1. Section 7. Lipid Metabolism
Fats: fatty acid biosynthesis
Last time, summary: the mobilization of fat to produce fatty acids, and then their degradation and other tissues in order to provide energy. Know lots of details, but keep in mind the framework which is, this is a way to provide energy, and in order to get energy outo f biological molecules, burn them, the way you get energy out of most fuels, burning means oxidizing. Saying this because the every increasing number of details that you need to remember are more easily remembered if you put them in a framework.
Today is reverse of last time
11/04/05

2. Oxidation of Fatty Acids Other Than Palmitate
Any even number of saturated carbons does not require any additional enzymes. Products are as for palmitate.
An odd number of saturated carbons does not require any additional enzymes. Same products plus one propionyl CoA.
Unsaturated fatty acids require additional enzymes. Same products, but less energy, compared to
saturated fatty acid the same length.
See table 12.1 for a list of common
fatty acids.
“loose ends” What happens if you get a non saturated fatty acid. Already has some oxidation, has double bonds, or if you get odd numbered fatty acid. Odd numbered case, when you get to the last cleavage, cuts of 2 carbons to make acetyl CoA, and remaining has only three carbons, your done. Propionyl CoA would then go and be utilized in some reaction, not to Krebs the way acetyl CoA does, some other scheme. Synthesis or modified and further degraded. Question of double bonds, general notion that if you start with something that’s already partially oxidized, get less energy out, true and applies. In some cases its very straight forward, what matters is where the double bond appears in the structure. This is an example of what’s an essential fatty acid, some that we must have, essential for human life, rare for people to not get enough—don’t worry about it, almost in everything. Arachidonate acid, four double bonds, all over the place with regard to structure. Might just skip one of the steps in beta oxidation cycle, since you put double bonds in there with beta oxidation, get a little less energy, but not that simple, depends on location 1

3. Double Bonds in Odd-Numbered Positions
As acetyl CoA’s are removed from an unsaturated fatty acid, double bonds move into or near the active sites of the -oxidation cycle enzymes.
16:1 cis-D9
An odd-numbered double bond moves into the 3-position, which is not a substrate for the principal enzymes of the cycle.
An isomerase moves it to the 2-position.
Moving the double bond is energy neutral, but one less FADH2 is made because one acyl CoA dehydrogenase step is “skipped.”
Starting material, in which we’ve got the double bond at the 9 position. 16:1 cis delta 9. First three rounds of beta oxidation, enzymes don’t see that double bond, each of the three arrows would produce FADH2, NADH, and acetyl CoA. Then you get to this molecule, has a double bond on the number 3 carbon, remains an odd numbered carbon. This is not a substrate for beta oxidation cycle. Neither a substrate nor a product. If we didn’t have some auxiliary enzyme to modify this—but we do, isomerase that moves the double bond from the 3-4 position to energy neutral reaction. Don’t need to activate it, don’t need to put energy in, don’t get energy out, move the double bond from this position to the next. This is now a substrate for hydration, and subsequent oxidation, that provides an NADH. What we missed, the first oxidation step, that put in the FADH2. This fatty acid, compared to saturated fatty acid of the same length, would give us one less FADH2 molecule, one and a half less high energy phosphate bonds. Once this has been done, then everything else is normal. This is the product of the first oxidation that would occur in saturated fatty acid, then would go through subsequent steps of cleavage.
(p 610) 2

