Lipid Metabolism Student Edition 7/3/14 version

Lipid Metabolism Student Edition 7/3/14 version
Dr. Brad Chazotte 213 Maddox Hall Pharm. 304 Biochemistry Fall 2014 Web Site: Original material only ©2014 B. Chazotte

LIPID METABOLISM GOALS
Learn how lipids in vertebrates are transported and enter metabolic pathways from the diet Learn how fatty acids undergo β-oxidation Learn how fatty acids are biosynthesized. Understand the control of fatty acid metabolism and apply it to metabolic regulation Ketone body anabolism and catabolism Have a general knowledge of the synthesis of other lipids Learn the metabolism of cholesterol

A Very Brief Recap from Previous Lipid Lectures
Lipids Are A Diverse Class of Compounds Biologically Relevant Fatty Acids . Horton et al 2012 Fig 9.2 Voet, Voet, & Pratt 2013 Table 9-1

Summary of Lipid Metabolism
Horton et al., Figure 16.XX

Lipid Digestion, Absorption & Transport
Triacylgylcerols Comprise ~90% dietary lipids Major form of energy storage in humans – highly reduced structures Hydrophobic molecules Consist of glycerol triesters of fatty acids Lipid Sources fats obtained from the diet fats obtained from lipid droplet storage in cells, e.g. adipocytes fats synthesized in one organ for transport to another Voet, Voet, and Pratt., 2013 Page 678

Overview of Dietary & Storage Lipid Processing in Vertebrates
Major Bile Salts Chylomicron Voet, Voet, and Pratt, 2013 Figure 20.1 Lehninger, 2005 Figure 17.1, 17.2

Mobilization of Traiacylgycerols Stored in Adipose Tissue
Need 2012 figure! Missing other lipases) Lehninger, 2005 Figure 17.3

Destinations of Storage Triacylglycerol Catabolism
Berg, Tymoczko & Stryer 2012 Figure 22.7

Glycerol Oxidation Where does glycerol from triacylglyerols go?
Berg, Tymoczko & Stryer 2012 Figure 22.7 Glycerol released from the catabolism of triacylglycerol is phosphorylated by glycerol kinase The resulting glycerol-3-P converted by glycerol-3-P dehydrogenase to dihydroxyacetone phosphate. Triose phosphate isomerase does the final conversion to D-glyceraldehyde-3-P which proceeds through glycolysis or, if needed, gluconeogenesis Lehninger, 2005 Figure 17.4

Human Plasma Lipoprotein’s Major Classes Characteristics
Lipids need to be transported in the circulatory system in complexes with proteins. These lipoprotein carriers are micelle-like globular structures. The hydrophobic core (thermodynamics!) is composed of triacylglycerols and cholesteryl esters. The core is surrounded by an amphiphilic coat comprised of protein, phospholipid, and cholesterol. You need to be familiar with the different classes of human lipoproteins and their full names but not details of the % components In human lipoproteins there are at least nine varieties that distributed among the 5 lipoprotein classes in this table. Be familiar with the descriptions of the apolipoproteins but not which lipoprotein they are in unless I request specifically it. That being said please note and remember that apolipoprotein b-100 is the sole one identified in LDLs Chylomicrons – at the stage of chylomicrons they are delipidated in peripheral tissue capillaries such as adipose tissue and skeletal muscle. The extracellular enzyme, lipoprotein lipase, hydrolyzes triacylglycerols with the tissues taking up the resultant glycerol and fatty acids. Voet, Voet, and Pratt, 2013 Table 20-1 Lehninger, 2005 Figure 17.2 Voet, Voet, and Pratt, 2013 Figure 20-5

Overview of Plasma Triacylgycerol & Cholesterol Transport in Humans
Cholesterol is transported in the blood via lipoprotein particles. HDL are involved in cholesterol transport from the tissues to the liver. VLDLs, IDLS, and LDLs are involved in transport of cholesterol from the liver to the tissues via the circulatory system and they are synthesized in the liver In this illustration we add a more detailed picture to the transport of triacylgycerols and cholesterol compared to the previous slide on dietary and storage lipid processing. In the digestive process the dietary fat is gradually broken down to large lipid droplets and with the action of bile acids into micelles Mucosal cells process the dietary fatty acids into triacylglycerols and pack them into chylomicrons with cholesterol. This is shown in the lower right part of the schematic. They are transported via the intestinal lymphatic system (green vessels) to the large veins (purple). They are than transported via the circulatory system throughout the body (arrows leading out from the blood vessel. Cholesterol is transported into the blood via lipoprotein particles. There are different types of these lipoprotein particles, as we have seen in a previous table. HDL are involved in cholesterol transport from the tissues to the liver via the circulatory system. Whereas VLDLs, IDLS, and LDLs are involved in transport of cholesterol from the liver to the tissues via the circulatory system and they are synthesized in the liver. Looking at the lipoprotein particle in the lower right illustration – it has been found that each lipoprotein has just enough protein, cholesterol, and phospholipid to form a approximately 20Å thick surface monolayer. NOTE: LIPOPROTEIN DENSITY INCREASES WITH DECREASING PARTICLE DIAMETER, i.e., THE DENSITY OF THE OUTER COATING IS GREATER THAN THE INNER CORE. Hence HDL are both the smallest and most dense of the lipoproteins. (CHECK THIS!!!) Know which lipoproteins go to or from the tissues! Voet, et al., 2008 Figure 20-5 Voet, et al., 2008 Figure 20-7

Low Density Lipoprotein (LDL) Receptor-mediated Endocytosis
Receptors synthesized on ER Processed by Golgi Inserted into plasma membrane and located at clathrin-coated pits Upon binding LDL receptors bud in to form clathrin-coated vesicles Vesicles fuse with endosome after depolymerization of clathrin coat Endosome pH (~5.0) causes dissociation of LDL and receptor Receptors concentrate in membrane and LDLs concentrate in vesiclular portion Endosome fuses with lysosome Cholesterol either to ER or converted to cholestryl ester Specific receptors for low density lipoproteins are used in the process called receptor-mediated endocytosis to take LDLs out of the circulatory system and bring them into cells where the cholesterol is used. LDL receptors are synthesized on the ER and undergo processing in the Golgi before insertion into the plasma membrane. The LDL receptors on the PM are located in the clathrin-coated pits. In the previous slide the table showed that apolipoprotein b-100 was LDL’s sole apolipoprotein. B-100 specifically binds LDL receptors to the PM clathrin-coated pits. These pits bud into the cell to form clathrin-coated vesicles that we have mentioned in the lecture on membranes. The clathrin coating depolymerize to yield uncoated vesicles that fuse with (pH 5.0) endosome vesicles. The low pH causes the LDL particle to dissociate from the receptor. The LDL particles concentrate in the vesicular portion while the receptors concentrate in the membrane of an attached tube-like structure. The receptors are recycled to the plasma membrane. A secondary lysosome is formed by the fusion of endosome with a lysosome. In the secondary lysosome the b-100 protein and the cholesteryl esters are hydrolyzed. From here cholesterol can either proceed to the ER or be converted to cholesteryl esters via acyl-CoA:cholesterol acyltransferase, also abbreviated ACAT. High concentrations of cholesterol in the ER cause HMG-CoA reductase synthesis to decrease and LDL receptors while ACAT synthesis is increased. (HMG-CoA reductase is the rate-limiting step in cholesterol synthesis. Voet, et al., 2008 Figure 20-8

Subcellular Location of Lipid Metabolism
The animal cell on the left dealing with the locations of lipid metabolism is something you should familiarize yourselves with. Transport of lipids across cellular and subcellular membrane frequently entails the use of transport systems, particularly for longer chain lipids. We want to remember that Fatty Acid β-oxidation occurs in the mitochondrial matrix whereas fatty acid synthesis occurs in the cellular cytosol – the spatial separation facilitates control of these opposing pathways. The mitochondrion is also responsible for the synthesis of ketone bodies and the ELONGATION of fatty acids (C16 palmitic acid is the typical FA of biosynthesis). The cellular cytosol also provides molecules for the early stages of isoprenoid (example? – ubiquinone and sterol (example? - cholesterol) biosynthesis. The ER is involved in a number of aspects of lipid metabolism including the elongation of fatty acids and the desaturation of fatty acids (produces what ?– double bonds in FA). The ER is also where phospholipids are synthesized and sterol synthesis is finished up. Lehninger, 2005 Figure 21.8

Fatty Acids Must Be “Activated”
Fatty Acid Activation Mechanism Acyl-CoA Synthetase Rx Voet, Voet, and Pratt, 2013 Figure 20-10 Acyl-CoA Synthetases (thiokinases) specific for short, intermediate, & long carbon chain FA’s catalyze the formation of a thioester linkage between fatty acid COOH group and CoA thiol group. Two step reaction. Fatty acyl CoA hydrolysis ∆G’˚ = -31 kJ mol-1 Synthesis ∆G’˚ = -34 kJ mol-1 with formation driven by hydrolysis of PPi. Fatty acyl- CoA esters formed at cytosolic side of mitochondrial outer membrane. Transported into mitochondria for energy production (oxidation) or used in cytosol for membrane lipid syntheses. Fatty acids must be primed for their catabolism. This is accomplished by first activating the fatty acids by adding Coenzyme A. The reaction is fatty-acid chain length dependent with at least 3 Acyl-CoA synthetases (short, medium & long chain) involved working on the ER or the outer mitochondrial membrane. CoA is added to a carbonyl carbon via a thioester link. The overall reaction is thermodynamically driven to completion by the exergonic hydrolysis of the pyrophosphate (PPi) from ATP by inorganic pyrophosphatase (seen on the lower left). Intermediate Lehninger, 2005 Figure 17.5

Fatty Acids Need to be Transported into the Mitochondrion
Voet, Voet , and Pratt, 2013 Figure 20-11 Lehninger, 2013 Figure 17.6 Needed for transmembrane transit of long chain fatty acids, e.g. palmitic acid Activated fatty acids can be transported across the mitochondrial inner membrane via the carnitine shuttle. Enzymes can either be called carnitine palmitoyl transferase or carnitine acyltransferase. Transferase one is on the cytosolic side of the outer mitochondrial outer membrane. Transferase II is on the matrix side of the mitochondria INNER membrane. This system is needed for the transport of long chain fatty acids whose transit of the inner membrane is thermodynamically unfavorable. Note that Medium chain fatty acids (8-10) and shorter DO NOT require this transport system to cross the inner membrane. [Refers to palmitoyl transferase II: The zwitterionic nature of carnitine make the reverse reaction thermodynamically favorable. ] Note that the CoA does not cross the inner membrane giving rise to two pools of CoA. The mitochondrial pool of CoA is primarily involved in the oxidative degradation of fatty acids, pyruvate and some amino acids. The cytosolic CoA pool is involved in the synthesis of fatty acids. Rate-limiting step for fatty acid oxidation in mitochondria and REGULATORY point. Voet, Voet, and Pratt, 2013 Page 666 Steps: Esterification to CoA (cyto.) Transesterification to carnitine + transport across IM Transesterification back to CoA (mito) Rate-limiting step for fatty acid oxidation in mitochondria and REGULATORY point.

