COX-1

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Introduction

The cyclooxygenase isoenzymes COX-1 and COX-2 are responsible for catalyzing the formation of prostaglandins, thromboxane, and levuloglandins. The prostaglandins are autocoid mediators whose reversible interactions with G-protein coupled membrane receptors can affect nearly all known physiological and pathological processes, including cardiovascular, neuronal, renal, immune, gastrointestinal, and reproductive systems. The levuloglandins are a newer class of products that appear to act through irreversible covalent attachment to a variety of proteins. COX enzymes play an important role clinically because they are inhibited by aspirin and many other non-steroidal anti-inflammatory drugs (NSAIDs), and this inhibition can relieve m inflammatory, pyretic, thrombotic, neurodegenerative, and oncological maladies. About a hundred years have passed since Hoffman designed and synthesized acetylsalicylic (aspirin) as an agent designed to reduce the gastrointestinal irritation of salicylates while maintaining its efficacy.  

Catalytic mechanisms of cyclooxygenase

About 95% of interactions involve hydrophobic residues. Site-directed mutagenesis analyses have positioned the residue that abstracts the proS hydrogen from C-13 of arachidonate (Tyr-385); the residue responsible for electrostatic interaction with the carboxylate anion of arachidonate (Arg 120); and the residues that govern the orientation of arachidonate so that it is optimally arranged to yield PGG2 (Val-349, Trp-387, and Leu-534).  Fig. 1 depicts the interaction and catalytic reaction between arachidonic acid and amino acid residues in the active site of COX-1. The two isoforms of COX share similar amino acid sequences and catalytic mechanisms. The crystal structure of murine apo-COX-2 also confirmed that arachidonic acid can assume a catalytically incompetent orientation within the active site. For example, the anionic carboxylate end of the substrate can form hydrogen bonds with Tyr 385 and Ser 530, but not the predicted ion pair with Arg120. Instead, the Arg 120 residue contacts the hydrophobic ω-end of the substrate. In its catalytically active state, COX-2 is similar to COX-1, i.e., the carboxylate anion of arachidonic acid is coordinated by Arg 120 and Tyr 355, and the C13 pro(S)-hydrogen atom of the substrate is located near Tyr 385.

Figure 1. Amino acid residues in COX 1 that govern substrate positioning and catalysis (Fitzpatrick, F. 2004)

Bi-functional catalysis in a single enzyme protein: integrated actions of peroxidase and cyclooxygenase

From the initial reports of the important role of radicals in COX catalysis until now, there have been several insights. For example, purified COX-1 and COX-2 enzymes reconstituted with manganese protoporphyrin IX and probed with peroxide produce electron paramagnetic resonance (EPR) spectra reflecting radical-dependent oxidation. This study shows that peroxide-generated radicals in COX 2 and MnCOX-1 each form the same arachidonic acid radical, which subsequently reacts with oxygen to form lipid hydroperoxides. The EPR data also suggest that the metastable arachidonate radical contains a plane pentadienyl radical, as has been widely speculated.

Given the similarities between their primary sequences, crystal structures, and catalytic mechanisms, the compositional regulation of COX-1 and COX-2 catalysis is also similar, with only a few differences. For example, the catalysis of COX-2 isoenzymes requires 10 times less hydroperoxide level than when COX-1 catalysis is promoted. Scientists speculate that this may be due to a mechanism by which COX-2 preferentially produces PGs when both isoforms are present in the same intracellular compartment, but it is unclear why cells find this advantage. The structural stability of COX-2 is lower than that of COX-1 in both the active site region and the subunits overall, which may be due to their different preferences for fatty acid substrates and inhibitors and the presence of the larger size pocket of COX-2. A valine residue at position 509 in COX-2 corresponds to the isoleucine residue at position 523 in COX-1, but mutational studies in both COX-1 and COX-2 suggest that the exchange of valine and isoleucine at position 509/523 does not account fully for all the differences in inhibitor specificity between the isoforms. Therefore, differences in structural stability may contribute to the selectivity of ligands at the respective enzyme active sites.

Figure 2. Effect of acetylation on substrate access to COX-1 and COX-2. Speculative effect of acylation on COX-2 (Fitzpatrick, F. 2004)