted to oxaloacetate in the pyruvate carboxylase reaction. NADPH and Acetyl-CoA are the two precursors if you will for this biosynthesis.

3. This shows the overall balance of oxidation or catabolism, depending on the circumstances. If energy charge is high, glucose will go through the pentose phosphate pathway to produced NADPH, so there is one resource of high energy electrons to drive biosynthesis. If citrate is abundant, it will build up in the cytoplasm and be split up into acetyl-CoA and oxaloacetate, so the acetyl-CoA is now available and ready to be condensed into fatty acids. Oxaloacetate itself can be oxidized into pyruvate to generate more NADPH. So we have everything we need in the cytoplasm.

4. Fatty Acid Biosynthesis
Similar to b-oxidation in reverse
Several notable differences:
1. Location occurs in cytoplasm instead of mitochondria and peroxisome
2. Enzymes significantly different than in b- oxidation
This is a first glimpse of fatty acid synthesis. This happens on a huge enzyme called fatty acid synthase which is a very large complex kind of like PDH in that it is a collection of several enzyme functions all in one big multi-subunit structure which coordinates the systematic condensation of acetyl groups into longer and longer chain fatty acids. It basically consists of a priming stage where acetyl-CoA goes under a transacylase reaction to load the acetate group onto the first acyl carrier protein which is a sulfhydryl containing group that is a lot like CoA. The big difference (really is significant) is the acyl carrier protein is anchored down to fatty acid synthase, so it becomes part of the enzyme in that way it fixes the acetate groups on the surface of the enzyme instead of as an acetyl-CoA it would float off and go somewhere else. So acyl carrier protein takes the CoA and takes the acetate part first onto the acyl carrier protein and then it transfers it to ketosynthase which is the condensation reaction (what actually synthesizes the fatty acid is the synthase enzyme) so notice that the acetate group moved from the acyl carrier protein to a cysteine residue in the synthase and now its loaded and ready to go for biosynthesis. So this happens once for each fatty acid molecule. An acetyl-CoA comes in and starts the process by first coming to acyl carrier protein and then moving to the active site of the enzyme.
Now in order to get elongation, you have a high energy precursor to drive the biosynthesis of the fatty acid called malonyl-CoA. Malonyl-CoA is like acetyl-CoA except it has one extra carbon. So there is an enzyme called acetyl-CoA carboxylase that takes acetyl-CoA and carboxylates it to get to 3-carbon Malonyl-CoA. So we have an activated precursor for biosynthesis. And then the malonyl-CoA gets transferred to acyl carrier protein (just as the acetyl group of acetyl-CoA did previous to that) so it comes on to the acyl carrier protein and you have malonyl acyl carrier protein. So now on the surface on the enzyme right next door to each other you have an acetate group on the reactive site and a malonyl group on acyl carrier protein. They brought these two guys together in order to get them to condense and form a fatty acid chain.
Once you get this condensation you get a ketoacyl acyl carrier protein (ACP) that is you have the 2 carbons of the orginial acetate plus 2 of the 3 carbons of malonyl-CoA. The thrid carbon comes off as CO2, its decarboxylated. Now we have a 4 carbon acetoacetyl-ACP, this gets transferred to the acyl carrier protein, what happens is the group that is on the active site moves to the acyl carrier protein so you increase the length of the chain by those 2 carbons that came in on malonyl-CoA. It kind of grows the fatty acid from the inside out, leaving the acetyl acyl group bound to ACP and now the other subunits of the fatty acid synthase do the business of reductive biosynthesis here. Remember when we oxidize fatty acids we have oxidation, hydrate, oxidation. We are going to do the exact opposite here and reduce, dehydrate, reduce. And then keep going each round in order to build up the reduced fatty acid chain.
The first step is ketoacyl reductase (this is reducing the carbonyl group that is in the terminal acetate, reducing it to an alcohol so you get 3-hydroxybutaryl-ACP (butaryl is a 4 carbon fatty acid) then you dehydrate the bond to create a double bond (just the opposite of oxidation where you create a double bond and then hydrated it and the oxidize again). So another reductase (this one is Enoyl-ACP reductase) again it uses NADPH to reduce that double bond to a single bond, in other words a fully reduced hydrocarbon chain. You end up with butaryl-ACP, in other words a fatty acid (a very short fatty acid) but you have a fatty acid product. This cycle will continue, in other words this butaryl-ACP will go back and become acetyl-ACP, it will get transferred to the synthase so now you have butaryl synthase and a new malonyl subunit can come in and repeat this cycle so you grow this chain 2 carbons at a time. Ultimately you will have a palmitate which is released by an enzyme called thiolase which breaks the thioester bond between ACP and the fatty acid.