Carnitine & Carnitine Palmitoyl Transferase (CPT) Deficiencies
Several diseases result from problems in transport of Long Chain –Fatty Acids across mitochondrial inner membrane. Can be due to: DEFICIENCIES IN CARNITINE LEVELS Primary: defect in high-affinity PM carnitine transporter in muscle, kidney, heart & fibroblasts (but not liver). Treat with dietary carnitine Secondary: associated with β-oxidation defects giving rise to accumulation of high levels acylcarnitines which are excreted DYSFUNCTION IN CPT SYSTEM CPT I: few reports hepatic – likely lethal CPT II: mutations with partial loss of activity. mutations (>90% loss) affect early infancy- periods of fasting precipitate problems Clinical Symptoms: mild recurrent muscle cramping to severe weakness & death Clinical Symptoms: muscle weakness during prolonged exercise What happens when the trans mitochondrial transport of fatty acids does not work properly. The diseases mentioned here illustrate how the impaired flow of a metabolite from one compartment to another can give rise to a pathological condition [Berg et al p. 646]. Clinical Symptoms: hypoketotic hypoglycemia, hyperammonemia, cardiac malfunction, sometimes death Devlin , ed p. 384

Fatty Acid Oxidation β-oxidation: process of successive removal of 2-carbon fragments from fatty acids . In mitochondria, also in peroxisomes. ω-oxidation: in liver ER of some vertebrate species - starts at “opposite” end of FA. Minor pathway in mammals unless β-oxidation is defective. Fatty acid oxidation occurs via the removal of two carbon units in a process determined as beta-oxidation (beta carbon of fatty acid). The original experiments by Dr. Franz Knoop in dogs analyzed urine metabolites depending on whether the dogs were given chemically labeled odd or even chain fatty acids in their diet . ω-oxidation utilizes different enzymes than in B-oxidation Lehninger, 2013 Table 17.1

Stages of Fatty Acid Oxidation
Ultimately energy is obtained from the complete oxidation of a fatty acid which proceeds via electron transport and oxidative phosphorylation to yield ATP Previously different stages of metabolism have been presented. Likewise for fatty acid catabolism there are three stages with the first being the process know as beta oxidation. In stage 2 the two carbon fragments, acyl CoA molecules, enter the citric acid cycle where reducing equivalents are transported to electron transport where the energy is used to generate ATP via oxidative pho sphorylation. The complete oxidation of the 16-carbon saturated palmitic acid yields 106 ATP. [10.5 from seven FADH2; 17.5 from seven NADH; 80 from 8 acetyl CoA; =108 minus 2 ATP for activate palmitate, thus 106 ATP] {DISAGREES WITH SLIDE IN METABOLISM OVERVIEW OF COMPLETE USE TO MOLECULAR OXYGEN} Lehninger, 2005 Figure 17.7

β-Oxidation Pathway for Fatty acyl-CoAs (Overview)
Comprised of 4 Reactions: Acyl Co A Dehydrogenase (AD): causes the formation of a trans-α-β double bond Enoyl CoA hydratase (EH) hydrates the double bond to form 3-L-hydroxyacyl CoA 3-L-hydroxyacyl CoA dehydrogenase (HAD) drives an NAD+-dependent dehydrogenation to form a β-ketotacyl-CoA β-ketotacyl-CoA thiolase (TK) or thiolase, causes a cleavage of a Cα-Cβ in a thiolysis reaction to produce acetyl-CoA and a new 2-carbon fewer acyl CoA KNOW THESE! The chemical/logical problem to be solved is for a carbonyl functional group to be created at the beta methylene group to the original carbonyl group so that both product molecules will be acyl CoAs. That is, you want to have the two carbon fragments produced to be conjugated to CoA and the remaining molecule minus the two carbon fragment to be conjugated to CoA to continue the degradation. This permits the cycle degradation of a fatty acid. Also and just as important the C2-C3 (α-β) bond is relatively stable [Walsh, 1979 p. 905] – need to make it less stable to break it. 1) Voet, Voet & Pratt Figure 20-12 The C2-C3 (α-β) bond is relatively stable – need to make it less stable to break it. Part of β-oxidation rationale.

Acyl-CoA Dehydrogenases
Four acyl-CoA dehydrogenases (isozymes) in mitochondria: SCAD short chain acyl CoA dehydrogenase specificity highest for C4 to C6 MCAD medium chain acyl CoA dehydrogenase specificity higher for C6 to C10 LCAD long chain acyl CoA dehydrogenase specificity higher for C12 to C18 VLCAD very long chain acyl CoA dehydrogenase 1st Reaction step in β-oxidation. Acyl-CoA dehydrogenases send reducing equivalents to the ETF dehydrogenase on the mitochondrial inner membrane and from there to ubiquinone in electron transport. This route in β-oxidation constitutes a major energy flux. In mammalian mitochondria there are four acyl-CoA dehydrogenases that function in the first step of β-oxidation. These are what enzymologists call isozymes. Each of these dehydrogenases have a range of specificity that depends on the length of fatty acid. Their activity is highest for a certain fatty acid chain length. Consequently, their names reflect this change length specificity. The short chain acyl CoA dehydrogenase has it highest activity with FA chains of 4 to 6 carbons. The medium chain Co A dehydrogenase has its highest specificity from 6 to 10 carbons, LCAD from 12 to 18 and VLCAD > carbons. Logically the process of β-oxidation starts with the longer chain dehydrogenase and works to the shorter.

Acyl-CoA Dehydrogenase Deficiencies
Either SCAD, MCAD, and LCAD can be deficient as a result of an autosomal recessive genetic defect and impair β-oxidation. MCAD deficiency is one of the best characterized. Typically manifests itself within 2 years of birth, usually after 12+ hours of fasting. Symptoms can include: vomiting, lethargy and frequently coma accompanied by hypoketoic hypoglycemia and dicarboxylic aciduria. .Devlin 1997 p.385 MCAD Deficiency has also been found in 10% of sudden infant death syndrome (SIDS) . Declines in glucose catabolism a while after eating leads to an increase in fatty aid oxidation. Imbalance between glucose and fatty oxidation in such infants may give rises to SIDS Voet, Voet & Pratt, 2013 p.668 The absence of starvation ketosis is due to the block in fatty acid oxidation which also causes a slowdown in gluconeogenesis. Adding also fatty acid oxidation impairment in muscle tissue which drives up glucose consumption (need energy in muscle switch to glucose), these give rise to profound HYPOglycemia. There is accumulation of medium chain acyl CoAs in tissues which forces alternative pathways such as ω-oxidation and transesterification to glycine or to carnitine. [Devlin 1997] “older” Patients with MCAD deficiency can do well by avoiding prolonged periods of starvation which drive fatty acid oxidation to supply energy during carbohydrate deprivation [Devlin 1997]. MCAD deficiency might explain cases previously termed Reyes-like syndrome. Also Reyes syndrome concerns are why we do not give aspirin to children. Voet Voet & Pratt 2013 p.. 668; Devlin 1997, p. 385

β-Oxidation (1): acyl-CoA dehydrogenase Rx
~stable bond Dehydrogenation to form a trans double bond. Bond is α-β to the carbonyl, i.e. between C2 and C3 (∆2). Reducing equivalents are transferred via FAD to the ETF dehydrogenase in the mitochondrial inner membrane – hence to electron transport Product: called trans-∆2-Enoyl-CoA As mentioned just previously: the chemical problem to be solved is for a carbonyl functional group to be created at the beta methylene group to the original carbonyl group so that both molecules will be acyl CoAs. The first reaction in the sequence of β-oxidation involves the formation of a double bond beta(?) to the carbonyl. Want a carbonyl at the β—methylene group of the original molecule to have a “new” carbonyl to conjugate to CoA after cleavage/removal of 2 carbon fragment (that contained the original carbonyl group conjugated to a CoA). But the double bond formed is in the trans configuration. This is opposite of the cis double bond configuration found naturally in “unsaturated” fatty acids. The resulting compound is called trans-∆2-Enoyl-CoA Voet, et al., 2013 Figure 20-12

(For long chain fatty acids)
Mitochondrial Trifunctional Protein (TFP) (For long chain fatty acids) β-oxidation steps 2-4 also involve chain-length dependent (short, medium & long) enzymes For fatty acids of 12 or more carbons TFP heterooctamer of α4β4 subunits; each α subunit contains an enoyl-CoA hydratase & β-hydroxyacyl-CoA dehydrogenase each β subunit has the thiolase activity Permits efficient substrate channeling Fatty acids less than 12 carbons are then handled by soluble enzymes in the mitochondrial matrix There are different macromolecular structural organizations for the β-oxidation depending on the species and in the case of humans which is our primary concern on fatty acid chain length. For humans the shorter, i.e. <12 carbons are handled by a soluble system whereas longer chains fatty acids are oxidized on a macromolecule complex built on mitochondrial trifunctional protein. If there is a single complex with all the enzymes present then the substrates do not have to travel “long” distances - substrate channeling. Long chain fatty acids would be less soluble in an aqueous environment – thermodynamics. Lehninger (p.674) talks of 2 sets of enzymes, one for 12 or more (medium) in trifunctional enzyme; 12 or less go for soluble enzymes . Voet, Voet and Pratt (p.668-9), seem to talk of three enzyme systems . Long chain version of enzymes on TFP. Whereas short chain and medium chain with soluble enzymes.

