BCH Graduate Survey of Biochemistry

Transcription

1 BCH 5045 Graduate Survey of Biochemistry Instructor: Charles Guy Producer: Ron Thomas Director: Glen Graham Lecture 58 Slide sets available at:

2 David L. Nelson and Michael M. Cox LEHNINGER PRINCIPLES OF BIOCHEMISTRY Fifth Edition CHAPTER 10 Lipids 2008 W. H. Freeman and Company

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5 Von Euler initially described prostaglandins from preparations of semen in the 1930s by their ability to lower blood pressure and stimulate other physiological responses. Prostaglandins belong to a group of compounds known as eicosanoids, which are C 20 compounds that include the related compounds leukotrienes, thromboxanes and prostacyclins. Eicosanoids have some hormone-like properties in that they incite physiological effects when they are present at very low concentrations. They appear to be active in the regulation of blood pressure and clotting, reproductive functions, cycles of sleep and awaking, fever and pain, and inflammatory responses of various kinds.

6 Prostaglandin structures: Prostanoic acid, the prostaglandin parent compound.

7 Prostaglandins are produced in virtually all types of tissues, although the type may vary from tissue to tissue. In the last 30 years, throngs of biochemists have been in pursuit of understanding how these compounds function in such a wide variety of responses. They have been after the enzymes that produce these compounds, their targets, signaling mechanisms by which they activate the physiological responses as well as the materials that will interfere or inhibit their responses. However, eicosanoids are unlike hormones in that they are mediators of "local responses." Once outside the cell, C 20 compounds are rapidly degraded.

8 Arachidonic acid is a precursor for leukotrienes, thromboxanes and prostacyclins. Prostaglandins belong to the so called "cyclic pathway" while leukotrienes are produced by the so called "linear pathway." Thromboxanes and prostacyclins belong to the cyclic pathway and share a common intermediate with prostaglandins. The first step on the cyclic pathway is catalyzed by a bifunctional enzyme PGH 2 synthase. The first part of the activities catalyzed by PGH 2 synthase is the addition of two molecules of oxygen (a cyclooxygenase) to arachidonate forming PGG 2, a cyclic hydroperoxide. The second enzymatic function, a peroxidase, converts the hydroperoxide to an endoperoxide functional group forming PGH 2. PGH 2 synthase belongs to the grouping of cyclooxygenase enzymes known as COX-2.

9 Fig. 1. Schematic overview of the cyclooxygenase and lipoxygenase pathways. Fig. 2. (A) The 5-lipoxygenase/leukotriene pathway. (B) The leukotriene biosynthetic pathway and transcellular metabolism of lipoxins. Haeggström, Rinaldo-Matthis, Wheelock and Wetterholm (2010) Biochem Biophys Res Comm 396:

10 The linear pathway leading to leukotriene production begins with 5- lipoxygenase catalyzed oxidation of arachidonate to 5-HPETE. After forming an unstable epoxide, A 4, glutathione (Glu-Cys-Gly) is added to the molecule and then the Glu residue is removed yielding D 4. The glycine is removed from D 4 forming E 4. Alternatively A 4 can be converted to B 4 by the addition of water that converts the epoxide to a hydroxyl group. Leukotrienes are active in inflammatory, hypersensitivity reactions, chemotaxis of inflammatory cells and related disorders, for example asthma. Recently, leukotriene synthesis inhibitors have come into wide use as anti-asthma drugs. The 5-lipoxygenase (5-LO) of white blood cells, mast cells, requires an activating protein known as FLAP (5-lipoxygenase-activating protein) to be functional.

11 Arachidonic acid can be released by phospholipase A 2 in response to a variety of stimuli, and can be oxygenated form bioactive molecules called eicosanoids. Arachidonate can be metabolized by the cyclooxygenase (COX) pathway, involving COX-1 and COX-2, to generate prostaglandins (PG) and thromboxanes (TX), which activate G-protein-coupled receptors. Arachidonate can also be oxidized by the lipoxygenase pathway involving two lipoxygenases, 5-lipoxygenase and 12/15-lipoxygenase to form leukotrienes and lipoxins. Eicosanoids are mediators of acute inflammation, fever, and several disease states such as thrombosis, cancer, atherosclerosis, asthma and rhinitis. Many drugs that alter eicosanoid synthesis and function are widely used: including, (lowdose aspirin; Thrombyl Cytotec ), (misoprostol and celecoxib; gastroprotective NSAIDs), Singulair (montelukast) for asthma and Xalatan for glaucoma.