4. Double Bonds in Even- Numbered Positions.
An even-numbered double bond moves to the 4-position.
Acyl CoA dehydrogenase oxidizes it producing FADH2 and the non-substrate dienoyl CoA.
The additional enzyme 2,4-dienoyl CoA reductase uses NADPH to produce a position double bond.
The additional enzyme cis D3-enoyl CoA isomerase (see above) moves the double bond from the 3- to the 2-position.
NADH is produced after hydration, and then acetyl CoA, as usual (not shown).
The net effect for the cycle is the equivalent of one less NADH.
If double bond in even numbered position, more enzymes are required, and scheme shown here. Haven't shown you the first cycles to get to the molecule that is not a substrate, nor is it a substrate for the isomerase that would take care of an odd numbered double bond already present. In this case, the first thing that happens is a double bond is introduced in the three four position, so we get an FADH2 out of this, but this is not a substrate, and it is reduced to give us a fatty acid, with double bond in odd numbered position, uses equivalent of NADH, oxidize it, getting FADH2, reduce it using equivalent of NADPH, then get 3’ and isomerase moves it over. Energetically, they are equivalent, have double bond already there. If you look at complete fatty acid, difference when what you et out of it, oxidize it using the enzymes we have, because we have to go through this step. End up one less NADH worth of energy. Get the FADH, but this one that we use, in the subsequent step, would make one but we use it. If we burn them in a chem lab experiment, get the same amount of energy out. Whereas in this case you get one less equivalent of ATP because have to go through one existing pathway, and the intermediates waste some of the energy. 3

5. Example: Linoleoyl CoA C18:2 cis-D9,cis-D12
Fig
modified
+ 3 acetyl CoA
+ 3 FADH2
+ 3 NADH
Example: Linoleoyl CoA C18:2 cis-D9,cis-D12
Example, shows both of those cases using a real fatty acid that has two double bonds, and after we get down to where we have a double bond in the 3 position, isomerase moves it over, goes through rest of the cycle, get acetyl CoA, miss FADH2. Rehash of two slides ago, gives a product that has a double bond in even numbered position, so the sequence here would be to oxidize it, provide FADH2 then reduce it, then we have the isomerase and this would go through the rest of the cycle. Ought to be able to transfer what's on the previous two slides into this sequence, and if we tallied up the energy we get from this fatty acid that has two double bonds and compare it to the same fatty acid that is completely saturated, get one less FADH2, and one less NADH from the rest of the sequence, all else would be identical.
+ acetyl CoA
+ NADH (no FADH2) 4

6. Ketone Bodies
The liver normally converts acetyl CoA to ketone bodies that are used by peripheral tissues.
See fig
During anesthetization (covered in pharmacology, Q7) as respiration decreases, ketone bodies up, pH down (CO2 up).
Ketone bodies, produced in most cells. Acetyl Coenzyme A in the primary metabolic functions of cells, would go to the Krebs cycle and it would enter the Krebs by condensing with oxaloacetate, heard about the reaction weeks ago. If you don’t have enough oxaloacetate in the cell, then Krebs cannot accommodate as much acetyl CoA and there is an alternative pathway that provides a different energy rich molecule which can either be used by the cell or distributed to other itssues in te blood. Those molecules are Ketone bodies, made from acetyl coenzyme A. This is an alternative pathway, instead of this going on to condensing with oxaloacetate. Two acetyl COA’s can be condensed, then third acetyl coA, then cleave off one get acetyl acetate. Would occur in any cell. This can then be reduced further with NADH to give three hydroxy butyrate. In humans, the liver produces these ketone bodies for distribution. The liver makes ketone bodies. Circulated in the blood and there are some tissues that prefer them, kidney and heart, prefer over glucose as primary source of energy. Nothing pathological about it, required. Brain prefers glucose. Supply of glucose and glucose precursors that you can provide, prolonged starvation the brain will adapt. Water soluble, circulated in the blood. Some pathological or temporarily unusual situations where the ketone bodies predominate. Starvation, another is diabetes, inadequate levels of oxaloacetate which you get from glucose will be the case in tissue of diabetics. They depend more one ketone bodies as a way to provide energy to their cells. Hypoventilation, cut back oxygen, cut back on aerobic degradation, cut back glycolysis, depend more on ketone bodies. Under general anesthetic, ketone bodies go up, as does the level of CO2, and acidity. (no coherent explanation for why this is the case)
Also true that acetylacetate will spontaneously decarboxylate and form acetone. Acetone is volatile, comes out in breath from lungs. In blood as acetone, but its exhaled and you can smell it, people who are ketotic, often have the smell of acetone on their breath. Non enzymatic, not metabolic, just happens.
When abundance of energy, mechanisms for storing the energy, one is to make fatty acids to adipose cells, glycogen from glucose, and this is a smaller contribution, the 3-D-hydroxybutyrate
Production is enhanced by low carbohydrate (diabetes, starvation) and/or low O2 (hypoventilation)
General anesthesia: CO2 up, pH down, ketone bodies up.
Volatile acetone formation is non-enzymatic. 5