β-Oxidation (2): Enoyl-CoA Hydratase Rx
Three systems of isozymes enoyl-CoA-hydratase that are chain length dependent (short, medium, or long chain). Reaction involves the addition of water to the double bond. Forms the L stereoisomer. In this second step water is added to the trans double bond forming the L stereoisomer called 3- L-hydoxyacyl-CoA. The reaction is catalyzed by enoyl-CoA hydratase. It is a stereospecific addition! Similar to the fumarase reaction in the Citric Acid cycle wherein water is added across an α-β double bond. Mitochondrial trifunctional protein Voet, et al., 2013 Figure 20-12

β-Oxidation (3): 3-L-hydroxyacyl-CoA Dehydrogenase Rx
Dehydrogenation Rx Enzyme absolutely stereospecific for L-stereoisomer of hydroxyacyl CoA NAD is the electron acceptor The third reaction removes hydrogens from the β- or C3 carbon. Why have a carbonyl at C3? Oxygen is electron withdrawing so carbonyl carbon is delta positive. Can also help Thiol group can attack carbonyl carbon. Also have set up to have a 2C acetyl-CoA at end of fatty acid and a “new” carbonyl available to e conjugated with CoA. This is the β-ketoacyl CoA created. Why is it called β-ketoacyl ? 2nd carbonyl, a ketone, is β to the “first” or original carbonyl group. The enzyme 3- L-hydoxyacyl-CoA dehydrogenase (HAD) uses NAD+ as the electron acceptor. The enzyme is absolutely stereospecific for the L stereoisomerof hydoxyacyl-CoA. The reaction produces β-ketoacyl-CoA Analogous to the malate dehydrogenase in the Citric Acid cycle The NADH produced transfers its electron to NADH dehydrogenase in mitochonrial electron transport. Voet, et al., 2013 Figure 20-12

β-Oxidation (4): β-ketoacyl-CoA thiolase Rx
Rx yields an acetyl CoA and an acyl CoA with two carbons less than the previous cycle. Now β-ketoacyl-CoA C2-C3 bond much less stable due to two carbonyls. A Claisen ester cleavage Has acetyl CoA carbanion intermediate. This last reaction in β-oxidation yields an acetyl CoA and an acyl CoA with two carbons less than the previous cycle. It is catalyzed by acyl-CoA acetyltransferase commonly called thiolase. Claisen ester cleavage opposite of Claisen condensation. Reaction has the enzyme active site thiol adds to the β-keto group. Cleavage of the C-C bond occurs- yields thioester between acyl-CoA and enzyme thiol. Also have an acetyl-CoA carbanion intermediate stabilized via electron withdrawal to the thioester carbonyl. (how? why?) Enzyme’s acidic group protonates the acetyl Co A carbanion to produce acetyl CoA (how does this work – carbanion negative & proton positive. CoA’s thiol group displaces enzyme thiol group to form an acyl CoA. Similar to Citric Acid Cycle malate dehydrogenase reaction. β Voet, et al., 2013 Figure 20-12

Various Fatty Acid Types Must be Catabolized Somewhat Differently
Saturated Fatty Acids “normal” β-oxidation via 2-carbon acetyl CoA fragments Horton et al., 2013 Chapter 9 Unsaturated Fatty Acids Problem: double bonds Solution: Two additional reactions Voet: 4 additional enzymes Three main cases: Saturated, even chain fatty acids undergo the “normal” sequence of β-oxidation removing 2-carbon acetyl CoA fragments. However, if the fatty acid is unsaturated, i.e. has one or more double bonds, other enzymes are needed to handle the double bonds. Odd chain fatty acids present a different problem since you cannot remove only two carbon fragments at some point. Voet, Voet, and Pratt, 2013 Page 669 Problem: removing 2-carbon fragments eventually left with 3-carbon fragment Odd Chain Fatty Acids Solution: Three additional reactions to convert to citric acid cycle intermediate

The β-Oxidation Pathway
This is a recap of the normal β-oxidation pathway showing the four enzyme (each has isozymes) on the left and on the right a schematic of the successive removal of two carbon fragments of a C16 fatty acid (palmitic) to end with al as acetyl CoA. Lehninger, 2005 Figure 17.8

Oxidation of Unsaturated Fatty Acids
Goal: convert a molecular structure to one catabolizble by normal β-oxidation enzymes 3,2-enoyl-CoA isomerase (mammals) reversibly converts ∆2 & ∆3 bonds 3,4-2,4 dienoyl-CoA isomerase isomerizes the 3-5 diene to a 2,4 diene. 2,4 dienoyl-CoA reductase reduces the double bond 3,2-enoyl-CoA isomerase isomerizes the double bond Enoyl CoA isomerase converts the cis ∆3 to a trans ∆2. NADPH-dependent 2,4-dienoyl CoA reductase reduces the ∆4 double bond. Mammalian enzyme yields trans-3-enoyl CoA DO NOT NEED TO KNOW ALL THE ENZYME NAMES HERE, BUT KNOW DIFFERENT PROCESS NEEDED. We have considered the beta oxidation of saturated fatty acids. What happens if the fatty acid is unsaturated or polyunsaturated? (Unsaturated meaning what? - Double bonds) In nature virtually all the unsaturated fatty acids that are of biological origin have cis double bonds and these double bonds typically are between C9 and C10 (∆9). When there are multiple double bonds, these occur at 3-carbon intervals. Conjugated doubles are not found. Your text uses linoleic acid as an example We can summarize that the overall goal is convert a “problematic” molecular structure to one catabolizble by normal β-oxidation enzymes. This will allow the molecule to continue through successive cycles to remove acetyl-CoA fragments after solving the double bond problem. Problem 1: The existence of fatty acid β,γ double bond. In this linoleic acid example β-oxidation could proceed for three cycles before the double bond is encountered. The solution for the cis-β,γ double bond-containing enoyl-CoA cannot be a substrate for the enzyme enoyl-CoA hydratase in β-oxidation which uses the ∆2 double bond as a substrate. Therefore, Enoyl-CoA isomerase converts the cis ∆3 to a trans-∆2 so β-oxidation can continue Problem 2: Continuing with our linoleic acid example, the presence of a ∆4 double bond would become a problem in the 5th turn of β-oxidation. The presence of a double bond on an even carbon, that is having 2,4-dienoyl-CoA, presents a poor substrate for the β-oxidation enzyme enoyl-CoA hydratase. The NADPH-dependent enzyme 2,4-dienoyl CoA reductase reduces the ∆4 double bond, with the mammalian enzyme yielding trans-3-enoyl CoA (E. Coli: product is trans-2-enoyl-CoA) which requires a 3,2-enoyl-CoA isomerase, a second enzyme, to create the normal trans-2 substrate. Problem 3: A slightly more complicated problem that arises due to 3,2-enoyl-CoA hydratase’s isomerization of 2,5-Enoyl-CoA. The mammalian enzyme 3,2-enoyl-CoA isomerase . Due to the mechanism 20% of the time 3,5-2,4-dieneoyl CoA isomerase isomerizes the 3,5diene to the needed 2,4 diene. The latter is subsequently reduce by 2,4-dienoyl-CoA reductase and is subsequently isomerized (viz Problem 2). Voet, Voet, and Pratt, 2013 Figure 20-15

β-oxidation of odd-numbered fatty acid (Proprionyl-CoA oxidation )
Problem: Successive removal of acetyl-CoA 2-carbon fragments for odd chain fatty acids eventually leaves a 3-carbon fragment, propionyl-CoA. Solution: Convert propionyl-CoA to succinyl-CoA, a citric acid cycle intermediate via 3 enzymes So far we have dealt with even chain carbon fatty acids which predominate in nature. What happens if you have an odd number of carbons in a fatty acid? The regular process of β-oxidation can proceed until the last three carbons are reached, i.e. propionyl CoA. Step 1: Propionyl-CoA carboxylase (w/ biotine prosethetic group) adds a CO2 α to the carbonyl group driven by ATP hydrolysis. Step 2 Methylmalonyl-CoA racemase converts (S)-methylmalonyl-CoA to (R) form. Step 3 Methylmalonyl-CoA mutase rearranges the (R)-methylmalonyl-CoA carbon skeleton to Succinyl-CoA. The enzyme prosethetic group requires vitamin B12. NOTE: In order for the succinyl-CoA to undergo a NET OXIDATION in the citric acid cycle it must first be converted to pyruvate and then acetyl-CoA. – Why? As your text state C4 intermediates of the cycle function as catalysts. Remember that in the citric acid cycle acetyl-CoA adds to citrate in the cycle and then 2 decarboxylations. So to have a NET oxidation the carbon skeleton has to go through pyruvate rather than enter directly as succinyl-CoA. NOTE: In order for the succinyl-CoA to undergo a NET OXIDATION in the citric acid cycle it must first be converted to pyruvate and then acetyl-CoA Lehninger, 2005 Figure 17.11

Comparison of Mitochondrial β-oxidation to that in Peroxisomes
Peroxisomal β-Oxidation Involved in shortening very long (>22 carbons) fatty acids that are subsequently degraded via the mitochondrion. Only slightly different than mitochondrial β-Oxidation Comparison of Mitochondrial β-oxidation to that in Peroxisomes An electron micrograph of peroxisomes is shown in the upper right corner. Although the bulk of fatty acid β-oxidation occurs in the mitochondria of mammalian cells, peroxisomes also oxidize fatty acids, but primarily very long chain or branched chain ones. Interestingly mammalian peroxisomes also play a role in the synthesis of certain lipids including the bile acids. We will not need to remember the details of peroxisomal oxidation, only the location and that it functions for very long chain fatty acids that subsequently enter mitochondrial β-oxidation after shortening. Voet, et al., 2008 Figure 20-20 Lehninger, 2005 Figure 17.13

