title: The reaction kinetics of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1 provide an understanding of the para-hydroxylation enzyme catalytic cycle.
abstract: 3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that catalyzes the para-hydroxylation of 3-hydroxybenzoate (3HB) to form 2,5-dihydroxybenzoate (2,5-DHB). Based on results from stopped-flow spectrophotometry, the reduced enzyme-3HB complex reacts with oxygen to form a C4a-peroxy flavin with a rate constant of 1.13 ± 0.01 × 10(6) m(-1) s(-1) (pH 8.0, 4 °C). This intermediate is subsequently protonated to form a C4a-hydroperoxyflavin with a rate constant of 96 ± 3 s(-1). This step shows a solvent kinetic isotope effect of 1.7. Based on rapid-quench measurements, the hydroxylation occurs with a rate constant of 36 ± 2 s(-1). 3HB6H does not exhibit substrate inhibition on the flavin oxidation step, a common characteristic found in most ortho-hydroxylation enzymes. The apparent kcat at saturating concentrations of 3HB, NADH, and oxygen is 6.49 ± 0.02 s(-1). Pre-steady state and steady-state kinetic data were used to construct the catalytic cycle of the reaction. The data indicate that the steps of product release (11.7 s(-1)) and hydroxylation (36 ± 2 s(-1)) partially control the overall turnover.
authors.affiliation: From the Department of Biochemistry, Faculty of Dentistry, Chulalongkorn University, Henri Dunant Road, Patumwan, Bangkok 10330, Thailand.
authors.forename: Jeerus
authors.initials: J
authors.lastname: Sucharitakul
authors.forename: Chanakan
authors.initials: C
authors.lastname: Tongsook
authors.forename: Danaya
authors.initials: D
authors.lastname: Pakotiprapha
authors.forename: Willem J H
authors.initials: WJ
authors.lastname: van Berkel
authors.forename: Pimchai
authors.initials: P
authors.lastname: Chaiyen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D006900:Hydroxylation; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D012240:Rhodococcus / Q000201:enzymology*; D013053:Spectrophotometry
chemical_list: D001426:Bacterial Proteins; D009243:NAD; D006899:Mixed Function Oxygenases; C023315:3-hydroxybenzoate-6-hydroxylase
keywords: Enzyme Kinetics; Enzyme Mechanisms; Flavin; Flavoproteins; Hydroxylase