Info

COX Activity

km-AA

in the presence

Peroxidase activity

Mutated amino acids

(nmol/min x g)

(lM)

of aspirin (min)

(nmol/min x g)

Serine530 acetylated Serine530 $ alanine530 Serine530 $ asparagine530

450 7

388 8

30 70

Effects of aspirin: Acetylation of Ser530 in the COX protein (wt) results in complete loss of COX activity without inhibition of peroxidase activity of the complex. Replacement of Ser530 by alanine does not change the COX or peroxidase activity of the complex but makes the enzyme resistant against aspirin, suggesting that Ser530 is the acetylation target of aspirin. Replacement of Ser530by asparagine inhibits the cyclooxygenase activity completely but increases the peroxidase activity, suggesting that both activities can be regulated independently (after DeWitt et al. [108] and Smith WL [personal communication]).

channel that causes sterichindranceoftheaccess of arachidonic acid to the catalytic site. Salicylic acid and traditional NSAIDs prevent binding of arachi-donic acid by a mechanical channel blockade as a result of their lipophilic properties. Arg120 fixes the salicylate group by hydrogen bounds in the correct stericposition [115,116]. Figure 2.10 demonstrates the current view on the COX-1 substrate channel and active site of COX-1, as well as the modification by aspirin.

Another recently discovered effect of acetylation of COX-1 in platelets is the inhibition of the release of sphingosine-1-phosphate from human platelets. The inhibition parallels the inhibition of thrombox-ane formation and persists for 3 days in healthy men after ingestion of one single dose (1 g) of aspirin. Sphingosine phosphate does apparently not modify the platelet function but might change the function of sphingosine-derived lipid signaling [117] (Figure 2.11).

Time after oral ASA intake

Figure 2.11 Inhibition ofthrombin-induced release of sphingosine-1-phosphate (S1P) from human platelets by oral aspirin (ASA) (500 mg) and aspirin in vitro. The numbers in the columns indicate the remaining thromboxane (TX) formation. No inhibition is seen with salicylate (not shown) (modified after [117]).

Acetylation of COX-2 by ASA Transfer of the acetyl moiety toward one critical location in the COX enzyme(s) - the serine530 in COX-1 and COX-2 (Note: the COX-1 numbering system is used by convention that is also for COX-2: Ser516 in COX-2 is structurally identical with serine530 in human COX-1) has chemically the same reaction but has different consequences for COX-1 andCOX-2. Acet-ylation of COX-2 modifies the steric structure of COX- 2 protein in a way to generate 15-( R)-HETE. As already seen with COX-1, replacement of Ser530 with alanine not only prevented the aspirin acetylation reaction but also reduced the enzymatic activity to only 50% whereas replacement ofTyr385 by phenyl-alanine completely prevented any COX activity. All of these procedures left the peroxidase activity unchanged [113]. This is presumably due to the cyclo-oxygenase side pocket of COX-2 that allows fatty acids and inhibitors to bind more tightly at the COX-2 active site [116]. In contrast to COX-1, Arg120 plays only an accessory role for the binding of arachidonic acid and inhibitors in COX-2 - a deletion mutant was still active though at markedly reduced affinity.

Thus, in addition to the inhibition of prostaglandin formation, acetylation of COX-2 by aspirin results in the generation of a new product, 15-(R)-HETE [118-120]. 15-(R)-HETE is made by COX-2 in much higher proportions than the cyclo-oxygenase product PGE2. Interestingly, the production of 15-(R)-HETE by aspirin-acetylated COX-2 was inhibited by most traditional NSAIDs [112] and selective COX-2 inhibitors [121]. The significance of this finding for the anti-inflammatory action of these compounds remains to be determined.

This particular property of aspirin led to the hypothesis that the generation of 15-(R)-HETE by acetylated COX-2 may not just be a removal of metabolic "waste" but might serve specific purposes. Charles Serhan was the first to show that 15-(R)-HETE was the precursor of a class of new products the "aspirin-triggered lipoxins" by syner-gistic interaction of acetylated COX-2 with 5-lipox-ygenase from white cells [122] (Figure 2.12). ATL not only contributes to the anti-inflammatory actions of aspirin (Section 2.3.2) but might also be

2.2 Cellular Modes of Action j 59 Aspirin

Arachidonic acid ♦

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