ω-Oxidation of FA in the ER
Found in Vertebrates – located in ER of liver and kidney. Preferred substrates: 10 or 12 carbon. Works from opposite end of fatty acid compared to β-oxidation Normally a minor pathway in mammals Can be important if β-oxidation is defective The ω-oxidation of fatty acids found in vertebrates is typically a minor pathway that is found in the ER of liver and kidney. In this pathway the first step involves adding a hydroxyl group on the ω-carbon of the fatty acid. The oxygen comes from molecular O2 and involves cyt-P450 and NADPH via a “mixed function oxidase”. Alcohol dehydrogenase acts on the ω-carbon oxidizing the hydroxyl to an aldehyde followed by aldehyde dehydrogenase oxidizing the aldehyde to a carboxylic acid. The molecule can have either end then attached to Co A and enter β-oxidation IN THE MITOCHONDRION. These are dicarboxylic acids undergoing beta oxidation. Take home: beware this occurs and where but not the individual enzymes. Lehninger, 2005 Figure 17.16

Ketone Bodies & Ketogenesis
Acetyl-CoA can be converted to acetoacetate or D-β-hydroxybutyrate called “ketone bodies”. “Alternate fuels” Occurs in liver mitochondria (matrix) – supplied to peripheral tissues, e.g. heart & skeletal muscle, also brain under special conditions. Voet, Voet, and Pratt, 2013 Page 678 Acetyl-CoA acetyl transferase condenses 2 acetyl CoA’s to acetoacetyl-CoA. HMG-CoA synthase condenses a 3rd acetyl CoA to form β-hydroxy-β-methylglutaryl-CoA HMG-CoA lyase degrades HMG-CoA into acetoaetate and acetyl CoA Acetoacetate underoges nonenzymatic degradation to acetone & CO2. The top left panel shows the molecules termed “ketone bodies: acetoacetate, acetone, and D-β-hydroxybutyrate. The process of making ketone bodies. “ketogenesis”, occurs in the liver and the ketone bodies are supplied to peripherl tissues as an alternate fuel. Ketone bodies can be prominent in starvation and in diabetes. ( “Bodies” was historically applied to insoluble particles, however the above compounds are soluble in blood and urine) In the lower panel the reactions of ketogenesis are shown. The first step in ketogenesis reverses last step of β-oxidation and two acetyl CoA are condensed together “freeing” on CoA and is carried out by thiolase. One of the acetyl-CoAs is shown in red in the illustration. In the second step a third acetyl-CoA (green color) is condensed with the freeing of a CoA and is carried out by HMG-CoA synthase. The third reaction catalyzed by HMG-CoA lyase yields one molecule of acetyl-CoA and one acetoacetate that has no acetyl CoA Acetone is formed in very small amounts from acetacetate in normal, healthy people. Untreated diabetics produce large quantities of acetoacetate and their blood , therefore, contains significant acetone, which is toxic. Acetone, being volatile, is exhaled. (Breath of diabetics can smell of acetone.) Voet, Voet, and Pratt, 2013 Figure 20-21

Ketone Boyd Formation and Export from the Liver
One advantage of ketone body production and export from the liver is that oxidation of fatty acids can continue with minimal oxidation of acetyl-CoA such as when Citric acid cycle intermediates are siphoned off for gluconeogenesis. Ketone bodies are overproduced in diabetes, as mentioned before, and also during starvation for use as an alternative fuel. If diabetes is not treated and the insulin level is not sufficient then extrahepatic tissues do not efficiently take up glucose from the blood. Consequently the levels of malonyl-CoA (needed as the starting material for FA synthesis) falls, which in turn reduces inhibition of carnitine acyl transferase I. Fatty acids enter the mitochondria for degradation to acetyl- CoA which is unable to pass through the Citric Acid cycle since its intermediates have been drawn off for gluconeogenesis. The resultant increase in acetyl-CoA increases ketone body formation at levels extrahepatic tissues cannot keep pace with. The ketone bodies in the blood lower the pH resulting in acidosis – can lead to coma and death. “ketosis” Lehninger, 2005 Figure 17.20

Metabolic Conversion of Ketone Bodies to acetyl CoA
Metabolism of ketone bodies in PERIPHERAL tissues. D-β-hydroxybutyrate is converted to acetoacetate and produces NADH. The acetoacetate product (also a ketone body) is converted to acetoacetyl-CoA and also yields “free” succinate. Two acetyl-CoA’s are produced by the action of thiolase. The acetyl-CoA’s are available for use in the peripheral tissues. In the peripheral tissues the ketone bodies produced in the liver are metabolized to get energy (NADH) The CoA is provide by succinyl CoA (Citric Acid cycle) which does cost the GTP that would otherwise be produced in the Citric acid cycle. NOTE: Ketone bodies permits the continued oxidation of fatty acids with the minimal oxidation of acetyl CoA. Liver has a limited amount of CoA and when most Co A tied up as acetyl-CoA β-oxidation slows - by exporting ketone bodies frees up CoA in the liver permitting continued fatty acid oxidation. Voet, et al., 2008 Figure 20-22

Fatty Acid Biosynthesis
Occurs mainly in liver and adipocytes (mammals) FA synthesis and degradation occur by two completely separate pathways When glucose is plentiful, large amounts of acetyl CoA are produced by glycolysis and can be used for fatty acid synthesis Glucose oxidation in the pentose phosphate pathway provides NADPH for FA synthesis The synthesis of fatty acids is an energy-consuming anabolic process that for mammals occurs mainly, but no exclusively in the live and adipocytes. We remember from the glycolysis that acetyl-CoA is produced. This is the 2-carbon fragment key to lipid biosynthesis.

Comparing Fatty Acid β-oxidation and Fatty Acid Biosynthesis
Different: locations acyl carrier group electron donor/acceptor hydration/dehydration Rx stereochemistry form of C2 units produced/donated Voet, Voet, and Pratt, 2013 Figure 20-23 The two opposing pathways involving fatty acid metabolism are shown in a side-by-side comparison. β-oxidation is shown on the left side of the panel, and, as we know, occurs (mainly) in the mitochondrion. Fatty acid synthesis, which occurs in the cytosol, is on the right side of the panel. We have seen this cellular separation of pathways before. The illustration at top is only for comparison purposes not an actual cellular view. Both pathways involve 2-carbon units. So to highlight fatty acid biosynthesis: Malonyl-CoA is the donor of the 2-carbon units NADPH (seen this before) is the electron donor The D isomer instead of the L isomer of β-hdydroxyacyl group is involved in the biosynthesis. Acyl carrier protein (ACP) is the acyl carrier. By being “different” each can be thermodynamically favorable. Each can be separately regulated. Berg, Tymoczko & Stryer 2012 Figure 22.2

Tricarboxylate Transport System
One of reasons needed: mitochondrial inner membrane impermeable to acetyl-CoA. When low ATP demand: acetyl-CoA oxidation and oxidative phosphorylation are low. Save mitochondrial acetyl-CoA as fat. Must transport acetyl-CoA out of the mitochondrion to store as fat. Transport acetyl-CoA out as citrate On the left side of the panel is the mitochondrion, the inner membrane is in the center, and the cytosol on the right. One of reasons the tricarboxylic acid carrier is needed in this case is that the mitochondrial inner membrane impermeable to acetyl-CoA. When the demand for ATP is low the rate of acetyl-CoA oxidation is also low and also oxidative phosphorylation. Not wanting to waste energy and material the mitochondrial acetyl-CoA can be saved as fat. In order to do this acetyl CoA is transported out of the mitochondrion as citrate using the tricarboxylate (3 carboxylate groups) transport system. The enzyme citrate synthase (where have we seen this before? TCA) conjugates acetyl CoA to oxaloacetate to form citrate a tricarboxylic acid. The enzyme citrate lyase in the cytosol is responsible for releasing acetyl CoA into the cytosol in an ATP consuming reaction. Voet, Voet, and Pratt, Figure 20-24

Biosythesis of Fatty Acids: Reaction Sequence Overview
7 reactions located in cytosol Palmitic acid is a main product Reactions are endergonic & reductive ATP used for energy, Reduced electron carries, e.g., NADPH, for reductive power Mammalian Fatty Acid Synthase: Enzyme Structure In all organisms the long chain fatty acids are assembled in a repetitive 4-step sequence, a system collectively referred to as fatty acid synthase. In animals it is a 534kD multifunction enzyme (with two identical polypeptide chains). Note the enzyme abbreviations on the left panel and the same abbreviation in the lower right panel. An illustration of the fatty acid synthase complex is shown on the right side. The seven reactions in animals are localized to six discrete active sites on the complex. The take-home message is that these reactions are located together on a large, multi-activesite complex. Not all of the domains represented by the illustration on the bottom right have enzyme activity. Bacteria have soluble enzymes, other organisms also have variations in fatty acid biosynthesis occurs. Since it is a dimer it can synthesize two fatty acids simultaneously. Take-home message mammalian fatty acid synthesis occurs in a dimer super complex. Fatty acid biosynthesis generally proceeds at a low rate in well-nourished individuals. On malignancies and in certain tissues fatty acid synthesis proceeds at a high rate due to high levels of fatty acid synthase expression. There is some thought that in these cases inhibitors of fatty acid synthase may have a role as anticancer agents. Voet, Voet, and Pratt, 2013 Figure 20-26 Voet, Voet, and Pratt, 2013 Figure 20-27a