5. Acyl groups linked to ACP through phosphopantetheine prosthetic group
Comparison of the Phosphopantetheine Group in Acyl Carrier Protein (ACP) and in Coenzyme A (CoASH)
3. Thioester linkage - intermediates are linked through a thioester linkage to acyl carrier protein (ACP)
Acyl groups linked to ACP through phosphopantetheine prosthetic group
4. Electron carriers consume NADPH
This differs from oxidation are: it is in the cytoplasm, the enzymes are different instead of dehydrogenases we have reductases, there is the thioester linkage just as in oxidation with acetyl-CoA there but now it is a thioester linkage to ACP. They both have a pantatheine group (phosphopantatheine) which anchors it to whatever parent molecule there are on to a phosphate bridge and then the reactive part of the molecule (the sulfhydryl group) that can attack the fatty acid ester and convert it to a thioester. The big difference is that acetyl-CoA is a small soluble molecule, the pantatheine group is covalently boudn to the surface of the enzyme, limiting its options.
Then finally the carrier her is NADPH rather then NADH.

6. So this is a picture showing the difference between ACP (the big protein that serves to anchor the phosphopantetheine group with its sulfhydryl reactive group) CoA is much smaller and soluble.

7. This chart here lists the series of reactions and the enzymes that do it (sort of the flip of the beta oxidation chart before) we can see the sequence of events here and the key enzymes. First of all there are the transacylases will start with the formation of malonyl-CoA to begin with. Malonyl-CoA is produced by an important enzyme ACC (Acetyl-CoA carboxylase which is the major regulatory enzyme of fatty acid synthesis and also how we made malonyl-CoA out of acetyl-CoA) so the reaction here is driven by hydrolysis of ATP, its energy requiring, your essentially making this high energy precursor malonyl-CoA to drive biosynthesis, in order to do that you take ATP and hydrolyze it to ADP. With the breakage of that high energy phosphate bond, a free carbonic acid group (CO2 basically) is brought in and condensed on to the acetyl group to give a malonyl group (go from 2 carbon to 3 carbon) that is the fixing of CO2 into the reaction. Not to be included in the fatty acid because it isn’t, the CO2 is then removed later on, but by adding that CO2 you can make a high energy malonyl-CoA that can drive the condensation of the remaining two carbons. Then there are loading of the constituents onto the fatty acid synthases this is accomplished by transacylases the initial one is acetyl transacylase (where a CoA group donates its acetate group to ACP and acetyl transacylase makes this happen) Malonyl transacylase does the exact same thing only in this case it is moving a malonyl group onto ACP so it moves the stuff from soluble CoA adducts to fixed ones on the surface of the fatty acid synthase via ACP. Then we have the condensation reaction where the acetyl group on the ACP is combined with the malonyl group on ACP to create 4 carbon acetoacyl ACP and CO2 (2 carbons from acetate, 2 carbons from malonyl and the CO2 from malonyl coming off) So that’s the condensation steps. Load up the system with precursors and condense them together
So then the chain needs to be reduced so we have the reductase steps
Acetoacetyl ACP is reduced to hydroxybutaryl ACP. The carbonyl group goes to an alcohol and the NADPH is the reductant here NAD+ is reproduced so it will have to go get regenerated in the pentose phosphate pathway or from malic enzyme. Then there is the hydrotase where water is removed. So the hydroxbutaryl ACP becomes the Enoyl ACP, create the double bond by removing water then the double bond is reduced by a reductase (enoyl ACP reductase) which reduces it down to butaryl ACP. Those are the enzymatic steps.

