Lipids: synthesis and degradation

1. Lipids: synthesis and degradation Biochem I Lecture 4 Tues. Sept. 19/06 Chapter 22.0-22.2, 22.4, 22.6

2. First, a word about tests to determine the nature of FAs • You will use some of these tests in the first lab • We have seen / will see that the chemical makeup of the FAs (degree of unsaturation, chain length) affects their properties (Tm, biological activity) • This will be covered in the lab, but briefly these tests include • Degree of unsaturation: Iodine number • I can be used to saturate unsaturated FAs • Amount of I can be determined by titration • Chain length: Saponification number • Hydrolyze FAs in KOH • Longer chains  lower saponification numbers • These properties are useful to determine from biological samples (e.g., entering a manufacturing process)

3. Stored fats allow amazing physical feats p. 619 • The energy “richness” of triacylglycerols are the reason they were chosen over storage carbohydrate (polysaccharides) as our main energy stores during evolution • Adipocytes (fat-storing cells) serve as sites where fatty acids are imported, stored, and mobilized when needed by the organism for energy generation • Importance of fat stores as energy sources readily demonstrated in nature in 2 ways: • Migrating birds: many fly for days (3800 km straight) without eating, e.g., American golden plover • Hibernating mammals (bats, woodchucks) sleep for weeks http://www.state.nj.us/dep/fgw/ensp/somoct.htm

4. A few words about enzymes Change molecules from one form to another without being transformed themselves Enzymes are biological catalysts • All are p________ • “Out of sequence” discussion but we must address them briefly because they do all of the work in the cell • When we discuss synthesis and degradation of macromolecules, we mean a series of transforming steps converting substrates (of all 4 types of macromolecules!) to the ultimate desired product • These steps are performed by enzymes • Synthesize and degrade all macromolecules • The action of individual enzymes working in sequence to convert a substrate to a product is called a p________ (e.g., glycolysis) • Enzyme nomenclature: ---ase • Often can glean clues about an enzyme’s substrate (sucrose synthase) or product (pyruvate kinase) from its name • An enzyme reaction can be • close to equilibrium (K~1), often represented by double arrows ( ); these reactions occur very slowly • far away from equilibrium, favoring substrate (0<K<1, ) or product (K>1, ) formation; these are very fast reactions

5. Coupled reactions link enzyme activity with energy metabolism • Enzyme reactions that are far from equilibrium are interesting • Very energetically favorable reactions (K>>1) can be harnessed (=linked, or coupled) to energy production that the cell can “spend” elsewhere in metabolism in the form of • ATP, or • Reducing power (molecules that readily contribute hydrogens to reduce other molecules): NADH, FADH2; can also oxidize other molecules by removing their hydrogens (FAD, NAD+) • Why waste all the stored energy of a strongly exergonic reaction as heat when it can be preserved in ATP? Fight entropy! For example, pyruvate kinase • By contrast, energetically unfavorable reactions (K<<1) can be “pushed” to occur by coupling the reaction to ATP lysis or reducing power utilization • e.g., ADP-glucose pyrophosphorylase Glucose-1-phosphate + ADP-glucose + inorganic pyrophosphate (PPi) Enough about enzymes, back to fat metabolism… Adenine nucleotides! What kind of biomacromolecule? ADP + Pi ATP (a key reaction in glycolysis) Pyruvate Phosphoenolpyruvate ATP

6. How we store and degrade dietary fat • To be stored in adipocytes, ingested fats (mainly triacylglycerols) must be hydrolyzed to FAs • This occurs through the action of digestive enzymes secreted by the pancreas called lipases • But triacylglycerols are hydrophobic! Enzymes (biological catalysts) need an aqueous environment to function, so lipids must be solubilized into aqueous solution • Solubilization uses bile salts • cholesterol backbone • synthesized in liver, stored in and secreted by gall bladder • Contain both hydrophilic and hydrophobic domains: a_________ • Forms triacylglycerols into small spheres (micelles) with ester bonds oriented towards surface (e.g., Fig. 22.4)