Acetyl CoA Carboxylase
1st committed step in fatty acid biosynthesis – irreversible process Rate controlling step – allosteric & hormonal control This reaction, which is the first committed step in the synthesis of fatty acids, is comprised of two steps. The first step is a CO2 activation and the second a carboxylation. (Similar to the pyruvate carboxylase reaction we have seen before  in gluconeogenesis) Allosteric control: citrate stimulates long chain fatty acid feedback inhibitors Phosphorylation of Ser79 inactivates the enzyme – This is done by AMP dependent protein kinase (AMPK) which is part of the cAMP-independent pathway. Hormonal control: glucagon & epinephrine which act via protein kinase A (PKA) promote Ser79 phosphorylation. Insulin stimulates dephosphorylation of acetyl-CoA carboxylase which activates the enzyme. Mammals  two isoforms ACC1 in adipose tissue Tissues, such as liver, that both synthesize and oxidize fatty acids contain both isoforms. ACC2 in tissues that oxidize FA but do not synthesize them. E.g. heart muscle. Regulatory role – malonyl CoA product strongly inhibits mitochondrial import of fatty acyl-CoA for FA oxidation – major control point for the process. Voet, Voet, and Pratt, 2013 Page 682

OXYGENASES Oxygenases: catalyze oxidative reactions in which oxygen atoms are directly incorporated into the substrate molecule forming a new hydroxyl or carboxyl group. Dioxygenases: both oxygen atoms are incorporated into the organic molecule Monooxygenases: (more abundant & complex actions) one oxygen incorporated into the organic molecule and the other reduced to water. They require two substrates to serve as reductants. Also called hydroxylases since in most reactions the main substrate becomes hydroxylated. Sometime called mixed-function oxygenases – oxidize two different subsrates simultaneously. Mixed-Function Oxidases (monooxygenases), e.g. cytochrome P-450 (most numerous & complex monooxygenases). Different classes of monooxygenases depending on nature of cosubstrate: FMNH2 or FADH2 used by some NADH or NADPH used by some α-ketoglutarate used by some DO I NEED THIS? WHERE TO PUT? IF NEEDED MUST ADD TEXT HERE FROM LEHNINGER Enzymes that carry out redox reactions in which molecular oxygen is a participant. In our study of enzymes we studied various functional group :OTHLIL. Oxidase is a general name for enzymes that catalyze oxidations where molecular oxygen is the electron acceptor but oxygen atoms themselves are not found in the oxidized product. Oxygenases catalyze reactions in which oxygen atoms do appear in the oxidized product. We can define several classes of oxygenases in this slide. Oxygenases are involved in a number of lipid biosynthetic pathways. We will look at several types of oxygenases briefly here. Cytochrome P-450s, a class of many enzymes, are involved in the oxidation of molecules in biosynthetic pathways and the oxidation of xenobiotics. Lehninger 2005 Chap 21 p 798

Biosythesis of Fatty Acids: Reaction Sequence I
Acyl Carrier Protein (ACP) esterified to acyl group instead of CoA during synthesis of fatty acid. Priming Reaction: Rx1: carried out by malonyl/acetyl CoA-ACP transacylase [MAT]. Initiated with malonyl-CoA. Transfers malonyl CoA to ACP Likewise acetyl-CoA transferred to ACP for cyclic additions. To start initial synthesis of a fatty acid Where carbanion will form Where carbanion will attack Reaction #1 The synthesis of fatty acids begins with the formation of malonyl CoA –right side of figure. Subsequently malonyl/acetyl-CoA-ACP transacylase transfers an acetyl group from CoA to acyl carrier protein. Acyl carrier protein, used in fatty acid biosynthesis, remains attached to the growing fatty acid. For the synthesis of a fatty acid the enzyme transfers either a malonyl CoA To “Prime” (right side 1B) or an acetyl group “to extend” (left side of figure designated 1A) to Acyl carrier protein, abbreviated ACP. Reaction #2 The role of β-ketoacyl-ACP synthase (KS) “condensing enzyme” first STEP is to transfer the acetyl group from ACP to enzyme’s cysteine and then malonyl-CoA is decarboxylated with the resultant carbanion attacking the acetyl thioester to form a 4-carbon acetoacetyl-ACP. Why add CO2 and then cleave it off from malonyl-CoA? - The reaction uses the decarboxylation to energetically drive itself in the forward direction, i.e. make the reactions thermodynamically favorable. First “Cycle” Reaction: Rx2: β-ketoacyl-ACP synthase [KS] “condensing enzyme” Voet, Voet, and Pratt, 2013 Figure part 1

Biosythesis of Fatty Acids: Reaction Sequence II
Reload ACP with malonyl group each cycle to be able to add 2-carbon fragment Biosythesis of Fatty Acids: Reaction Sequence II Rx3: β-ketoacyl-ACP reductase [KR] use NADPH’s reductive power to convert a carbonyl to a hydroxyl group Rx4: β-hydroxyacyl-ACP dehydrase [DH] removes water to form a double bond beta to the carbonyl group. Reaction #3 Continuing with the sequence of fatty acid synthesis, the third reaction involves the reduction of the C-3 carbonyl group to form a hydroxyl group. The reaction is catalyzed by β-ketoacyl-ACP reductase and uses the reducing power of NADPH. The reaction forms D-β-hydroxybutyryl-ACP Reaction #4 Still attached to ACP, the fourth reaction catalyzed by β-hydroxyacyl-ACP dehydrase removes a water from the C2 and C3 carbons to yield a double bond. (Why a carbonyl was converted to a hydroxyl group.) The product is α,β-trans-Butenoyl-ACP. Reaction #5 The fifth reaction catalyzed by enoyl-ACP reductase, completes the first run through the “cycle” where the double bond is reduced using NADPH to yield butyrl-ACP (first time). This product will go through the cycle 6 more times starting at β-ketoacyl-ACP synthase and is elongated by 2 carbons each time till it is 16-carbons long (palmitic acid). Rx5: enoyl-ACP [ER] reductase uses the NADPH’s reductive power to reduce the double bond. Voet, Voet, and Pratt, 2013 Figure part 2

Biosythesis of Fatty Acids: Reaction Sequence III
Rx6: palmitoyl thioesterase [TE] After completion of the cyclic “growth” the mature/complete fatty acid is removed from ACP by hydrolyzing the thioester bond. Overall process: 8 acetyl-CoA + 7 ATP + 14 NADPH +14 H+ → palmitate + 8 CoA + 7 ADP + 7Pi + 14 NADP+ + 6H2O Still not well mechanistically understood but chain elongation generally stops at 16 carbons and free palmitate is released from ACP by the enzyme palmitoyl thioesterase (in the multifunctional fatty acid synthase). It has been found in nonphotosynthetic eukaryotes XETYL-Coa produced in the mitochondrion must be transported (part of citrate) to the cytosol and consumes 2 additional ATP per acetyl-CoA to get it to the cytosol for fatty acid synthesis. Voet, Voet, and Pratt, 2013 Figure part 3

Fatty Acid Elongation Mitochondrial
FA elongase system present in mito & ER – (but are different) Mito elongation a reverse of β-oxidation (but last Rx uses NADPH) Palmitate precursor of longer chain fatty acids Stearate (C18) a major product As we have seen fatty acid synthase’s main product is palmitic acid, a 16 carbon molecule. So how are longer fatty acids produced? There are systems in the mitochondrion and the ER. Since FA biosynthesis occurs in the cytosol, palmitate must be transported into the mitochondrion across the inner membrane in order for mitochondrial-based elongation to occur. The panel on the left shows the mitochondrial elongation system. Elongases. The more active ER elongation system extends the 16-carbon chain of palmitoyl-CoA forming steroyl-CoA. Coenzyme A functions as the acyl carrier rather than ACP in the ER and the enzymes, located on the cytoplasmic face of the membrane, are different but the mechanism is otherwise identical to the malonyl-CoA based synthesis of palmitate. Voet, Voet, and Pratt, 2013 Figure 20-28

Terminal Desaturases to produce Unsaturated Fatty Acids
Palmitate and stearate serve as precursors for the two most common animal monounsaturated fatty acids: palmitoleate (C 16:1 ∆9) and oleate (C 18:1 ∆9) Fatty acyl-CoA desaturase Mixed function oxidases needed forfatty acid desaturation. Voet, Voet, and Pratt, 2013 Page 689 Electron Transfer in Desaturation Fatty acid synthase produces a saturated fatty acid, palmitic acid, but many fatty acids are unsaturated. Remember, e.g., how membrane phospholipids contain one saturated and one unsaturated fatty acid. So how are unsaturated fatty acids produced? Palmitate and stearate serve as precursors for the two most common animal monounsaturated fatty acids: palmitoleate (C 16:1 ∆9) and oleate (C 18:1 ∆9) that have a cis double bond between carbons 9 and 10. Fatty-acyl-CoA desaturase, a mixed-function oxidase, oxidizes the fatty acid to introduce a double bond. In the lower panel the fatty acid and NADPH simultaneously undergo two electron oxidations with electrons flowing to cytochrome b5 and a flavoprotein cyotchrome b5 reductase both located in the smooth ER along with the enzyme. Note: Plants have greater ability to insert double bonds than animals. Thus those plant polyunsaturated fatty acids are Essential Fatty Acids and must be in an animal’s diet Lehninger, 2005 Figure 21.13

Mammalian Terminal Desaturases
Four terminal desaturases of broad chain-length specificity ∆9-, ∆6-, ∆5-, and ∆4- fatty acyl-CoA desaturases Generic reaction below right: For the desaturases to function “x” must be ≥ 5 and (CH2)x can contain one or more double bonds but (CH2)y portion is always saturated. Double bonds inserted between existing double bonds in (CH2)x portion and CoA so that the new double bond is 3-carbons closer to the CoA, i.e. not conjugated. Note: In animals never beyond position C9. What is the message to be taken from this? Double bonds can only be inserted at certain positions, not next to double bonds in mammalian system. Fatty acids that do not conform to these rules must be obtained from other sources in the diet, e.g. plants and are termed essential fatty acids. Voet, Voet, and Pratt, 2013 Page 689

Routes of Synthesis of Other Fatty Acids
Mammals cannot convert oleate to linoleate or α-linolenate - hence they are ESSENTIAL FATTY ACIDS in the mammalian diet NOTE: Linoleic acid (∆9,12 –octadecadienoic acid) is a required precursor for prostaglandin and other eicosanoids and is an essential fatty acid. There are other fatty acids that are made from the biosynthesized palmitate. An overview of this is present in this diagram. You do not need to memorize this diagram There is a problem for animals since palmitic acid is the shortest fatty acid produced by mammals a ∆12 double bond in linoleic acid is not possible to create. Lehninger, 2005 Figure 21.12

Lipid Metabolism: Summary
In individuals that are well nourished fatty acid synthesis functions at a low rate. Whereas in certain tissues, particularly malignancies, fatty acid synthase is expressed at high levels and fatty acids are produced at a high rate. Your text speculated that inhibitors of fatty acid synthase might serve as anticancer agents. (p.687). Voet, Voet, and Pratt, 2013 Figure 20-30

Fatty Acid Metabolism Regulation
Fatty acid oxidation and synthesis regulated by hormones and cellular factors It is the case in mammals that glycogen and triacylglycerols are primary fuels supplying energy to many processes. They are synthesized when nutrients are abundant to have for future use. Since these fuels are needed by the whole organism the bloodstream is the transport system interconnecting the tissues and organs.