8. Fatty acid synthesis begins with the carboxylation of
Fatty acid synthesis begins with the carboxylation of acetyl-CoA to form malonyl-CoA
This is an activating reaction requiring ATP for the carboxylation of biotin (Acetyl-CoA carboxylase, ACC1)
The ACC enzyme is important, it catalyzes this reaction (the production of malonyl-CoA) and the way this happens is that acetyl-CoA makes an attack on carboxybiotin (remember biotin is one of your vitamins/ coenzyme and is a carrier of carboxyl groups. The biotin group can be covalently linked to CO2 to create carboxy biotin. This strucure has an electrophile, a carbon that is partially positively charged because of the oxygens pulling the electrons away from it so these carbanion of acetyl-CoA came make a nucleophilic attack on that carboxyl group leading to the transfer of CO2 onto acetyl-CoA to create malonyl-CoA, you just added a CO2 out there and this regenerates the decarboxylated form of biotin or biotinate which then needs to be recharged so the biotin needs to be carboxylated (the business of ACC 1) it takes up free CO2 hydrolizes ATP which drives the synthesis carboxybiotin bond to the CO2. Now your ready to go around again.

9. This summarizes the reaction without showing the biotin, of course the biotin is bound to the enzyme and is regenerated so you don’t see it as a reactant in the pathway its just making it happen. Again to look at this as a energy consuming process in order to create a high energy bond to CO2 indicates why it would highly regulated and reversible due to the cleavage of ATP

10. Regulation of Acetyl-CoA Carboxylase 1
ACC enzymes are highly regulated by allosteric modulators and phosphorylation
Glucagon and AMPK lead to phosphorylation of ACC which inactivates the enzyme
Insulin activates phosphoprotein phosphatase 2A which activates ACC
As I mentioned before ACC is the main regulatory enzyme of fatty acid synthesis and again think logically about this, fatty acid synthesis is going to occur when things are good when materials are abundant. It is not going to occur when there is low blood glucose and energy is needed in anticipation of activity like epinephrine.
ACC is a polymerizable protein. When it is active it forms these polymers, when it sticks together and polymerizes its active. When it gets depolymerized its deactivated. The mechanism for controlling that polymerization are basically phosphorylation (this creates the monomer, destabilizes the polymer and inactivates it so when ACC gets phosphorylated it inactivates it kind of like glycogen synthases. When it is dephosphorylated it polymerizes and is active, so dephosphorylating ACC activates it. The things that activate ACC are phosphatases that are produced in response to insulin, these are the same phosphatases that regulate glycogen synthesis. These phosphatases will remove the inhibitory phosphate groups activating ACC 1 which will then now mobilize acetyl-CoA for the biosynthesis of fatty acids, it will commit to malonyl-CoA and biosynthesis instead of getting oxidized in the TCA cycle. If the energy charge is low then an enzyme AMP kinase (which is activated by low energy charge) will phosphorylate the enzyme to cause it to depolymerize and make it inactive. So again when energy charge is low you don’t want to be producing fatty acids you want to use that CoA for oxidation to get more ATP out. Also recall that glucagon and epinephrine through the phosphokinase A pathway can phosphorylate these targets which will also depolymerize and inactivate the ACC1 enzymes, so either a hormonal signal from glucagon or epinephrine or a cellular signal (an intracellular energy charge indication) can inactivate this enzyme and dedicate the CoA to oxidation instead of polymerization. Indications that things are good that is insulin indicating a high glucose concentration in the blood or just the absence of glucagon and epinephrine activate phosphatases which dephosphorylate ACC activate it and off you go with fatty acid synthesis.

12. glucagon
Also this shows the relationship with active carboxylase and inactive carboxylase. We already talked about AMP activated protein kinase as an indicator of intracellular energy charge. If AMP is high then the one of the right is activate and it inactivates. Quite separately this cell can respond to glucagon which is also saying that the energy state in the body is low and it is not a good time to be synthesizing fatty acids so glucagon will lead to down stream phosphorylation as well through protein kinase A. Insulin on the other hand activates protein phosphatases leading to activation of the ACC enzyme.
insulin

13. Citrate partially activates hormonally inactivated carboxylase
There is a second allosteric control that is created by citrate (remember what citrate is, it is a compound when energy needs are low it will build up because of the condensation of acetyl-CoA and oxaloacetate in the mitochondrial matrix, citrate gets made but not oxidized so all this acetyl-CoA is sitting around doing nothing and citrate can bind to the phosphorylated inactivated enzyme (the monomers) in the presence of phosphate group (even if hormonal signals are saying don’t synthesize fatty acids it can activate the ACC) So this allows a cell that is rich in citrate to synthesize fatty acids even when the rest of the body is wanting to use them. This might make sense for an adipoctye for example to generate fatty acids that can be dumped into the blood so that the fatty acids can go to the rest of the body to provide fuel.
Citrate partially activates
hormonally inactivated carboxylase

14. We see that tendency here
We see that tendency here. The phosphorylated enzyme is less active, the dephosphorylated is very active but even the inactive phosphorylated enzyme can be activated by rather high concentrations of citrate, and this can not fully activate as dephosphorylation would but about 50% of the activity can be realized. That is regulation again you see hormonal sensitivity as well as intracellular energy charge and in this case the indication that there is biosynthetic potential in the formation of a lot of citrate.