7. Acetoacetate Utilization
In peripheral tissues, the ketone body acetoacetate is activated, and converted back to acetyl CoA.
3-hydroxybutyrate and acetoacetate are favored over glucose by the renal cortex and cardiac muscle.
When the ketone bodies get to the cell
Acetoacetate is then activated to acetoacetyl CoA and converted back to acetyl CoA, that can be metabolized by the cell.
All cells can have these reactions going on, liver lacks this enzyme, so acetoacetate in the liver does not get converted to acetyl CoA, it leaks out, diffuses into the blood and the liver becomes a producer of ketone bodies.
Fig 6

8. Summary of Fatty Acid Biosynthesis
When the cell energy level is high, rather than being used by the Krebs cycle, acetyl CoA is transferred from the mitochondrial matrix to the cytosol.
In the cytosol, acetyl CoA is converted to malonyl CoA, which is used by fatty acyl synthase (FAS) for the synthesis of palmitate.
Palmitate is transported to adipose tissue and used to synthesize triacylglycerol.
The palmitate synthetic reactions are reversals of the degradative reactions, but the enzymes, cofactors and locations are different.
Next topic, biosynthesis. Flip side of fat metabolism. When energy level is high, acetyl coA, in addition to being used by Krebs or becoming ketone bodies, used to make palmitate. Gets distributed back to adipose, made in adipose, also distributed back to adipose cells where its used to make fat. The enzyme, complex of several enzymes that makes palmitate is fatty acyl synthase. Efficient system where all the enzymes are covalently attached to one another, and it does all of the steps combining acetyl coAs into palmitate. This is the framework, opposite for framework of oxidation, condensing not cleaving, reducing not oxidizing, molecules getting bigger, more reduced, contain more energy. 7

9. Reactions on the right are catalyzed by FAS in the cytosol. Compare
degradation
and
synthesis
structures.
Last time, start off with some fatty acid, activated. Oxidize with double bond, hydrate, then oxidize again, then cleave. Oxidation and cleavage provides energy. The fatty acyl synthase, catalyzes the reversal of these reactions. Condenses, reduces, dehydrates, and reduces. Condensation requires activated precursors, reductions require that NADPH be converted to NADP+. The co reactants will be oxidized but the product is subsequently reversed. The first thing with fatty acid degradation, less detailed, with 2 units being cut off, this is the detailed part, taken some activated acyl moiety, and increased its length by two carbons.
Fig. 22.2 8