Fatty Acid Metabolism: Regulation Sites
Fatty acid oxidation is largely regulated by fatty acids concentration in the blood.- controlled by rate of triglyceride hydrolysis in adipose tissue via hormone-sensitive triacylglycerol lipase (lower figure) . Glucagon/Insulin ratio determines the rate and direction of fatty acid metabolism. During FA biosynthesis malonyl-CoA inhibits CPT1 (mito)– reducing FA oxidation. AMPK activated by AMP and inhibited by ATP. When active it phosphorylates ACC inactivating ACC thus decreasing malonyl-CoA concentration. Voet, Voet, and Pratt, 2013 Figure 20-31 Citrate role: Central to diverting cellular metabolism from metabolic fuel oxidation to storage as fatty acids. When mitochondrial acetyl-CoA & ATP increase citrate is transported out of the mitochondrion. Becomes a precursor for acetyl-CoA & allosteric activator for acetyl-CoA carboxylase; inhibits phosphofructokinase-1 reducing glycolysis. The top figure is a liver cell and the bottom figure an adipose cell. They are “connected” via the circulatory system. In vertebrates, palmitoyl-CoA, the product of FA synthesis is an inhibitor of acetyl-CoA carboxylase (top figure left side) and citrate is an allosteric activator increasing Vmax. Glucagon, epinephrine, norepinephrine - indicators of metabolic energy demand – increase adipose tissue cAMP. The molecule cAMP allosterically activates phosphokinase A which subsequently phosphorylates certain enzymes – this activates hormone-sensitive lipase which give rise to higher FA levels in the blood – this increases β-oxidation in other tissues. Insulin, which indicates high blood glucose levels, stimulates glycogen and triacylglycerol synthesis. Insulin also activates acetyl-Co A carboxylase ACC2 Fatty Acid oxidation can also be inhibited, when FA synthesis is stimulated, by malonyl CoA inhibiting CPT1 – RED symbol by mito on right side of liver cell schematic. Special case of heart – no FA biosynthesis –isoform of acetyl-CoA carboxylase, ACC2 – believed it functions to synthesis of malonyl-CoA to regulate heart’s FA oxidation. AMP-dependent protein kinase (AMPK) whose phosphorylation of ACC inactivates it. AMPK activated by AMP and inhibited by ATP. When AMPK is activated it phosphorylates ACC which inactivates it and decreases malonyl-CoA concentrations decreasing FA biosynthesis in adipose tissue while FA oxidation increases in muscle (yield more ATP in muscle) Lehninger, 2005 Figure 17.12

Hormonal Regulation Summarized
Fed state: Insulin (levels increase) Inhibits hydrolysis of stored Triglycerides Stimulates formation of malonyl CoA, which inhibits CPT I Fatty acids remain in cytosol (Fatty acid oxidation enzymes are in the mitochondria) Fasted state: Epinephrine and glucagon increase (insulin decreases) Epinephrine activates lipase enzyme to produce more fatty acids Glucagon inactivates malonyl CoA synthesis enzyme (leads to increased transport of fatty acids into mitochondria and the b-oxidation pathway) As with other pathways we have studied, hormones effect fatty acid synthesis tying it into the needs of the body – i.e. integrating with energy demands and availability. Horton et al Chapter 16

Gene Expression in Long-Term Regulation of Fatty Acid Metabolism
Stimulation by insulin Adipose tissue lipoprotein lipase amount increased by insulin fatty acid synthesis fatty acid storage in adipocytes Inhibition by starvation acetyl-CoA carboxylase synthesis and fatty acid synthase amounts decreased lipoprotein lipase amount decreased Gene expression Long Term regulation of fatty acid metabolism occurs at the level of gene regulation. Short-term regulation can involve such factors as substrate availability, allosteric effects on enzymes, covalent modification of enzymes – these occur on a time frame of minutes. What is meant by long term is responses that requires days or hours and is the result of changes in the amount of enzymes present in the pathway. The abundance of glucose which gives rise to higher insulin levels promotes fatty acid synthesis and adipocyte fatty acid storage. In the figure on the right (shown previously) the top image is of a liver cell and the bottom one an adipocyte. Here the blue arrows in the diagram itself are the points of long-term regulation – these are emphasized by the two large added green arrows in this slide. Gene expression Voet, Voet, and Pratt, 2013 Figure 20-31

Triacylglycerol Biosynthesis
Synthesized from glycerol & fatty acyl-CoA esters In mitochondrion and ER Glycerol-3-P comes from DHAP using a single step In peroxisomes from DHAP in a 3-step sequence Glyceroneogensis (part of gluconeogenesis) necessary during starvation to provide glycerol. Triacylglycerol 1st stage is the acylation of the two free hydroxyl groups of glycerol-3-P by fatty acyl-CoAs to yield diacylglycerol-3-P. Diacylglycerols converted to triacylglycerol by transesterification with 3rd fatty acyl-CoA ER & Peroxisomes Mito & ER Humans can synthesize and store large amount of triacylglycerols (triglycerides) – have highest energy content of stored nutrients: > 38 kJ/g. Vast majority of Glycerol-3-P is derived from DHAP via the cytosolic enzyme glycero-3-P dehydrogenase in liver and kidney. – small amount from glycerol kinase. Fatty acyl-CoAs formed from fatty acids by acyl-CoA synthases. Rate of triacylglycerol biosynthesis is affected by several hormones. Insulin promotes the conversion of carbohydrate to triacylglycerol Voet, Voet, and Pratt, 2013 Figure 20-29

Regulation of Triacylglycerol and Glyceroneogenesis
Glucocorticoid hormones stimulate glyceroneogenesis & gluconeogenesis in liver while suppressing glyceroneogenesis in adipose tissue. Insulin stimulates conversion of dietary carbohydrates and proteins to fat. Diabetics (low insulin secretion or action) have diminished FA synthesis and acetyl CoA from catabolism shunted to ketone body production. Thiazolidinediones used to treat type-2 diabetes- activate a nuclear receptor PPARγ which induces activity of PEP carboxykinase; the latter largely controls the flux through triacylglycerol cycle in liver –limits rate of glyceroneogensis & gluconeogenesis Lehninger, 2013 Figure Lehninger, 2013 Figure

Phospholipid & Sphingolipid Syntheses
General Structures Lipids synthesized mostly on cytosolic side of ER 1,2 diacylglycerol and phosphatidic acid are the precursors of most glycerophospholipids Palmitoyl-CoA and serine are the precursors of sphingolipids. For our purposes in this course we will not delve into the details of glycerophospholipid syntheses or of sphingolipids. Herein are only the most general details. The polar headgroups are linked to glycerol’s C3 via a phosphodiester bond. In mammals the headgroups are activated by phosphorylation of a hydroxyl group. Most sphingolipids are the sphingoglycolipid type with their polar headgroup consisting of carbohydrate units. There are the cerebrosides which are ceramide monosaccharides. There are gangliosides that have sialic acid-containing ceramide oligosaccharides . Voet, Voet, and Pratt, 2013 Figure 20-32

Plasmogen & Alkylacylglycerophospholipid Structures (Eukaryotic membrane lipids)
Also included in the glycerophospholipids are the plasmalogen and the alkylacylglycerophospholipids. Remember that the plasmalogens contain a hydrocarbon chain linked to glycerol C1 via a vinyl ether linkage (Lipids lecture), whereas in akylacylglycerophospholipids there is an ether linkage at the glycerol C1 with an alkyl substituent. Some 20% of mammalian glycerophospholipids are plasmalogens, but there is variation among species AND tissues. Voet, Voet, and Pratt, 2013 Page 697

Sphingolipids & C20 Lipid Syntheses
Most are glycolipids (carbohydrate polar headgroups) Includes cerebrosides and gangliosides (Lipid Lectures) Built from palmitoyl-CoA and serine C20-based lipids Prostaglandins, leukotrienes, thromboxane, lipoxin (Lipid lectures) Derivatives of C20 compounds such as arachidonic acid Berg, Tymoczko & Stryer 2012 Figure 22.32

Ceramide Biosynthesis
4 Steps Synthesized by the attachment of carbohydrates units to the C1-hydroxyl of ceramide 3-Ketosphinganine synthase – condenses palmitoyl-CoA with serine 2-Ketosphinganine reductase – uses NADPH to reduce keto group on 3-ketosphinganine Acyl-CoA transferase - transfers an acyl group from acyl-CoA to form an amide bond wit sphinganine’s 2-amino group Dihydroceramide dehydrogenase – uses an FAD-dependent oxidation to convert dihydroceramide to ceramide DELETE THIS? CANNOT HAVE THEM MEMORIZING ALL THESE ENZYMES Voet, Voet, and Pratt, 2013 Figure 20-35