15. Fatty Acid Synthase Structure
The remaining reactions of fatty acid synthesis take place on fatty acid synthetase multienzyme complex
Site of seven enzyme activities
Two fatty acids constructed simultaneously
The fatty acid synthetase complex is a very large multi-enzyme complex. Its got the business end where you load the precursors, the reduction enzymes that reduce them down to alkyl groups and then a thiolase or thioesterlase that cleaves off the fatty acid ultimately once its produced. We see in the acyl and malonyl binding domain. The place for the KS activity (ketoacyle synthetase: that’s where the actual synthesis occurs, where the condensation occurs at the KS site) then there is the malonyl transacylase which brings the incoming malonyl group from malonyl-CoA and transfers it to malonyl ACP as well as the substrate level which is either acetyl-CoA initially or the growing fatty acid chain later on. The enzyme exists as a dimer so it is carrying all these things twice and of course as an aggregate there can be multiple dimers together so you have these synthase complexes that are producing lots and lots of fatty acids at one time.
Fatty Acid Synthase Structure

16. Substrate entry leads to condensations where the acyltransferase and the malonyl transacylase affect the condensation of the incoming malonyl group with the acyl group that is already present. that is the first part to form the new bond. Then the bond needs to be reduced that’s the reductases, the dehydration, and then finally the thiolase that cleaves it off at the end.

17. Formation of Acetyl-ACP
During the first three reactions, acetyl-CoA is used to form malonyl-ACP, which is converted to acetoacetyl-ACP by KSase
Malonyl-CoA comes in (this is the entering precursor to synthesis) and bound to the enzyme is acetylated (an acetate group from acetyl-CoA or this could be the fatty acid chain it could be the butatryl-CoA after the first round of synthesis) the incoming malonyl-CoA makes the nucleophillic attack the carbanion on malonyl-CoA attacks this nucleus and you end up with a bond formed between the malonyl-CoA and the acetate that’s on the enzyme and that’s the business of this synthase to form acetoacetyl ACP at this point. It will become butaryl after reduction. So notice its all attacked to ACP at this point it needs to move back to the enzyme site to allow ACP to pick up another malonyl group.

18. Fatty Acid Biosynthesis
During the next three steps (two reductions and a dehydration), a butyryl group is formed
Butyryl is then transferred to KSase and the ACP-SH group can bind another malonyl group
Eventually synthesizes palmitoyl-ACP
This is kind of a complicated diagram. They show don’t show the loading of acetyl-CoA onto ACP they just show it initially showing up on the cysteine site (the KS site on the enzyme) in reality acetyl-CoA comes in, acetyltransacylase moves it from ACP to the enzyme. So you have the enzyme reactive site primed with the acetate group of the first acetyl-CoA. Then malonyl-CoA comes in and binds to ACP. So now you see malonyl group bound to the ACP and you see the “pant” there is the pantethenic acid that it is present on the ACP. These guys are now right next door to eachother so they can do that nucleophillic attack, the acetyl group attacks the malonyl group and CO2 is removed (the third carbon of malonyl is removed) the two carbons from malonyl-CoA and 2 carbons from acetyl-CoA condense together to get a 4 carbon keto structure notice the carbonyl structure which makes it a keto. That is the condensation (that lengthens the chain by 2) We now need to reduce the chain so there is a recducatse the acetoacetylreductase. There is dehydration to make that bond less stable and be reduced a second time from crotonyl ACP to butaryl ACP so now you have 4 carbons that are fully reduced. At the end of all of this, stuff is bound to ACP needs to move back to the reactive site to essentially load this fatty acid waiting for the malonyl to do it all over again.
Fatty Acid Biosynthesis

19. Condensation is associated with decarboxylation and binding to ACP to anchor these guys down. The acetoacetyl ACP is reduced dehydrated and reduced a second time (opposite what we saw with oxidation and really very similar but opposite of what we saw in the TCA where saw succinate oxidized to fumarate which was hydrated to malate which was then oxidized to oxaloacetate, same reaction scheme)