Open AccessConservative Secondary Shell Substitution In Cyclooxygenase-2 Reduces Inhibition by Indomethacin Amides and Esters via Altered Enzyme Dynamics
Author(s) -
Mary E. Konkle,
Anna L. Blobaum,
Christopher W. Moth,
Jeffery J. Prusakiewicz,
Shu Xu,
Kebreab Ghebreselasie,
Dapo Akingbade,
Aaron T. Jacobs,
Carol A. Rouzer,
Terry P. Lybrand,
Lawrence J. Marnett
Publication year - 2015
Publication title -
biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1520-4995
pISSN - 0006-2960
DOI - 10.1021/acs.biochem.5b01222
Subject(s) - chemistry , active site , enzyme , cyclooxygenase , moiety , stereochemistry , mutagenesis , amide , molecular dynamics , residue (chemistry) , biochemistry , mutant , computational chemistry , gene
The cyclooxygenase enzymes (COX-1 and COX-2) are the therapeutic targets of nonsteroidal anti-inflammatory drugs (NSAIDs). Neutralization of the carboxylic acid moiety of the NSAID indomethacin to an ester or amide functionality confers COX-2 selectivity, but the molecular basis for this selectivity has not been completely revealed through mutagenesis studies and/or X-ray crystallographic attempts. We expressed and assayed a number of divergent secondary shell COX-2 active site mutants and found that a COX-2 to COX-1 change at position 472 (Leu in COX-2, Met in COX-1) reduced the potency of enzyme inhibition by a series of COX-2-selective indomethacin amides and esters. In contrast, the potencies of indomethacin, arylacetic acid, propionic acid, and COX-2-selective diarylheterocycle inhibitors were either unaffected or only mildly affected by this mutation. Molecular dynamics simulations revealed identical equilibrium enzyme structures around residue 472; however, calculations indicated that the L472M mutation impacted local low-frequency dynamical COX constriction site motions by stabilizing the active site entrance and slowing constriction site dynamics. Kinetic analysis of inhibitor binding is consistent with the computational findings.