Sphingomyelin Structure
An important structural lipid in nerve cell membranes Formed from the reaction of phosphatidylcholine with the C1-OH group of N-acylsphingnosine. transfer of choline group from the phospholipid to the sphingomyelin. Voet, Voet, and Pratt, Page 698

Eicosanoids Phospholipase A2, reacting to hormonal signals, cleaves arachidonic acid-containing membrane phospholipids to release arachidonic acid which is the precursor to eicosanoids. Prostaglandins – e.g. PGE C-8 and C-12 of arachidonate (pH 7 form) are joined to form the characteristic 5-membered ring. The name derives from the prostrate gland from which they were first isolated. Two prostaglandin groups were originally defined: PGE (ether soluble) PGF (phosphate buffer soluble) Each group contains numerous subtypes indicated by a numerical subscript. Prostaglandins have an array of functions: some stimulate uterine smooth muscle contraction during menstruation and labor others effect blood flow to specific organs wake-sleep cycle responsiveness of certain tissues to glucagon and epinephrine third group elevate body temperature and cause inflammation and pain. Thromboxane A2 - C-8 and C-12 are joined plus an oxygen atom is added to form the 6-membered ring. Produced by platelets and in the formation of blood clots and reduction of blood flow to the site of a clot. Leukotriene A4 – has a series of three conjugated double bonds. First found in leukocytes. They function as powerful biological signals. Overproduction of leukotrienes causes asthmatic attacks. Strong contraction of the lung’s smooth muscle during anaphylactic shock. Lehninger Figure 10.18b

Prostaglandin H2 Synthase Rx
Eicosanoid Synthesis Prostaglandin H2 Synthase Rx Eicosanoids: * Potent short range signaling molecules * Hormonal or other stimuli trigger phospholipase A2 * phospholipase A2 releases arachidonic acid from middle carbon of membrane phospholipid glycerol Formation of prostaglandin H2 (PGH2) first step (precursor of many prostaglandins and thromboxanes) 1st step catalyzed by cyclooxygenase (COX) prostaglandin H2 synthase inserts molecular O2 to form PGG2 2nd step catalyzed by COX’s peroxidase activity to form PGH2 COX enzymes affected by NSAIDS! Eicosanoids are a family of very potent biological signaling molecules. Act over short-range affecting tissues near the cells that produce them. Hormonal or other stimuli trigger phospholipase A2, present in most mammalian cells, which interacts with membrane phospholipids to release arachidonic acid (C20) from the middle carbon of the glycerol backbone. Enzymes in the smooth ER convert arachidonate to prostaglandins starting with prostaglandin H2 Voet, Voet, and Pratt, 2013 Figure 20-36

Aspirin & Two NSAID’s Inhibition of Cyclooxygenase Rx in Prostaglandin Synthesis
Aspirin – Acetylates a specific Ser residue of prostaglandin H2 synthase which blocks arachidonate from the active site. (Effects on heart attacks and strokes too.) NSAIDS – bind noncovalently to the enzyme and also block the enzyme active site: e.g. acetaminophen & ibuprofen Low dose, daily ~81 mg aspirin has been found to significantly reduce the incidence of heart attack (What is it?) and stroke (what is it?) by selectively inhibiting platelet aggregation and, therefore, blood clot formation. Platelets are enucleated cells that have an approximate 10-day lifetime in the circulation cannot synthesize more enzyme, whereas vascular epithelial cells have a nucleus and are exposed to essentially an even lower aspirin dose can synthesize the enzyme. Nonsteroidal anti-inflammatory drugs (NSAIDS) include some of the most widely used drugs such as acetaminophen (Advil) and Ibuprofen (Tylenol). Interestingly aspirin and ibuprofen are found to be relatively nonspecific and can have side effects such as gastrointestinal ulceration (and bleeding). COX COX-2 Acetaminophen, one of the most commonly used analgesics/antipyretic, in acutality does not bind well to COX-1 and COX-2 (also is not an anti-inflammatory agent). Actually effects “COX-3” which is expressed at high levels in the CNS – target of drugs deceasing fever & pain Voet, Voet, and Pratt, 2013 Page 699a Lehninger, 2005 Box 21.2 Figure 2a

COX Isoforms and NSAIDS
Two NSAID COX-2 Inhibitors of Prostaglandin H2 Synthesis (Vioxx and Celebrex) Chemists designed inhibitors to COX-2 called coxibs based on 3-D structures of COX-1 & COX Analgesics Were considered important for treating inflammatory diseases viz. arthritis Withdrawn 2004 Use more restricted now Bextra withdrawn ~2004 Have not well understood cardiac side effects. COX-1: constitutively expressed in most mammalian tissues to maintain prostaglandin synthesis needed to maintain homeostasis in organs and tissues. COX-2: expressed only in certain tissues in response to certain stimuli – responsible for elevated PG levels that cause inflammation. COX-3: high expression levels in CNS, re: acetaminophen These are two of the drugs that were marketed in the 1990’s with great hopes of reducing the pain from inflammatory arthritis. These coxibes were designed by pharmaceutical chemists to interact with the active site of COX-2 and not COX-1 by taking advantage of the somewhat (20%) large active site of COX-2. Voet, Voet, and Pratt, 2013 Page 699b

Prostaglandins , prostacyclins, thromboxanes and leukotrienes are termed eicosanoids as they contain 20 carbon atoms. They are built/modified from the 0 carbon acid arachidonate, shown in yellow highlight. They function as local hormones because they are short-lived. Theses agents alter the activity of the cells in which they are synthesized and neighboring cells acting via 7TM receptors. Interestingly their effects can vary from one cell type to another in contrast to such global hormones insulin and glucagon. Prostaglandins stimulate inflammation, regulate blood flow to particular organs, control ion transport, modulate synaptic transmission and induce sleep. Berg, Tymoczko & Stryer 2012 Figure 22.32

THROMBOXANES Thromboxane synthase (in platelets) coverts PGH2 to thromboxane A2. From thromboxane A2 others are produced. Thromboxanes contain a ring of 5 or 6 atoms Thromboxanes induce blood vesicle constriction and platelet aggregation. Regular low doses of aspirin reduce thromboxane production – reduces heart attack and stroke risk The name comes from the fact these molecules have a thrombus forming potential. Thromboxane A synthase in present in the ER of platelets and lungs. Enzymes catalyzes the rapid conversion of PGH2 to TXA2. TXA2 ‘s half-life in very short in water ~1 min as it is rapidly converted to the inactive thromboxane B2. [Devlin 1997, p435].

Arachidonate to Leukotrienes (Brief Overview)
Leukotrienes are linear compounds. Their synthesis begins with the action of several lipooxygenases that catalyze molecular oxygen incorporation. Enzymes are found in leukocytes, heart, brain, ling and spleen are mixed function oxidases of the cytochrome P450 family. (Not inhibited by aspirin or NSAIDS.) Arachidonate is the immediate 20-C precursor molecule shown in the top center of the illustration. Lipoxygenase, which adds molecule oxygen, form the intermediate 12-HPETE which is subsequently converted to other leukotrienes. Alternatively lipoxygenase forms 5-HPETE which through a series of steps is converted to leukotriene A4. Know lipoxygenase converts arachidonate to leukotrienes buy adding oxygen to LINEAR molecules. Lehninger, 2005 Figure 21.16

Spingolipid Degradations and Lipid Storage Diseases: Diagram
What happens when sphingoglycolipids are not properly degraded? We call those diseases – know the names but not the specific enzymes and pathway. IN a series of lysosome-based hydrolytic reactions the degradation of Ganglioside GM1, globoside, and sulfatide are shown. A hereditary, i.e. genetic, defect in one of these enzymes results in sphingolipid storage disease where the substrate for the enzyme accumulates Voet, Voet, and Pratt, 2013 Box 20-4 figure 1

Cholesterol Metabolism
Despite falling into popular disfavor, cholesterol has a number of important functions and is synthesized in the body naturally. We have seen it function as a component of membranes in earlier lectures. Cholesterol is a precursor of the steroidal hormones. Cholesterol is also converted to bile acids in the liver which are used in the digestive process for emulsifying fats. Cholesterol not recycled from the bile acids in the liver is metabolized by the intestinal microorganisms and excreted! THIS IS THE ONLY ROUTE FOR THE EXCRETION OF CHOLESTEROL “In a healthy organism, an intricate balance is maintained between biosynthesis, utilization, and transport of cholesterol, keeping harmful deposition to a minimum.” Voet, Voet and Pratt, 2013 p. 701

Summary of Cholesterol Biosynthesis
Made from acetyl-CoA There are four major stages: Condensation of 3 acetate units to form mevalonate. Conversion of mevalonate to activated isoprene units. Polymerization of six 5-carbon isoprene units to form 30-carbon linear squalene molecule. Cyclization of squalene to form the four rings of the steroid nucleus with additional modifications Lehninger, 2005 Figure 21.32 Cholesterol is synthesized in 4 major steps using acetate as its precursor. In the upper left corner the structure of the planar steroid molecule, cholesterol, is shown with the red carbon indicating where the carboxyl carbon if acetate winds up. Be familiar with these 4 steps as you will need to understand where and how various cholesterol lowering pharmaceuticals function. Lehninger, 2005 Figure 21.33

1. Synthesis Mevalonate from acetyl-CoA
Two molecules of acetyl-CoA are condensed to form acetoacetyl-CoA A third molecule of acetyl-CoA is condensed with acetoacetyl-CoA to form β-hydroxy-β-methylglutaryl-CoA (HMG-CoA) HMG-CoA: a key cholesterol precursor. We previously saw how cholesterol was comprised of multiple acetate molecules. Considering the first major stage of cholesterol biosynthesis, one starts with acetyl-CoA. The cytosolic enzymes thiolase and HMG-CoA generate the HMG-CoA for cholesterol biosynthesis. (In liver the mitochondrial enzymes are used to provide HMG-CoA for ketone body synthesis.) The third reaction in stage I is a committed step: the reduction of HMG-CoA to mevalonate involving two electrons each from two NADPH molecules. The reduction of MHG-CoA to mevalonate is a committed step that utilizes the electrons from 2 NADPH molecules.. Major regulation point in cholesterol synthesis!. Lehninger, 2005 Figure 21.34