10. Citrate Shuttle Transfers Acetyls to Cytosol
ATP
+ CO2
This occurs in the cytosol. Wouldn’t be a good idea to degrade and make fatty acids in the same compartment, while taking advantage of compartmentalization is easier way to do it. Mitochondrial inner membrane shown here. Inside is this acetyl CoA that is being synthesized by various sources, metabolic sources. When its concentration is high, in stead of being used citrate that is being produced when it combines with ocaloaxetate, go across membrane. Citrate shovel, in cytosol get back acetyl coA. Reversal of the reaction shown on the other side, then oxaloacetate is oxidized to malate and then reduced to pyruvate, pyruvate goes back across the shuttle. Translocase molecule will take citrate in this direction and pyruvate in this direction, pyruvate converted to oxaloacetate, get the cycle going around. Two anabolic virtues, aspects to this cyle, one is that we are moving energy rich out of mitochondria to make palmitate. Converting NADH to NADPH. NADH is an energy rich molecule produced by catabolism, NADPH is energy rich, used for biosynthesis. In addition to bringing out the carbons that are going to be used to make palmitate, we are also redistributing the reductive power in the cytosol into the molecule that is always involved in biosynthetic reduction. Remember that when the palmitate, acyl groups were being transferred from cytosol into mitochondria, carnitine was the carrier, translocase carrying acyl group but not coenzyme A group. The reason for that is that the coenzyme A concentration inside and outside mitochondria are different, and to maintain that, don’t have them intermingle. That is also true moving the acytyl groups. COA doesn’t cross the membrane, when acyl COA combines with oxaloacetate, get coenzyme A as product, stays in the cytosol, and on the other side this citrate combines with cytosolic coenzyme A to give acetyl.
Fig
+CO2
High [ATP] inhibits the Krebs cycle; [citrate] increases.
Citrate translocase enables citrate and pyruvate to cross the mitochondrial inner membrane. CoA does not cross (remember acyl CoA / acyl carnitine).
NADPH is made at the expense of NADH in the cytosol. 9

11. Activation by Acetyl CoA Carboxylase
(p 617)
In the cytosol, acetyl CoA is carboxylated to make the activated precursor, malonyl CoA.
This is the committed step in fatty acid biosynthesis.
ATP provides energy.
Biotin is a cofactor.
Two sequential reactions occur in the active site.
Acetyl CoA is the source of carbons for fatty acids. Is activated on carboxyl end, but needs to be activated at carbon that will act in biosynthetic scheme, attach carboxyl group to make malonyl coA. This is the activation, catalyzed by acetyl CoA Carboxylase. Biotin requiring enzyme. Biotin is cofactor, covalently attached in active site, first part of reaction is for ATP to react with biotin and make it reactive so that it will covalently attach itself to CO2, and we get chemical volatility, reactive CO2 group, hydrolyzed ATP. Acetyl molecule binds and CO2 is transferred to give malonyl. This reaction shown here as single arrow, actually ahs a couple of steps.
(p 618)
biotin-Enz + ATP + HCO3-  CO2~biotin-Enz + ADP + Pi
CO2~biotin-Enz + acetyl CoA  malonyl CoA + biotin-Enz 10

12. Biotin: a CO2 Carrier
ATP reacts first providing energy to bind and activate HCO3-.
Next acetyl CoA binds and the activated CO2- is transferred to the acetyl group.
Figs and (Stryer 4th)
Some steps here. Biotin, attached to a carrier protein. In this scheme ATP not shown but energy from ATp activates the biotin at the nitrogen, end up with CO2 covalently attached to it, then transferred, bottom picture depicts what occurs over time, in the active site, first ATP binds then carbonate binds, ADP comes off Pi comes off. Then acetyl CoA binds and get malonyl, and that comes off. Ping-pong mechanism. Things bouncing in and out of active site, before you get product. 11

13. Fatty Acid Synthase ReactionsCE
(KR)
CE +
Activated intermediate, malonyl CoA. More detail on first sequence for fatty acid synth. First is condensation, Co2 released, get aceacetyl ACP, carrier like coenzyme A, condensation to four carbon unit, have a ketone group on carbon 3, reduce with NADPH, to hydroxyl level, next step
Fig
Condensation forms 4 carbon unit on acyl carrier protein (ACP).
Reduction of ketone to hydroxyl by NADPH. 12

14. Fatty Acid Synthase Reactions, con’t
(DH)
(ER)
Dehydrate it, energy neutral removal of water, now have double bond, and then we reduce that again, then have four carbon fatty acid that is completely saturated. This cycle can then occur again with another malonyl CoA, adding another two carbon unit. Exactly like beta oxidation in reverse, two carbon units are being manipulated with regard to attachment.
Fig
Dehydration produces a double bond.
Reduction to a saturated 4 carbon fatty acid chain. 13

