title: Evidence for flavin movement in the function of p-hydroxybenzoate hydroxylase from studies of the mutant Arg220Lys.
abstract: The isoalloxazine ring system of the FAD cofactor of p-hydroxybenzoate hydroxylase must be secluded from solvent at specific stages of catalysis in order to form and stabilize a flavin C4a-hydroperoxide. This species may then react with the activated phenolate of p-hydroxybenzoate. A number of crystal structures of the enzyme with alterations to active site substituents or complexes with analogue benzoates have revealed an alternate position for the isoalloxazine (Gatti et al. (1994) Science 266, 110-114; Schreuder et al. (1994) Biochemistry 33, 10161-10170). This new flavin conformation is 7 A "out" toward solvent and may open a passage for substrate entry to the active site. Arginine 220 is one of the few residues in the structure to demonstrate conformational changes when the flavin is "out". In this study we have made the Arg220Lys mutant to test the significance of this residue in flavin movement. The R220K mutation has brought about dramatic alterations to all aspects of catalysis. Stopped flow kinetic characterization of the mutant has revealed that, while the effector role for the substrate is maintained, there exists an order of magnitude decrease in the limiting rate of reduction, even though there is 40-fold increase in association with NADPH. The mutant enzyme has only a fraction of its reductive half-reaction coupled to product formation, and the hydroxylation process is slow. This occurs despite a higher proportion of the more activated substrate phenolate in the active site. Many of the observed changes can be attributed to a decrease in the stability of the "in" conformation of the flavin during the catalysis and indicate a role for flavin conformational states in many of the catalytic processes of the enzyme.
authors.affiliation: Department of Molecular and Cellular Biology, University of New England Armidale, New South Wales, Australia.
authors.forename: G R
authors.initials: GR
authors.lastname: Moran
authors.forename: B
authors.initials: B
authors.lastname: Entsch
authors.forename: B A
authors.initials: BA
authors.lastname: Palfey
authors.forename: D P
authors.initials: DP
authors.lastname: Ballou
mesh_terms: D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005415:Flavins / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010226:Parabens / Q000378:metabolism; D011487:Protein Conformation; D013053:Spectrophotometry
chemical_list: D005415:Flavins; D010226:Parabens; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D001120:Arginine; D015109:4-Hydroxybenzoate-3-Monooxygenase; C038193:4-hydroxybenzoic acid; D008239:Lysine