Cyclooxygenases

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Cyclooxygenases
Cyclooxygenases
Cyclooxygenase-2 dimer, PDB  1CVU
Enzyme classification
EC, category 1.14.99.1 dioxygenase
Substrate Arachidonic acid + AH 2 + 2 O 2
Products Prostaglandin H 2 + A + H 2 O

Cyclooxygenases (COX) are the essential enzymes at the beginning of a prostaglandin synthesis from arachidonic acid , dihomogammalinolenic acid (DGLA) or eicosapentaenoic acid (EPA). Since this first step is rate-determining, the COX have a central function in regulating the inflammatory process ; they are inhibited by non-steroidal anti-inflammatory drugs .

Cyclooxygenases are located inside the endoplasmic reticulum , inside the nuclear envelope and in the Golgi apparatus and adhere to the inside of the membranes of these cell compartments . They have been found in cells of animals since the early development of invertebrates , e.g. B. already in coral cells , but not in unicellular organisms, plants or insects. However, related enzymes from the superordinate family of pathogen-inducible oxygenases (PIOXs) occur here.

Very early in the evolution of cyclooxygenases there are two isoenzymes , cyclooxygenase-1 and cyclooxygenase-2 , which differ in their gene locus , have a slightly different structure, occur in different cell types, are regulated differently, show different substrate specificities and can be influenced differently pharmacologically.

History of exploration

Prostaglandins are among the eicosanoids and have been known since the 1930s. In the 1970s, purified cyclooxidases were produced for the first time from tissue homogenates of the seminal vesicles of cattle and sheep and recognized as the enzymes of prostaglandin synthesis with cyclooxygenase and peroxidase activity. In 1971 it was demonstrated that non-steroidal anti-inflammatory drugs already in use at that time inhibit cyclooxygenase activity. From 1972, due to the different kinetics of the enzyme reactions, it was speculated that there must be more than one cyclooxygenase.

The protein structures of cyclooxygenase-1 and cyclooxygenase-2 were sequenced in the 1990s and their tertiary and quaternary structure clarified and led to the development of other drugs that affect these enzymes.

The first cyclooxygenase-2 selective inhibitors were on the market in 1999.

Furthermore, a paradigm shift in prostaglandin research took place in the early 1990s , when it was recognized that the regulation of cyclooxygenases is the essential control point in prostaglandin synthesis (previously it was thought that these were the phospholipases , which among other things produce arachidonic acid) .

structure

Cyclooxygenases are globular proteins with around 600 amino acids. They have a molar mass of 67 to 72  kDa , are 65% the same in their amino acid sequence and have almost identical active centers . They each combine to form two dimers . With a hydrophobic region they float on / in the inside of the microsomal membranes e.g. B. the endoplasmic reticulum. This membrane-binding region forms a likewise hydrophobic, narrow opening in a blind-ended channel to the active site with the cyclooxygenase activity. This channel is narrower in cyclooxygenase-1 than in cyclooxygenase-2 (due to an exchange at position 523 from isoleucine to valine ).

In the inner part of the channel there is a tyrosine (at position 385) , which is activated to a tyrosyl radical before the cyclooxygenases can carry out their actual reaction. This is done with the help of the peroxidase activity of the cyclooxygenases, which is located in another active center (on the side of the enzyme opposite the membrane of the endoplasmic reticulum). Here, with the help of oxidants occurring in the endoplasmic reticulum , Fe 3+ bound to heme is oxidized to a ferryl-oxo-porphyrin radical (Fe 4+ = O ), which then removes an electron from the tyrosine OH in the center of the cyclooxygenase activity and so the active radical Tyrosyl-O forms. (For more details see pages 400 and 401 of).

function

Formation of prostaglandin H 2 from arachidonic acid in two steps catalyzed by cyclooxygenase-1 and -2. The first step can be inhibited by non-steroidal anti-inflammatory drugs .

