Download MendeleyArg-513 and Leu-531 Are Key Residues Governing Time-Dependent Inhibition of Cyclooxygenase-2 by Aspirin and Celebrex.
Dong, L., Anderson, A.J., Malkowski, M.G.(2019) Biochemistry 58: 3990-4002
- PubMed: 31469551
- DOI: https://doi.org/10.1021/acs.biochem.9b00659
- Primary Citation of Related Structures:
6OFY - PubMed 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 3+ -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.
Organizational Affiliation:
Department of Structural Biology, Jacobs School of Medicine and Biomedical Sciences , University of Buffalo, the State University of New York , Buffalo , New York 14203 , United States.
