Conservative Secondary Shell Substitution In Cyclooxygenase-2 Reduces Inhibition by Indomethacin Amides and Esters via Altered Enzyme Dynamics

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• 作者：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
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：January 19, 2016
• 年：2016
• 卷：55
• 期：2
• 页码：348-359
• 全文大小：676K
• ISSN：1520-4995

文摘

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.