Bioc 460 - Dr. Miesfeld Spring 2008 1 of 13 pages 1985 Nobel Prize in Medicine Figure 1. Lipid Metabolism 3 Key Concepts - Cholesterol biosynthesis - Metabolism of dietary fats and cholesterol - Steroid biosynthesis - Eicosanoid biosynthesis Cholesterol is synthesized from acetyl-CoA Cholesterol has been called a Janus-faced molecule, having both a critical role in the function of cell membranes and production of cell signaling molecules, but it is also a major contributing factor to cardiovascular disease. The problem with cholesterol is that it is made in high quantities in liver cells and must be transported through the circulatory system by lipoprotein particles. As long as it is safely delivered to cells and incorporated into membranes, or used as a substrate to make bile acids and steroids, it is harmless, and indeed vital for life. However, elevated levels of cholesterol transporting lipoproteins in the blood called low density lipoproteins (LDL), leads to the formation of atherosclerotic plaques in the lining of blood vessels. These atherosclerotic plaques contain large numbers of inflammatory cells, fibrous tissue and cholesterol which can lead to the formation of blood clots that restrict blood flow resulting in cardiac arrest or stroke. Humans synthesize as much as 1 gram of cholesterol per day and absorb ~0.3 grams per day from their diet. Cholesterol synthesis occurs in all cells with the highest levels being made in the liver. Because of this de novo cholesterol biosynthetic pathway, cholesterol is not an essential lipid in the diet. Figure 1 gives an overview of the cholesterol biosynthetic pathway which takes place in the cytosol and consists of four distinct stages. In the first stage, three molecules of acetyl-CoA are used to make mevalonate, a C6 compound that is the product of a reaction catalyzed by the highly-regulated enzyme HMG CoA reductase. Mevalonate is then phosphorylated and decarboxylated in stage 2 to form the activated C5 isoprenoid intermediate, isopentenyl pyrophosphate. This important metabolite is an intermediate in a number of other biosynthetic reactions including those of plant chlorophylls and plant hormones (gibberellic acid), a number of vitamins (A, E, K) and the quinones (ubiquinone and plastoquinone). In stage 3 of the pathway, three molecules of isopentenyl pyrophosphate are combined to form farnesyl pyrophosphate (C15) which is then used to generate squalene, a C30 cholesterol precursor. Farnesyl pyrophosphate is also used as a lipid anchor in some membrane-associated proteins, one of which is Ras, a cell signaling protein in the MAP kinase pathway. In the fourth and final stage, squalene cyclicizes to form a four-ringed molecule which is then modified by a number of reactions, ultimately resulting in the loss of three methyl groups to generate cholesterol (C27), a four-ringed sterol molecule.Bioc 460 - Dr. Miesfeld Spring 2008 2 of 13 pages Figure 2. Figure 3. Stage 1 - generation of mevalonate from acetyl-CoA. In the first reaction of this cytosolic pathway, two molecules of acetyl-CoA are condensed to form acetoacetyl-CoA through a reaction catalyzed by the enzyme thiolase as shown in figure 2. A third acetyl-CoA is used to generate the C6 compound β-Hydroxy-β-methylglutaryl-CoA (HMG-CoA) by the action of HMG-CoA synthase. In what turns out to be the rate-limiting step in the cholesterol biosynthetic pathway, the enzyme HMG-CoA reductase converts HMG-CoA to mevalonate in a reduction reaction that uses two molecules of NADPH and releases coenzyme A. Because of the central role of HMG-CoA reductase in cholesterol biosynthesis, inhibitors of HMG-CoA reductase activity, the so-called statin drugs, are very effective in treating atherosclerosis. The HMG-CoA reductase enzyme is localized to the smooth endoplasmic reticulum and functions as a homodimer with the catalytic site located at the dimer interface. Stage 2 - conversion of mevalonate to isopentenyl pyrophosphate and dimethylallyl pyrophosphate. Mevalonate is activated by the addition of two phosphates groups donated from ATP to generate 5-pyrophosphomevalonate (figure 3). The enzyme pyrophosphomevalonate decarboxylase then catalyzes an ATP-dependent reaction that removes the terminal carboxyl group to generate the C5 isoprenoid compound isopentenyl pyrophosphate which is readily isomerized to form dimethylallyl pyrophosphate. Stages 3 and 4 - formation of cholesterol (C27) from six C5 isoprenoids. The next set of reactions make use of the C5 isoprene units generated in stage 2 to build the C30 carbon scaffold of squalene which is then converted to lanosterol the precursor to cholesterol (figure 4). In the first reaction, prenylBioc 460 - Dr. Miesfeld Spring 2008 3 of 13 pages Figure 4. transferase catalyzes a condensation reaction in which isopentenyl pyrophosphate and dimethylallyl pyrophosphate condense in a head to tail fashion (pyrophosphate being the head) to form the C10 compound geranyl pyrophosphate. The same enzyme adds a second isopentenyl pyrophosphate to the head of geranyl pyrophosphate to generate the C15 intermediate farnesyl pyrophosphate. Two molecules of farnesyl pyrophosphate are then linked in a head to head arrangement by the enzyme squalene synthase to form squalene (C30) in a reduction reaction using NADPH. The conversion of a C30 hydrocarbon chain into a cyclic cholesterol molecule involves a complex set of reactions that begin with the oxygenation of squalene by the enzyme squalene monooxygenase. The product of this reaction, squalene-2,3-epoxide, is then subjected to an amazing cyclicization process in animal cells that is catalyzed by enzymes called cyclases that promote the formation of the four-ringed cholesterol precursor lanosterol. In the final steps of the pathway, lanosterol is converted to cholesterol by a series of 19 reactions that involve additional bond rearrangements, and ultimately, the removal of three carbons to generate the C27 product. Cholesterol synthesized in the liver has essentially three fates; 1) it can be esterified with a fatty acid by the enzyme acyl-CoA-cholesterol acyl transferase (ACAT) to make cholesterol esters that are stored in lipid droplets, 2) packaged into lipoprotein particles and exported to the peripheral tissues, or 3) converted into bile acids whichBioc 460 - Dr. Miesfeld Spring 2008 4 of 13 pages Figure 5. are transported to the bile duct and
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