title: Evidence of Negative Cooperativity and Half-Site Reactivity within an F420-Dependent Enzyme: Kinetic Analysis of F420H2:NADP(+) Oxidoreductase.
abstract: Here, we report the very first example of half-site reactivity and negative cooperativity involving an important F420 cofactor-dependent enzyme. F420H2:NADP(+) oxidoreductase (Fno) is an F420 cofactor-dependent enzyme that catalyzes the reversible reduction of NADP(+) through the transfer of a hydride from the reduced F420 cofactor. These catalytic processes are of major significance in numerous biochemical processes. While the steady-state kinetic analysis showed classic Michaelis-Menten kinetics with varying concentrations of the F420 redox moiety, FO, such plots revealed non-Michaelis-Menten kinetic behavior when NADPH was varied. The double reciprocal plot of the varying concentrations of NADPH displays a downward concave shape, suggesting that negative cooperativity occurs between the two identical monomers. The transient state kinetic data show a burst prior to entering steady-state turnover. The burst suggests that product release is rate-limiting, and the amplitude of the burst phase corresponds to production of product in only one of the active sites of the functional dimer. These results suggest either half-site reactivity or an alternate sites model wherein the reduction of the cofactor, FO occurs at one active site at a time followed by reduction at the second active site. Thus, the data imply that Fno may be a functional regulatory enzyme.
authors.affiliation: Department of Chemistry and Biochemistry, University of Texas at Arlington , Arlington, Texas 76019-0065, United States.
authors.forename: Ebenezer
authors.initials: E
authors.lastname: Joseph
authors.affiliation: Department of Chemistry and Biochemistry, University of Texas at Arlington , Arlington, Texas 76019-0065, United States.
authors.forename: Cuong Quang
authors.initials: CQ
authors.lastname: Le
authors.affiliation: Department of Chemistry and Biochemistry, University of Texas at Arlington , Arlington, Texas 76019-0065, United States.
authors.forename: Toan
authors.initials: T
authors.lastname: Nguyen
authors.affiliation: Department of Chemistry and Biochemistry, University of Texas at Arlington , Arlington, Texas 76019-0065, United States.
authors.forename: Mercy
authors.initials: M
authors.lastname: Oyugi
authors.affiliation: Department of Chemistry and Biochemistry, University of Texas at Arlington , Arlington, Texas 76019-0065, United States.
authors.forename: Mohammad Shawkat
authors.initials: MS
authors.lastname: Hossain
authors.affiliation: Department of Chemistry and Biochemistry, University of Texas at Arlington , Arlington, Texas 76019-0065, United States.
authors.forename: Frank W
authors.initials: FW
authors.lastname: Foss
authors.affiliation: Department of Chemistry and Biochemistry, University of Texas at Arlington , Arlington, Texas 76019-0065, United States.
authors.forename: Kayunta
authors.initials: K
authors.lastname: Johnson-Winters
mesh_terms: D000465:Algorithms; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019611:Archaeoglobus fulgidus / Q000201:enzymology*; D055162:Biocatalysis; D020134:Catalytic Domain; D019281:Dimerization; D006860:Hydrogen Bonding; D008024:Ligands; D008958:Models, Molecular; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012256:Riboflavin / Q000031:analogs & derivatives* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D019843:Archaeal Proteins; D008024:Ligands; D011994:Recombinant Proteins; D009249:NADP; C015084:coenzyme F420; D009247:NADH, NADPH Oxidoreductases; C109941:F420-dependent NADP reductase; D012256:Riboflavin