Chem*4520 Metabolic Processes

Fall Semester 2000

Modified October 2000

schematic view of the enzyme citrate synthase,
with bound acetyl-CoA analog in green

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Lecture 23:


Fri Nov 3Voet , Chapter 23, pp. 708-713.
Mathews, Van Holde: Chapter 19, 700-704.
Stryer:Chapter 27, pp.705; Chapter 24, 624-625
Comment: there's a notable lack of information, and even some misinformation, on leukotrienes in the textbooks!

Lipoxygenases produce linear peroxides of arachidonate

5-HPETEA variety of lipoxygenases exist which catalyze peroxidation of the allylic systems of arachidonate. 5-lipoxygenase adds O2 at C-5 by the radiacl mechanism, shifting the 5 cis double bond to 6 trans, giving 5-hydroperoxyeicosatetraenoate, 5-HPETE.

Similar peroxidations may attack C-12 or C-15 to give 12-HPETE or 15-HPETE, but only 5-HPETE and 15-HPETE are currently known to proceed to physiologically active derivatives.

5-HPETE is the starting point for synthesis of Leukotrienes

Leukotriene A and BA simple dehydration with a proton removed from C-10 gives an epoxide ring at C-5 and C-6, and three conjugated double bonds between C-7 and C-12. This is leukotriene A4 LTA4.

The triene designation refers to the signature ultraviolet spectrum due to three conjugated double bonds. The subscript denotes the total number of double bonds - four when derived from arachidonate.

A fatty acid precursor lacking 5 won't enter this pathway, but eicosapentaenoate 20:55,8,11,14,17 can give rise to leukotrienes with an extra unconjugated double bond 17, designated LTx5.

Leukotriene B is obtained by hydrolytic opening of the epoxide, by OH- attack through the conjugated double bond system, which is shifted one place down, becoming all trans. The product is hydroxylated at C-5 and C-12.

Leukotriene B is an important chemoattractant for cells of the immune system, secreted by polymorphonuclear leukocytes (PMNs) (hence the name leukotriene), and acting on other PMNs, eosinophils, macrophages and monocytes. Cells respond to concentrations as low as 10-10M LTB4, making it the most active such factor known. This initiates localized inflammation.

At 10-8M LTB4, PMNs may release their lysosomal contents, triggering hydrolytic digestive attack at the site of inflammation. This is useful if cleaning up damage due to bacterial invasion, but excessive response could be damaging to host tissue.

A Leukocyte glossary:
PMNsare bone marrow derived cells from the granulocyte-monocyte stem cell, with a major role in inflammation. The name denotes the varied nuclear shape, and they are also known as neutrophils. PMNs are phagocytic cells, like macrophages, but do not act as antigen presenting cells, instead are most effective at killing ingested microorganisms.
Macrophagesare large phagocytic cells, may be motile or localized to a specific tissue. Ingested antigens are processed internally, and then fragments are presented to reactive lymphocytes to stimulate the immune response. They secrete interferon and lysosyme to upregulate inflammatory response.
Monocytesare immature macrophages.
Eosinophilsare granulocytes which can stain intensely with the dye eosin. The cytoplasm is filled with granules of basic substances such as histamine. Eosinophils are motile, phagocytic agents against parasites, but are also partly responsible for allergies.
Basophilsare granulocytes stained by basic dyes such as toluidine blue, carrying IgE receptors. As they mature, they settle into fixed tissue locations and become mast cells.
Mast cellsare tissue-resident granule containing cells with IgE receptors. Immunoglobulin E (IgE) produced by antigen stimulated lymphocytes is captured and displayed on the cell surface. Granules contain stored mediators of inflammation such as histamine, proteases, and proteoglycans such as heparin. Degranulation is stimulated by physical trauma, toxins or venoms, tissue proteases, binding of allergens to IgE or binding complement factor C5a.

Slow reacting substance of anaphylaxisDegranulating basophils and mast cells release a factor called Slow-reacting substance of anaphyllaxis, SRS-A. Unlike histamine, which is stored, SRS-A is produced at the time of IgE stimulation.

SRS-A was found to consist of the products of glutathione conjugation to LTA4. Simple addition of glutathione to C-6, which opens the epoxide ring, gives Leukotriene C, LTC4.

This reaction is catalyzed by a specific microsomal glutathione-S-transferase, distinct from the cytoplasmic soluble GST enzymes for xenobiotic detoxification.

Isopeptidase-catalysed removal of the -glutamyl gives Leukotriene D, LTD4, and subsequent removal of the glycine gives Leukotriene E, LTE4

SRS-A elicits contraction of smooth muscle, in particular bronchial and vascular tissues, and also increases vascular permeability. This results in constricted breathing, tissue swelling, oedema and mucus release, in particular the effects seen in asthma and allergies. These are similar to the effects of histamine, but LTC and LTD are 200 and 20000 times more active respectively than than histamine. Excessive release of SRS-A gives rise to life-threatening anaphylactic response.

The on-site conversion of LTC to the superactive LTD probably accounts for the characteristic gradual build-up of the response - hence the term Slow reacting substance.

Drug action:

Antihistamines are ineffective against SRS-A mediated allergic response, since histamine release is not the major factor. Aspirin and NSAIDS are specific for the cyclic prostanoids, and don't control leukotriene response, since they act on the cyclo-oxygenase.

Corticosteroids control arachidonate release, but by the time anaphylactic response is observed, it's too late. The standard emergency treatment is by intravenous injection of epinephrine, since cyclic AMP appears to oppose these responses.

The acetylenic analog of arachidonate, ETYA, eicosatetraynoic acid is an effective in vitro inhibitor of both cyclooxygenase and lipoxygenase, but its usefulness as a drug needs to be demonstrated.

In addition, the structure of the 5-lipoxygenase is also being studied to design drugs specifically effective against the non-cyclic eicosanoid pathway. The enzyme requires an 18 kDa membrane polypeptide FLAP (5-lipoxygenase activating protein), which binds and delivers the arachidonate substrate. Several drugs have been found to inhibit FLAP.

Dietary considerations:
LTX5 derivatives are significantly less active that the LTX4 compounds, suggesting that an increase in the diet of precursors leading to eicosapentaenoate 20:55,8,11,14,17, e.g. as found in fish oils, may be beneficial in keeping leukotriene activity under control.

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