title: A Revised Mechanism for Human Cyclooxygenase-2.
abstract: The mechanism of ω-6 polyunsaturated fatty acid oxidation by wild-type cyclooxygenase 2 and the Y334F variant, lacking a conserved hydrogen bond to the catalytic tyrosyl radical/tyrosine, was examined for the first time under physiologically relevant conditions. The enzymes show apparent bimolecular rate constants and deuterium kinetic isotope effects that increase in proportion to co-substrate concentrations before converging to limiting values. The trends exclude multiple dioxygenase mechanisms as well as the proposal that initial hydrogen atom abstraction from the fatty acid is the first irreversible step in catalysis. Temperature dependent kinetic studies reinforce the novel finding that hydrogen transfer from the reduced catalytic tyrosine to a terminal peroxyl radical is the first irreversible step that controls regio- and stereospecific product formation.
authors.affiliation: From the Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218.
authors.forename: Yi
authors.initials: Y
authors.lastname: Liu
authors.affiliation: From the Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218 jproth@jhu.edu.
authors.forename: Justine P
authors.initials: JP
authors.lastname: Roth
mesh_terms: D016718:Arachidonic Acid / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D051546:Cyclooxygenase 2 / Q000737:chemistry / Q000378:metabolism*; D003903:Deuterium / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D019787:Linoleic Acid / Q000378:metabolism; D013058:Mass Spectrometry; D050505:Mutant Proteins / Q000378:metabolism; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D012997:Solvents; D013696:Temperature; D014783:Viscosity
chemical_list: D050505:Mutant Proteins; D012997:Solvents; D016718:Arachidonic Acid; D019787:Linoleic Acid; D003903:Deuterium; D051546:Cyclooxygenase 2
keywords: dioxygenase; enzyme mechanism; fatty acid; isotope effect; prostaglandin