12 FLAP is an 18 kda integral protein that binds the arachidonate substrate of the lipoxygenase and helps it bind to the enzyme. Inhibitors that bind to FLAP and block its ability to bind arachidonate and activate the lipoxygenase enzyme, inhibit A 4 synthesis which in turn blocks B 4 as well as other leukotriene product production. While the clinical treatment of asthma has recently become possible with nonsteroidal drugs, steroids and glucocorticoid steroids in particular, have long been in use to treat inflammatory disease symptoms. However, long-term steroid therapy has frequently carried the potential for adverse side-effects, because the glucocorticoid steroids like cortisol and corticosterone have many diverse effects on protein synthesis, carbohydrate and lipid metabolism and other aspects of cell function.

13 5-LO catalyzed oxidation of arachidonic acid to LTA 4 via the intermediate 5-HPETE. Structure of prostaglandins E 1, E 2, F 2α.

14 Thus, many biochemists have worked to find a COX-2 specific inhibitor that will reduce pain and inflammatory responses but not damage the stomach lining. CELEBREX (celecoxib) is chemically designated as 4-[5-(4- methylphenyl)-3-(trifluoromethyl)-1hpyrazol-1-yl] benzenesulfonamide and is a diaryl substituted pyrazole.

15 Inactivation of PGH synthase by aspirin Any idea about the type of reaction mechanism? What type of enzyme inhibition would this be?

16 Interestingly, a drug that functions to reduce the inflammatory response has been in wide use for more than 100 years. That drug is none other than aspirin (acetylsalicyclic acid). In 1971, the mechanism by which aspirin reduces inflammatory responses was discovered by John Vane. He found that aspirin inhibited the synthesis of prostaglandins by blocking the function of PGH 2 synthase. Using nonsteroidal anti-inflammatory drugs (NSAIDs) it was possible to show the existence of the prostacyclins and thromboxanes and that they were products of the "cyclic pathway." Aspirin and other NSAIDs inhibit the synthesis of prostaglandins, prostacyclins and thromboxanes by inactivating the cyclooxygenase of PGH 2 synthase by acetylating the enzyme.

17 NSAIDs do not inhibit the synthesis of leukotrienes. When the PGH 2 synthase enzyme is acetylated, it is irreversibly inactivated. Other NSAIDs are thought to function in a similar way by inhibiting PGH 2 synthase. Some of the NSAIDs you will readily recognize. Can you give the over-the-counter name of these NSAIDs? Common NSAIDs are aspirin, ibuprofen, naproxen, ketoprofen, piroxicam, celcoxib and rofecoxin. Celcoxib is known as Celebrex and is considered to be a COX-2 specific inhibitor. Apparently a COX-1 type cyclooxygenase activity is necessary for regeneration and maintenance of the lining of the stomach. NSAIDs like aspirin are non-specific inhibitors of COX-1 and COX-2 cyclooxygenases, and hence the deleterious effect upon the stomach.

18 Schror K, Meyer-Kirchrath J (2000) Cyclooxygenase-2 inhibition and side-effects of nonsteroidal anti-inflammatory drugs in the gastrointestinal tract. Curr Med Chem 7: Inhibition of prostaglandin biosynthesis via inhibition of the fatty acid cyclooxygenase (COX) is the mechanism of action of non-steroidal anti-inflammatory drugs (NSAIDs). This results in an inhibition of the inflammatory and pain-producing activities of prostaglandins at a site of tissue injury but also in inhibition of prostaglandin production in the gastrointestinal tract (GI) and platelets, i.e. sites where endogenous prostaglandins are possibly involved in control of physiological functions. The discovery of two COX isoenzymes, COX-1 and COX-2, and the detection of their separate function and regulation, has initiated the search for new and putatively more selective inhibitors of prostaglandin biosynthesis. Specifically, selective inhibitors of COX-2 were developed in order to improve the anti-inflammatory and analgetic specificity and potency of the compounds and to reduce side-effects in the GI tract. Available experimental and clinical data of selective COX-2 inhibitors, including flosulide, celecoxib or rofecoxib, suggest improved gastric tolerance as compared to conventional, non-selective NSAIDs. However, experimental evidence suggests that both, the analgetic and antiinflammatory action of COX-inhibitors, might also require inhibition of COX-1. COX-2-selective compounds at anti-inflammatory doses might have other side-effects, and for example reduce vascular prostacyclin production. Evidence is accumulating that COX-2 might not only be considered as a putatively detrimental enzyme but rather a highly regulated enzyme that also contributes to tissue protection and is even constitutively expressed in healthy human stomach mucosa. This paper reviews some of these newer aspects of COX-2-selective inhibitors in clinical use and discusses their possible benefits and risks.