7. Making fats mobile in the body FAs + monoacylglycerol exit intestine lumen and enter mucosal cells and lymph system Fig 22.3 • FA mobilization relies on the action of lipases (enzyme:-ase) that release 2 free FAs and monoacylglycerol • FAs and monoacylglycerol are amphipathic! • What does this mean physiologically? They can cross amphipathic cell membranes! • Remember: like dissolves like • When you injest fats (triacylglycerides) • Break down to FAs and monoacylglycerols in small intestine • Present hydrophilic domain to enter membrane • Display hydrophobic domain to remain soluble inside membrane lipoproteins • Exit again into mucosal cells in small intestine • Triacylglycerols resynthesized • Complex with other lipids and proteins as chylomicrons • Exported to lymphatic system (space inside small intestine) Fig 22.5

8. Making fats mobile in the body • From the lymphatic system the chylomicrons • enter the bloodstream • circulate until they find their target storage tissue: muscle (metabolized to make ATP) and adipocytes (stored for future use) • bind to cell-surface membrane-bound lipoprotein lipases in target tissues • Again these degrade triacylglycerols to 2 FAs + monoacylglycerol • These are transported across the membrane, resynthesized as triacylglycerols and stored (adipocytes) or metabolized (muscles) Harnessing the energy stored in FAs • Tissues in other parts of the body gain access to stored triacylglycerol energy stores through 3 sequential processes • Mobilization to target tissues • FA activation and transportation to mitochondria within target tissue cells • Catalysis inside the mitochondrial inner membrane to acetyl CoA Guess which enzymes initially involved in FA mobilization? • Enzymes that degrade triacylglycerols =l_______s • Process is called lipolysis

9. FA catalysis links lipid and carbohydrate metabolism • Glycerol backbone of triacylglycerol metabolized in the liver to intermediates in CHO metabolism; process is reversible as well • Demonstrates how lipid and CHO metabolism are linked! Once the FAs arrive inside the destination cell, they must be activated before they can be oxidized for energy extraction • Activation means transforming the FAs into higher energy compounds that are more readily degraded p. 621

10. Activating molecules involves the use of energy Coenzyme A • Activating FAs involves transducing some of the stored energy in ATP’s high energy phosphodiester bond into useable work • Remember that not all will be used, some will be lost as e_______ • The enzyme acyl coA synthetase uses the stored energy in ATP to link coenzyme A (CoA) to the terminal methyl group of the FA • The activated FA is called an acyl CoA pyrophosphatase 2 Pi Acyl CoA synthetase Acyl CoA Activated FA ready for oxidation p. 622 • The reaction is freely reversible but is driven forward by hydrolysis of pyrophosphate (PPi), one of the products of the reaction, by the enzyme pyrophosphatase But initially you are using ATP! Isn’t the point to make ATP to power metabolism? • Common in important pathways: give up a little bit of energy to get a lot in return • Also very common to make enzymatic reactions irreversible by the hydrolysis of PPi

11. Activated FAs enter mitochondria for oxidation Remember mitochondrial structure? • outer membrane contains porins: freely permeable to small (<10 kDa) molecules • Thus activated FAs enter mitochondrial cytosol, need to get across inner membrane for hydrolysis in matrix • Inner membrane not permeable to activated FAs (a/k/a acyl CoA molecules) • They enter after conjugation to another molecule (carnitine) FA activation by acyl CoA synthetase takes place on Fig 18.2

12. Fig 22.9 Degradation of fatty acids 1 Why are we oxidizing FAs? • to provide energy to actively metabolizing cells (e.g., exercising [glucose-starved] muscles) • Use energy in stored FA molecules to make ATP for metabolism • Now that the FAs are inside mitochondrial matrix they can be degraded through a seqential series of steps • Sum is called the ß-oxidation pathway • Products from the 4 enzymatic reactions in the pathway are 2-carbon units:acetyl CoA • How will this make ATP? Where in metabolism is acetyl CoA is a substrate? Stage II of catabolism! 2 3 4 Note we are also making reducing power: FADH2 and NADH: all of these provide e- to o________ p________ to power ATP synthesis!

