Functional analysis of the molecular determinants of cyclooxygenase-2 acetylation by 2-acetoxyphenylhept-2-ynyl sulfide.
Selective inhibition of cyclooxygenase-2 (COX-2) leads to relief of pain and inflammation with reduced gastrointestinal side effects relative to nonsteroidal anti-inflammatory drugs. 2-Acetoxyphenylhept-2-ynyl sulfide (APHS) is a selective COX-2 inhibitor that covalently modifies the protein by acetylating Ser-530. We utilized site-directed mutants in the COX-2 active site to probe the molecular determinants of APHS acetylation of COX-2. Incorporation of acetyl groups into Ser-530 was monitored by HPLC and mass spectrometry. Mutations that introduce steric bulk into a channel at the top of the active site (e.g., G533A, G533V) lead to a significant reduction in APHS acetylation. Reduction in acetylation is also observed by mutation of the active-site tyrosine (Tyr-385) to phenylalanine. Mutations in the side-pocket region, into which diarylheterocycle inhibitors insert, do not affect the ability of APHS to acetylate COX-2. Surprisingly, mutation of Arg-120, which is located on the floor of the active site, strongly reduces acetylation. Based on these results, we propose that the heptynyl side chain of APHS inserts into the top channel and acetylates Ser-530 with the assistance of hydrogen bonding from Tyr-385. Arg-120 is proposed to fix the conformation of the active site to one that favors acetylation.
Phillip Hochgesang
G
G
Department of Biochemistry, Vanderbilt Institute of Chemical Biology, Nashville, TN 37232-0146, USA.
Nemeth-Cawley
Jennifer F
JF
Rowlinson
Scott W
SW
Caprioli
Richard M
RM
Marnett
Lawrence J
LJ
D000480:Alkynes; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000494:pharmacology; D051546:Cyclooxygenase 2; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006536:Heptanes / Q000494:pharmacology*; D006859:Hydrogen / Q000378:metabolism; D006860:Hydrogen Bonding; D007313:Insecta; D007527:Isoenzymes / Q000378:metabolism*; D008024:Ligands; D013058:Mass Spectrometry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D011487:Protein Conformation; D012694:Serine / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013440:Sulfides / Q000494:pharmacology*; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
D000480:Alkynes; D004791:Enzyme Inhibitors; D006536:Heptanes; D007527:Isoenzymes; D008024:Ligands; D013440:Sulfides; C112390:o-(acetoxyphenyl)hept-2-ynyl sulfide; D014443:Tyrosine; D012694:Serine; D006859:Hydrogen; D001120:Arginine; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D003488:Cyanogen Bromide