title: A dual substrate kinetic model for cytochrome P450BM3-F87G catalysis: simultaneous binding of long chain aldehydes and 4-fluorophenol.
abstract: OBJECTIVE
  To develop a model for binding and catalysis associated with the stimulation of 4-fluorophenol (4-FP) oxidation in the presence of long chain aldehydes by the enzymatic catalyst, cytochrome P450BM3-F87G.
  
  
  RESULTS
  A variation of the Michaeli-Menten kinetic model was employed to describe interactions at the active site of the enzyme, along with computer aided modeling approaches. In addition to the hydroquinone product arising from de-fluorination of 4-FP, a second product (p-fluorocatechol) was also observed and, like the hydroquinone, its rate of formation increased in the presence of the aldehyde. When only aldehyde was present with the enzyme, BM3-F87G catalyzed its oxidation to the corresponding carboxylic acid; however, this activity was inhibited when 4-FP was added to the reaction. A 3D computer model of the active site containing both aldehyde and 4-FP was generated, guided by these kinetic observations. Finally, partitioning between the two phenolic products was examined with an emphasis on the conditions directing the initial epoxidation at either the 2,3- or 3,4-positions on the substrate. Temperature, reaction time, substrate concentration, and the structure of the aldehyde had no substantial effect on the overall product ratios, however the NADPH coupling efficiency decreased when unsaturated aldehydes were included, or when the temperature of the reaction was reduced.
  
  
  CONCLUSIONS
  The unsaturated aldehyde, trans-2-decenal, stimulates BM3-F87G catalyzed oxidation of 4-fluorophenol through a cooperative active site binding mode that doesn't influence product distributions or coupling efficiencies, while 4-fluorophenol acts as a competitive inhibitor of aldehyde oxidation.
authors.affiliation: Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, Greensboro, NC, USA.
authors.forename: Chelsea
authors.initials: C
authors.lastname: Ledford
authors.affiliation: Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, Greensboro, NC, USA.
authors.forename: Monica
authors.initials: M
authors.lastname: McMahon
authors.affiliation: Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, Greensboro, NC, USA.
authors.forename: Ashley
authors.initials: A
authors.lastname: Whitesell
authors.affiliation: Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, Greensboro, NC, USA.
authors.forename: Ghalib
authors.initials: G
authors.lastname: Khan
authors.affiliation: Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, Greensboro, NC, USA.
authors.forename: Suneel K
authors.initials: SK
authors.lastname: Kandagatla
authors.affiliation: Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, Greensboro, NC, USA.
authors.forename: Dow P
authors.initials: DP
authors.lastname: Hurst
authors.affiliation: Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, Greensboro, NC, USA.
authors.forename: Patricia H
authors.initials: PH
authors.lastname: Reggio
authors.affiliation: Department of Biology and Chemistry, Liberty University, Lynchburg, VA, USA. graner@liberty.edu.
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Raner
mesh_terms: D000447:Aldehydes / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D007700:Kinetics; D010636:Phenols / Q000378:metabolism*
chemical_list: D000447:Aldehydes; D010636:Phenols; C106880:4-fluorophenol; D003577:Cytochrome P-450 Enzyme System
keywords: 4-Fluorophenol; Active site; Aldehydes; Benzoquinone; Cytochrome P450; De-fluorination