The cyclooxygenases catalyze the conversion of arachidonic acid to prostaglandin H 2 , or DGLA and EPA to the corresponding precursors of PG 1 and PG 3 . This happens in two steps in two different reaction centers of the enzyme:

  1. The first reaction step takes place in the catalytic center described above with the cyclooxygenase activity. A ring closure is achieved between the carbon atoms C 8 and C 12 and two oxygen atoms are inserted at C 9 and C 11 , which then form a covalent bond with each other, so that a peroxide bridge is formed in the prostaglandin G 2 (for more details see in). The resulting prostaglandin G 2 diffuses out of the channel.
  2. The second reaction step is catalyzed by the reaction center with peroxidase activity: here the prostaglandin H 2 is formed from the prostaglandin G 2 .

The various other prostaglandins are then synthesized from the prostaglandin H 2 produced , partly by spontaneous isomerization and partly with the help of various synthases or oxidases .

The cyclooxygenases are, however, the rate-determining step in prostaglandin formation , so they have a central position in the regulation of the inflammatory process. They have a half-life of 1–2 minutes when exposed to arachidonic acid at a concentration that maximizes the utilization of the enzyme.

Subforms (isoenzymes)

There are two subforms of cyclooxygenases. They separated from one another through gene duplication early in the evolution of invertebrates and have since followed their own evolutionary paths. They have 65% of the amino acid sequences in common, catalyze the same enzymatic reaction, but are distributed and regulated differently in the organism. An important difference between cyclooxygenase-1 and cyclooxygenase-2 is the exchange at position 523 of isoleucine for valine, which makes the active center of cyclooxygenase-2 somewhat larger and can also oxidize somewhat bulky substrates except arachidonic acid there. This is possible, for example, for endocannabinoids such as anandamide (see page 399 of). Another difference is the multiple regulation of the transcription of cyclooxygenase-2, which is mainly induced by inflammatory processes and other conditions of cell activation.

A splicing variant of cyclooxygenase-1 was described as COX-3 in 2002 . One intron less is expressed from the same gene, PTSG1 . In dogs, by omitting 93 base pairs, this leads to an enzymatically active cyclooxygenase with 31 amino acids fewer. But due to a frameshift mutation , the unexpressed intron in mice and humans is 94 base pairs long and this results in a completely different protein structure that has no cyclooxygenase activity. In addition to these two forms, a further nine splicing variants are described.

COX inhibitors

  • Because when cyclooxygenase is inhibited, more arachidonic acid is available for the lipoxygenase pathway , which results in the formation of leukotrienes , which increase inflammation and anaphylaxis , inhibitors of cyclooxygenase can trigger an asthma attack .
  • Acetylsalicylic acid (aspirin) leads to a transacetylation at the serine in position 530 in the catalytic center of the cyclooxygenase, which renders the enzyme inoperable until it is regenerated. The cyclooxygenase-1 for this is 10-100 times more sensitive than cyclooxygenase-2 .
  • Competitively acting NSAIDs compete in the cyclooxygenase center for the binding site for the arachidonic acid . Ibuprofen binds very quickly and is quickly washed out again. Diclofenac or indomethacin have a slower bonding behavior.
  • Selective COX-2 inhibitors mainly inhibit the activity of cyclooxygenase-2 (see there for details).
  • Analgesic / antipyretic substances such as paracetamol or metamizole are important drugs for pain and fever without anti-inflammatory properties. The mechanism and conditions of their rather weak inhibition of cyclooxygenases are still unexplored.

Treatment of inflammatory diseases

NSAIDs are currently used as the drugs of choice for the treatment of osteoarthritis , rheumatism , systemic lupus erythematosus (SLE) and other inflammatory diseases. Treatment is mostly palliative and does not change the course of the disease. NSAIDs inhibit inflammation and reduce pain.

Individual evidence

  1. a b c d e f Simmons, DL et al. (2004): Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition. In: Pharmacol. Rev. Vol. 56, pp. 387-437. PMID 15317910
  2. ^ Regina Botting: COX-1 and COX-3 inhibitors . Thrombosis Research 2003, Volume 110, Double Issue 5-6, June 15, 2003, pages 269,272; doi: 10.1016 / S0049-3848 (03) 00411-0 ; PMID 14592546
  3. ENSEMBL entry

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