2. Conversion of mevalonate to activated isoprene units Isopentenylpyrophosphate formation from HMG-CoA In stage 2 three Pi groups are transferred from ATPs. The 1st phosphorylation of the newly added OH group is performed by mevalonate-5-phosphotransferase. Phosphomevalonate kinase converts the added Pi to PPi. In the second major stage of cholesterol biosynthesis ATP is used to donate phosphates to create activated isoprene: isopentenylpyrophosphate. The mevalonate molecule is show in green, with the hydroxyl group undergoing undergoing to the 1st phosphorylation by mevalonate-5-transferase in RED. Note also the carboxylate anion in blue. Phosphomevalonate kinase is tasked with adding a second phosphate to form a pyrophosphate. The 3rd phosphate is used to aid in the decarboxylation reaction carried out by pyrophosphomevalonate decarboxylase. The small illustration shows the structure of an isoprene carbon skeleton. An ATP-dependent decarboxylation occurs via pyrophosphomevalonate decarboxylase yielding ∆3-isopentyl pyrophosphate (an activated isoprene). Isoprene carbon skeleton Voet, Voet and Pratt, 2013 Figure 20-37 Voet, Voet, and Pratt, 2013 Page 701

Pyrophomevalonate decarboxylase reaction in Cholesterol intermeidate biosynthesis
This illustration details the last reaction in stage 2 from the previous slide showing the reaction’s electron movements. The Hydroxyl oxygen’s electrons engage in a nucleophilic attack on the terminal phosphorus of ATP (electron poor) cleaving the ATP while the carboxyl carbon’s electrons are drawn to the hydroxyl carbon facilitating the decarboxylation. Voet, Voet, and Pratt, 2013 Page 702a

Intermediate Reactions in Squalene Biosynthesis
The conversion of a portion of isopentenyl pyrophosphate to dimethylallyl pyrophosphate provides the second activated isoprene for cholesterol biosynthesis. Preparation for stage 3. Voet, Voet, and Pratt, 2013 Page 702b

3. Formation of Squalene from Isopentenyl pyrophosphate & dimethylallyl phosphate
Activated isoprenes Head has PPi 1st head-to tail condensation to produce C10 geranyl pyrophosphate 2nd head-to tail condensation of geranyl pyrophosphate with isopentenyl phosphate to form C15 farnesyl pyrophosphate Head to Head condensation of two farnesyl pyrophosphates by squalene synthetase to form squalene. The first reaction in stage 4 involves the head to tail condensation of dimethylallyl pyrophosphate and isopentyl pyrophosphate, the activated isoprenes, catalyzed by prenyltransferase to form geranyl pyrophosphate, a C10 compound. The second reaction forms the C15 compound, farnesyl pyrophosphate from geranyl pyrophosphate and isopentneyl pyrophosphate in a head to tail condensation. Note that for mammals farnesyl pyrophosphate is the precursor molecule for other isoprenoid compounds such as ubiquinone and the isoprenoid tails of lipid-linked membrane proteins (Membranes lecture). The third reaction via squalene synthase is the head to head condensation of two farnesyl pyrophosphates forms squalene. Voet, Voet, and Pratt, 2013 Figure 20-38

4. Conversion of Squalene to the Four Ring Steroid Nucleus
Squalene Epoxidase Reaction Voet, Voet, and Pratt, 2013 Figure 20-39 In the left side panel we see in an oxidation reaction utilizing NADPH squalene oxidase converts squalene to 2,3 oxidosqualene with two hydrogens combining with one of the molecular oxygens to form water. In the panel on the right oxidosqualene cyclase cyclizes 2,3-oxidosqualene in a first step to a protosterol cation by protonating the epoxide oxygen added by the previous enzyme. The resultant electron-deficient center migration in turn drive a series of cyclizations. This is followed by a series of methyl and hydride migrations and then the elimination of the C9 proton of the sterol to form a double bond to yield lanosterol. Voet, Voet, and Pratt, Figure 20-40

Lanosterol Conversion to Cholesterol in 19 (simple?) Steps
Conversion of Lanosterol to Cholesterol acyl-CoA:cholesterol acyltransferase The really good news is we will not go into the 19 reactions to get from lanosterol to cholesterol – even your textbook chicken out on this one. In the lower right panel the structure shown is a cholesterol ester (why ester?) These highly hydrophobic compounds are formed from cholesterol in the liver by acyl-CoA:cholesterol acyltransferase (ACAT !) and are transported throughout the body in LIPOPROTEIN COMPLEXES. Transported from the liver in lipoprotein complexes Voet, Voet, and Pratt, 2013 Page 704a Voet, Voet, and Pratt, 2013 Page 704b

Overview of Isoprenoid Biosynthesis
Just to provide some perspective on the diverse fate of the “activated” precursor molecule ∆3 -isopentenyl pyrophosphate. Included in this are vitamins A, E, & K, dolichols (soluble lipid carriers in complex polysaccharide synthesis, ubiquinone. Plants and insects also use this molecule for other syntheses specific to their needs. Lehninger, 2005 Figure 21.48

Regulation of Cholesterol Synthesis
HMG-CoA reductase – rate limiting step for cholesterol biosynthesis – main regulatory site of pathway!! -short-term regulation: competitive inhibition, allosteric effects, reversible covalent modification by phosphorylation hormones effecting short-term phosphorylation: Glucagon stimulates phosphorylation (inactivation) Insulin promotes dephosphoylation (activation) -long-term regulation: feedback control of the amount of enzyme present in the cell “transcriptional regulation” *PRIMARY regulatory mechanism* The enzyme is less active when phosphorylated. Phosphorylation by AMP-dependent protein kinase (AMPK). This enzyme also inactivates acetyl-CoA carboxylase. Thought that this is a way of conserving energy when ATP levels fall and AMP levels rise by inhibiting biosynthetic pathways. Long-term regulation can vary the concentration of the HMG-CoA Reductase enzyme up to 200-fold. This is a result of an increase in its synthesis and a decrease in its degradation. We will discuss the role of statin drugs shortly. STATINS INHIBIT!

Cholesterol-mediated Proteolytic Activation of SREBP
Sterol Regulatory Element-Binding Proteins Cholesterol’s cellular concentration effects HMG-CoA reductase transcription. HMG-CoA reductase gene + 20 other genes encoding enzymes mediate the uptake and synthesis of cholesterol & unsaturated fatty acids. [Cholesterol] =high [Cholesterol] high: SREBP-SCAP remains in ER [Cholesterol] low: SCAP “escorts” SREBP to the Golgi via COPII-coated vesicles. In Golgi SREBP cleaved by proteases S1P and S2P – SREBP N-terminal domain goes to nucleus and interact with target genes’ SREs [Cholesterol] = low These 20 genes affected by cholesterol concentration all contain a specific regulatory sequence called a sterol regulatory element, SRE. SCAP has two domains: an N-terminal domain designated the sterol-sensing domain that is the interaction site with sterols. The C-terminal domain has 5 copies of a WD repeat motif that is involved in protein-protein interactions - it interacts with SREBP’s regulatory domain. When cholesterol is depleted in the ER membrane SCAP changes conformation and escorts SREBP to the Golgi Apparatus via COPII coated vesicles. SREBP is cleaved by proteases in the GOLGI and the cleaved N-terminal domain travels to the nucleus binding to the genes SREs causing an increase in transcription of those genes. Voet, Voet, and Pratt, 2013 Figure 20-41

Some Steroid Hormones Derived from Cholesterol
Cholesterol is also a precursor itself, involved in the synthesis of steroid hormones. Steroid hormones are effective at very low concentrations and are consequently synthesized at very in amounts, as such consumes little cholesterol. Adrenal Gland Cortex: two classes of steroid hormones are synthesized: mineralocorticoids – reabsorption of inorganic ions (Na+, Cl-, and HCO3- by the kidney. glucocorticoids – help regulate gluconeogenesis and reduce inflammatory response. Male and female gonads and placenta: sex hormones progesterone (female reproductive cycle), androgens and estrogens. Lehninger, 2005 Figure 21.46

Competitive Inhibitors of HMG-CoA Reductase for Hypercholesterolemia Treatment
STATINS Statins all contain HMG-like group – competitive inhibitor have much lower Km than regular substrate Bulky hydrophobic groups interfere with HMG-CoA reductase (lactones- active as hydroxy acids) Mode of action a tad more complex: Presence of statins initially causes a decrease in the cellular cholesterol level Causes induction (meaning?) of LDL receptor and HMG-CoA reductase However, increased LDL receptors cause increased removal of LDL and IDL (a receptor precursor) result in an appreciable decrease in serum LDL cholesterol levels Regular substrate Voet, Voet, and Pratt, 2013 Figure 20-42

Atherosclerosis Atherosclerotic Plaque in a Coronary Artery
Slow progressive disease Deposition of lipid in large blood vessel walls. Initial event may be association of lipoproteins with vessel wall proteoglycans Trapped lipids trigger inflammatory response with endothelial cells to express adhesion molecule to monocytes Monocytes burrow into vessel wall & become macrophages and engorge (oxidized) lipids – “foam cells” Foam cells attract more WBC Vessel forms a plague – cholesterol, cholesterol esters and dead macrophages surround by smooth muscle cells. (the latter can undergo calcification) Restricts or can block blood flow Voet, Voet, and Pratt, 2013 Figure 20-43

THE END “So long, farewell, auf wiedersehen, good-bye” Oscar Hammerstein II Have a nice life!

Lipid Metabolism Student Edition 7/3/14 version

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Lipid Metabolism Student Edition 7/3/14 version

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