15. FAS is a Dimer
Enzyme shown here in schematic fashion. Catalyst for all the reactions ,with exception of production of malonyl CoA. This FAS, fatty acyl synthase is a dimer. The various letters on here, indicate the location of where these reactions occur. What is particularly interesting about this series of events is that this acyl carrier protein has a long flexible chain which has the same structure as coenzyme A, long flexible chain but in this case chain anchored on one of the proteins of this FAS and it allows the growing chain to be moved from one active site to another. Efficient way to make fatty acids. This is the main reason, once you’ve got the acetyl CoA on this chain, it moves over to condensing site, pics up two carbons, reduction site, dehydration site, etc. Then you’ve got four carbons, and it keeps doing that till you get 16 carbons, then it binds to a site that releases it. It is a dimer because, rather than this one stretching over to here, cycle described occurs with these subunits and these subunits, minor nuance, don’t worry about. Maybe emphasizes the flexible chain has to be able to reach. This is schematic, it’s a big ball of enzyme-
Fig
Malonyl transfer (MT), acetyl transfer (ATP and condensation (CE) on one subunit.
Reduction (KR), dehydration (DH), reduction (ER) and thiolysis (TE) on the other subunit.
The growing FA chain is passed between subunits by ACP. 14

16. Acyl carrier protein (ACP)
Picture showing the flexible group onto which its attached, growing fatty acid attached, and even more schematic picture, showing acetyl group on the condensing enzyme.
Fig
ACP has a long flexible chain, derived from pantothenic acid, to which the growing fatty acid is attached. 15

17. Condensation Both subunits of FAS are involved.
Blue structure is a malonyl coA, on the acyl carrying protein. More detailed depiction of molecule being moved around from site to site. Showing reactions done at different sites. Condensation, loss of DCDO2, transfers this two carbon unit to this. Color coded, get rid of Co2.
Fig
Both subunits of FAS are involved.
Condensation is catalyzed by CE on upper subunit. 16

18. Reduction 2 NADPH are used.
Reduction, stays attached, moves to the site not shown, reduction dehydration reduction. Four carbon unit on acyl carrying protein
Fig
2 NADPH are used.
Reduction (reduction, dehydration, reduction see slides 12 &13) occur on lower subunit of the dimer. 17

19. Translocation and binding of a new malonyl CoA
Fig
The 4 carbon chain is transferred to CE.
A new malonyl CoA binds ACP on other subunit.
The cycle (condendation, reduction, dehydration, reduction) repeats until 16 carbon palmitate is formed (not shown).
Palmitate is released by TE (see slide 14).
Subsequent step would be to transfer it to the condensing enzyme. Then we bring in a new malonyl coA, and it goes around again. Look at the scheme, point being to realize that the three carbon unit is the new one coming in, gets decarboxylated, and then the other reactions occur by flipping it to the sites which catalyze. Eventually the chain, instead of being 4 carbons long is 16 carbons long. Affinity for the enzyme—esterase, goes way up and binds in that site. Whtn its shorter, doesn’t bind, if it does bind doesn’t activate the enzyme to react. For that reason, goes around cycle 7 times before you cleave it and free that fatty acid. 18

