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Arg-513 and Leu-531 Are Key Residues Governing Time-Dependent Inhibition of Cyclooxygenase-2 by Aspirin and Celebrex.
- Source :
-
Biochemistry [Biochemistry] 2019 Sep 24; Vol. 58 (38), pp. 3990-4002. Date of Electronic Publication: 2019 Sep 09. - Publication Year :
- 2019
-
Abstract
- Aspirin and Celebrex are well-known time-dependent inhibitors of the cyclooxygenases (COX). Molecular dynamics simulations suggest that Arg-513 and Leu-531 contribute to the structural mechanisms of COX inhibition. We used mutagenesis and functional analyses to characterize how substitutions at these positions influence time-dependent inhibition by aspirin and Celebrex. We show that substitutions of Leu-531 with asparagine and phenylalanine significantly attenuate time-dependent inhibition of COX-2 by these drugs. The introduction of side chain bulk, rigidity, and charge would disrupt the formation of the initial noncovalent complex, in the case of aspirin, and the "high-affinity" binding state, in the case of Celebrex. Substitution of Arg-513 with histidine (the equivalent residue in COX-1) resulted in a 2-fold potentiation of aspirin inhibition, in support of the hypothesis that the presence of histidine in COX-1 lowers the activation barrier associated with the formation of the initial noncovalent enzyme-inhibitor complex. As a corollary, we previously hypothesized that the flexibility associated with Leu-531 contributes to the binding of arachidonic acid (AA) to acetylated COX-2 to generate 15 R -hydroxyeicosatetraenoic acid (15R-HETE). We determined the X-ray crystal structure of AA bound to Co <superscript>3+</superscript> -protoporphyrin IX-reconstituted V349I murine COX-2 (muCOX-2). V349I muCOX-2 was utilized as a surrogate to trap AA in a conformation leading to 15R-HETE. AA binds in a C-shaped pose, facilitated by the rotation of the Leu-531 side chain. Ile-349 is positioned to sterically shield antarafacial oxygen addition at carbon-15 in a manner similar to that proposed for the acetylated Ser-530 side chain.
- Subjects :
- Animals
Arachidonic Acid chemistry
Arachidonic Acid metabolism
Arginine genetics
Arginine metabolism
Crystallography, X-Ray
Cyclooxygenase 2 chemistry
Cyclooxygenase 2 genetics
Cyclooxygenase 2 isolation & purification
Enzyme Assays
Histidine
Hydroxyeicosatetraenoic Acids chemistry
Hydroxyeicosatetraenoic Acids metabolism
Leucine genetics
Leucine metabolism
Mutagenesis, Site-Directed
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins isolation & purification
Recombinant Proteins metabolism
Sf9 Cells
Stereoisomerism
Substrate Specificity
Time Factors
Aspirin pharmacology
Celecoxib pharmacology
Cyclooxygenase 2 metabolism
Cyclooxygenase 2 Inhibitors pharmacology
Subjects
Details
- Language :
- English
- ISSN :
- 1520-4995
- Volume :
- 58
- Issue :
- 38
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 31469551
- Full Text :
- https://doi.org/10.1021/acs.biochem.9b00659