13. FA oxidation yields energy for cellular work • Each round of oxidation yields 1 FADH2, 1 NADH, and 1 acetyl CoA • The complete oxidation of palmitate (16:0) to acetyl CoA requires ____ reaction cycles • Note that the CoA molecule is not consumed or changed, merely passed between molecules during the rounds of oxidation • Remember that the NADH and FADH2 are destined for oxidation in oxidative phosphorylation • Energy yield: • 2.5 ATP per NADH • 1.5 ATP per FADH2 (lower energy electrons yield less ATP) • 10 ATP per acetyl CoA oxidized in the TCA cycle • ATP yield is 7(2.5) + 7(1.5) + 8(10) = 108 ATP per 16:0 FA oxidized • Recall that we “primed the pump” with 2 ATP to initially activate FA through complexing with acetyl CoA • Thus total energy yield is 108 – 2 = 106 ATP per 16:0 FA oxidized Will be completely oxidized via the ______ cycle and _______ _______

14. FA synthesis occurs using different enzymes in an anabolic pathway • Common when considering anabolic vs. catabolic pathways • Synthesis makes a more complicated molecule out of simple ones (acetyl CoA): a reducing pathway that requires reducing power • One major difference is location of pathways in the cell: synthesis occurs in the cytosol versus degradation in the m_______ • Step 1: make malonyl CoA from acetyl CoA by carboxylation by acetyl CoA carboxylase Follow the Cs: 2 + 2 -1 = 3 p. 635 Fig 22.21 • Step 2:Activate malonyl CoA and acetyl CoA by swapping CoA groups for acyl carrier protein (ACP) • ACP thus replaces CoA as activated prosthetic group during FA synthesis vs. degradation • In both molecules FAs attach to terminal sulfurs (FA degradation) (FA synthesis)

15. Like for FA degradation, the process is repeated to change chain length • Round 2: condense acetyl ACP + malonyl ACP to make butyryl ACP • Again decarboxylate to drive reaction forward • Rounds 3-7: Continues for 7 rounds to make C16-acyl ACP • A thioesterase esterifies the sulfur bond to remove the ACP and yield palmitic acid (16:0 FA) Overall: 2 C + 3 C – CO2 = 4 C Note the use of anabolism-specific reducing power!

16. How are other FAs made from palmitate? • The basic synthetic process forms palmitate (16:0), but there are many other FAs present in living cells Longer FAs • Eukaryotes form longer fatty acids by elongation reactions (2 Cs at a time) catalyzed by enzymes on the cytosolic face of the endoplasmic reticulum (ER) • Again, malonyl CoA is the C donor and is decarboxylated upon incorporation into FA acyl chains • Enzymes active on saturated and unsaturated FA substrates Unsaturated FAs • Also introduced by enzymes localized to the ER membrane • Catalyzed by oxidase enzymes, e.g., for C18 unsaturation • Other unsaturated FAs are derived from palimitoleate (16:1), oleate (18:1), linoleate (18:2) or linolenate (18:3) • Mammals lack enzymes to synthesize C=C bonds beyond C9 but require the products of substrate FAs linoleate (“omega-6”; cis,cis-Δ9, Δ12) and linolenate (“omega-3”; cis,cis,cis-Δ9, Δ12, Δ15 ) • These are thus dietary essential FAs: substrates for other unsaturated FAs

17. Animal vs. plant metabolic flexibility: which are more advanced? Two interesting things to note about animal versus plant FA metabolism • Both deal with how these kingdoms interface lipid metabolism with carbohydrate metabolism • Animals cannot make glucose directly from oxidized FAs (acetyl CoA) • Cannot convert acetyl CoA to gluconeogenic intermediates (pyruvate, oxaloacetate) • Plants have a glyoxylate cycle that can “steal” acetyl CoA away for glucose synthesis • Plants do not make FAs for transient energy storage, they make starch (~glycogen, both polysaccharides) • When plants make FAs, it is for oil deposition as food reserves for germinating seeds • Developmental/temporal and physical separation of FA synthesis and degradation in plants 1 2