20. Net Reaction for Palmitate Synthesis
8 acetyl CoA + 7 ATP + 14 NADPH + 6 H+ 
palmitate + 14 NADP+ + 8 CoA + 6 H2O + 7 ADP + 7Pi
For this reaction, there are 7 (for 7 ATP) + 35 (for 14 NADPH) = 42 ~P equivalents used.
Compare to 26 obtained from the palmitate conversion to acetyl CoA by fatty acyl CoA synthetase and the b-oxidation cycle.
(p 622)
If we review the energetics of this. We have 8 acetyl groups that will provide 16 carbons for palimate. 7 have to be activated by putting on CO2, converted into maolonyl coA, that will require these 7 ATPs. Go around that reduction dehydration reduction cycle 7 times, each cycle with 2 reducing steps, reach requires NADPH to reduce the acyl chain. Then we need hydrogens for balance, start with 8 acetyl coA’s, end up with palmitate, hydrolyzed 7 ATPs, and 14 NADPHs get back COA’s etc. If we look at acetyl COA level, not worry about making acetyl COA. The total number of high energy phosphate bonds, net number comes from—tally NADPH’s, those use some number of –2.5 each, then 7 actual ATP’s that are hydrolyzed, this gives us total of 42 high energy phosphate bonds used to convert acetyl CoA into plamitate. Compare with beta oxidation, we make 7 NADHs, 7 FADH2, only 26 high energy phosphate bonds. Typical of comparisons of synthetic to degradative. Pay to make it, don’t get as much out. Hard to get more than you put in. Difference is not negligible. 19

21. Control of Fatty Acid Synthesis is at the Committed Step
Major control site for this process. Next week all the control sites on one slide. This one is crucial for the synthesis of palmitate, acetyl coA carboxylase, conversion of acetyl coA to malonyl coA, responds to modulators, if we first look at the ones that increase, citrate, citrate shuttle is moving acetyl groups out into the cytosol, as the citrate concentration goes up, binds to this enzyme and gets it started at converting acetyl groups into maolnyl groups, citrate providing malonyl groups, things don’t happen instantaneously, when flux is out of mitochondria, a lot of acetyl coA and citrate in cytosol, so that would activate. Insulin would be present when there is an abundance of carbohydrate source energy, indicator to the cell that it should be not degrading fatty acid, but should be synthesizing fatty acid. Insulin would be indicator that there is high energy level in cytosol, so it makes sense to make palmitate. Other molecules are just the opposite, palmitoyl coA is the activated version of palmitate from adipose tissue that goes into mitochondria, activated, transfers to carnitine. When its high, high because the cell is using fatty acids as source of energy, so it would reduce the activity of acetyl coA carboxylase, so you aren't making palmitate at the same time you bring it in to degrade. Glucagon and epinephrine are flip of insulin, inhibit acetyl coA carboxylase, AMP is a common signal that the cell is low in energy, activates many pathways that will make more energy, not unique to fatty acid metabolism. When its concentration is high, the connection is its high because ATP is low so the cell would be needing more energy, would inhibit the enzyme that would be storing energy. Don’t store energy when you need it for cellular function
Fig Styer 4th 20

22. Control of Acetyl CoA Carboxylase Activity
Fig
Phosphorylation by kinase inhibits carboxylase (+AMP, -ATP).
Phosphatase activates carboxylase (+insulin, -glucagon, & epinephrine).
Citrate partially activates the inactive phosphorylated acetyl Co A carboxylase allosterically.
Palmitoyl CoA inhibits carboxylase and citrate translocase.
Inactive form is an octomer
Active form is filamentous. Citrate promotes transition. See p624 in Berg etal
This enzyme has its regulation, more complex, more interesting. If we start with the inactive form, its phosphorylated, covalent modification of enzymes by phosphorylation is a common way of regulating activity. Seen some cases with lipase, in mobilization of fats, phosphorylated to active this one is phosphorylated to inactivate, done by AMP activated kinase. Though AMP down regulates activity of this. It activates a kinase which phosphorylates and then inactivates it. Phosphatases would do the reverse. Typical kind of regulation, but in addition, citrate binds directly to inactive form and activates it partly. Don’t get complete activation. Arrival of citrate gets things going then phosphotase takes a little longer. Palmitoyl coA inhibition, talk about htat next time.
Meant to say before I started, exam on Monday, material up through today’s lecture, no questions on the rest of this section 7. 21

23. Next Topic: Membrane lipids.
Web links
Odd Chain Fatty Acids. The fate of propionyl CoA.
Unsaturated Fatty Acid Oxidation. The role of isomerase.
Next Topic: Membrane lipids.