Competition and allostery govern substrate selectivity of cyclooxygenase-2

Abstract

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and its ester analog, 2-arachidonoylglycerol (2-AG), to prostaglandins (PGs) and prostaglandin glyceryl esters (PG-Gs), respectively. Although the efficiency of oxygenation of these substrates by COX-2 in vitro is similar, cellular biosynthesis of PGs far exceeds that of PG-Gs. Evidence that the COX enzymes are functional heterodimers suggests that competitive interaction of AA and 2-AG at the allosteric site of COX-2 might result in differential regulation of the oxygenation of the two substrates when both are present. Modulation of AA levels in RAW264.7 macrophages uncovered an inverse correlation between cellular AA levels and PG-G biosynthesis. In vitro kinetic analysis using purified protein demonstrated that the inhibition of 2-AG oxygenation by high concentrations of AA far exceeded the inhibition of AA oxygenation by high concentrations of 2-AG. An unbiased systems-based mechanistic model of the kinetic data revealed that binding of AA or 2-AG at the allosteric site of COX-2 results in a decreased catalytic efficiency of the enzyme toward 2-AG, whereas 2-AG binding at the allosteric site increases COX-2’s efficiency toward AA. The results suggest that substrates interact with COX-2 via multiple potential complexes involving binding to both the catalytic and allosteric sites. Competition between AA and 2-AG for these sites, combined with differential allosteric modulation, gives rise to a complex interplay between the substrates, leading to preferential oxygenation of AA.

Inhibition of key enzymes in the inflammatory pathway by hybrid molecules of terpenes and synthetic drugs: In vitro and in silico studies

“…However, the compounds are not able to establish the same HB interactions with residues Arg120 and Tyr355. [21] Compound 3, 13, and 14 block the entrance to the main pocket of COX-2 in different extents. Compounds 6 and 7 are located outside the pocket, establishing some π-stacking interactions with aromatic side chains of residues Tyr115 and Tyr355.…”

Molecular Basis of Binding Interactions of NSAIDs and Computer-Aided Drug Design Approaches in the Pursuit of the Development of Cyclooxygenase-2 (COX-2) Selective Inhibitors

“…Recent studies revealed that only one monomer of the COX homodimer is active at a given time [29]. It has been postulated that these monomers can act additionally through an allosteric/catalytic couple, with AA oxygenation being controlled in the 'catalytic' monomer (E cat ) through the binding of non-substrate faty acids and nonselective NSAIDs to the opposite monomer, the 'allosteric' monomer (E allo ) [30,31]. Moreover, the major diferences between COX-1 and COX-2 are the substitutions of the bulkier amino acid residues Ile434, His513 and Ile523 in COX-1 by comparatively smaller residues Val434, Arg513 and Val523, respectively, in COX-2 at the main channel of cyclooxygenase binding site (Figure 3).…”

Signal integration and information transfer in an allosterically regulated network

“…One subunit in the dimer harbors the catalytically active site, while the other subunit contains an allosteric site that modulates the overall activity of the enzyme (Dong et al, 2013, 2011; Kulmacz and Lands, 1984). An array of substrates, inhibitors, and allosteric modulators can bind to, and thus compete for, either site, giving rise to highly complex reaction kinetics (Kudalkar et al, 2015; Kulmacz and Lands, 1985; Mitchener et al, 2015; Rimon et al, 2010; Yuan et al, 2009; Dong et al, 2016a). The various products from COX-2 activity drive multiple downstream pro- and anti-inflammatory processes that lead to diverse cellular fates including stress responses and apoptosis (Funk, 2001; Rouzer and Marnett, 2003; Smith et al, 2000).…”

“…Previously, most studies of COX-2 function have used simplified models based on Michaelis-Menten kinetics (Briggs and Haldane, 1925). Not surprisingly, these approaches have proved insufficient to capture the rich complexity of the COX-2 network of reactants, intermediates and products (Mitchener et al, 2015). We posit that a systems approach to understand COX-2 mechanism will improve inhibitor design to achieve desired outcomes in clinical settings.…”

“…Nonetheless, it is sufficiently complex that it represents a non-trivial example of how multiple inputs lead to multiple outputs in a physiological context. We focus our study on the allosteric regulation network of COX-2 by two important substrates, arachidonic acid (AA) and 2-arachidonoylglycerol (2-AG), which generate unusual dynamics in the COX-2 network when both are present (Mitchener et al, 2015). Levels of AA and 2-AG also vary widely in vivo (Seibert et al, 1997; Monjazeb, 2006; Sugiura et al, 2006), and it is unclear how such variation would influence COX-2 signal processing.…”