19 In nature, phosphorus is very often a limiting inorganic nutrient for plants. To conserve limited phosphorus, plants have substituted the phosphate group with carbohydrate for the extensive chloroplast membrane lipids.

20 Based on the stick structure of cholesterol, it is hard to realize its true 3-D structure. It looks like a mummy with the OH group where the head would be. Several steroids are derived from cholesterol.

21 Cholesterol Biosynthesis The structure of cholesterol was determined in the 1930s and was thought be derived from acetate. It was suggested that 3 acetates make a five carbon compound (isoprene), and later it was shown that multimers of this basic building block were the basis for the structural aspects of cholesterol. The final gap in the overall pathway was closed when mevalonic acid was identified as the isoprenoid precursor. The last enzyme in β oxidation, thiolase, that converts the 4C β- ketoacyl-coa in the presence of CoASH into two molecules of acetyl- CoA, catalyzes the formation of the initial precursor of sterol biosynthesis; only the reaction is the reverse of the last step of β oxidation. Another name for the reverse product of the thiolase reaction is acetoacetyl-coa.

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24 If a third molecule of acetyl-coa condenses with acetoacetyl-coa, the products are free CoASH and a six carbon-coa derivative generally known as HMG-CoA (β-hydroxy-β-methylglutaryl-coa). The enzyme that catalyzes this reaction is HMG-CoA synthase. The HMG-CoA is then cleaved and reduced forming mevalonate and CoASH. The enzyme that catalyzes this reaction is HMG-CoA reductase. Sterol biosynthesis is regulated at the HMG-CoA synthase and reductase steps, with the latter being the major rate-limiting step in the pathway leading to cholesterol.

25 Both enzymes are regulated at the mrna level, meaning, that the amounts of the enzymes present are proportional to the amounts of the messenger RNA that is produced from the respective genes. More mrna - more enzyme, and vice versa. Generally the amount of mrna produced is dependent on the levels of cholesterol present. The reductase is further regulated by the rate of degradation of the enzyme. A balance exists between synthesis and breakdown. Changing the rate of breakdown can lead to either an increase or a decrease in the amount of enzyme present if the rate of synthesis is not changed. The reductase is also regulated by phosphorylation / dephosphorylation. When the enzyme is phosphorylated it is inactive, and when it is not phosphorylated it is catalytically active.

26 Since HMG-CoA reductase is a rate-limiting step in sterol biosynthesis, inhibitors that reduce the activity of this enzyme can influence the endogenous production of cholesterol. Next the mevalonate is phosphorylated twice to yield a pyrophosphate compound. Following an ATP dependent decarboxylation, the five carbon intermediate isopentyl pyrophosphate (IPP) is produced. IPP can be isomerized to form dimethylallyl pyrophosphate (DMAPP). A relatively new biosynthetic pathway leading to IPP has been identified in microorganisms and plants in the last decade. This new pathway uses pyruvate and glyceraldehyde-3-phosphate as starting materials to synthesize IPP.

27 The first enzyme of the pathway is 1-Deoxy-D-Xylulose-5-Phosphate Synthase and it produces 1-Deoxy-D-Xylulose-5-Phosphate from pyruvate and glyceraldehydes-3-p. This gives rise to the name DOXP pathway. Lovastatin, a fungal metabolite is a very powerful competitive inhibitor of HMG-CoA reductase (KI < 1.0 nm). Therefore, therapy with lovastatin or similar materials can lower serum cholesterol levels. The statins form a class of hypolipidemic drugs used to lower cholesterol levels in people with or at risk of cardiovascular disease.

28 Inhibition of the HMG-CoA reductase enzyme in the liver stimulates LDL receptors, resulting in an increased clearance of lowdensity lipoprotein (LDL) from the bloodstream and a decrease in blood cholesterol levels. Schachter M (2004) Chemical, pharmacokinetic and pharmacodynamic properties of statins: an update. Fundam Clin Pharmacol. 19:

29 Vitamin D 3 is produced when 7- dehydrocholestero is illuminated in the skin by UV light to form cholecalciferol (vitamin D 3 ) which can be used to make the active form of vitamin D in the liver. Vitamin deficiency causes rickets. Active form of the vitamin.

30 β-carotene is the precursor of vitamin A 1 (retinol) and its derivatives like retinal and retinoic acid. A number of important biochemical molecules are isoprenoid compounds or isoprenoid derivatives.