title: Structural and kinetic study of an internal substrate binding site in dehaloperoxidase-hemoglobin A from Amphitrite ornata.
abstract: X-ray crystal structures of dehaloperoxidase-hemoglobin A (DHP A) from Amphitrite ornata soaked with substrate, 2,4,6-tribromophenol (2,4,6-TBP), in buffer solvent with added methanol (MeOH), 2-propanol (2-PrOH), and dimethyl sulfoxide (DMSO) reveal an internal substrate binding site deep in the distal pocket above the α-edge of the heme that is distinct from the previously determined internal inhibitor binding site. The peroxidase function of DHP A has most often been studied using 2,4,6-trichlorophenol (2,4,6-TCP) as a substrate analogue because of the low solubility of 2,4,6-TBP in an aqueous buffer solution. Previous studies at low substrate concentrations pointed to the binding of substrate 2,4,6-TCP at an external site near the exterior heme β- or δ-edge as observed in the class of heme peroxidases. Here we report that the turnover frequencies of both substrates 2,4,6-TCP and 2,4,6-TBP deviate from Michaelis-Menten kinetics at high concentrations. The turnover frequency reaches a maximum in the range of 1400-1700 μM, with a decrease in rate at higher concentrations that is both substrate- and solvent-dependent. The X-ray crystal structure is consistent with the presence of an internal active site above the heme α-edge, in which the substrate would be oxidized in two consecutive steps inside the enzyme, followed by attack by H2O via a water channel in the protein. The physiological role of the internal site may involve interactions with any of a number of aromatic toxins found in benthic ecosystems where A. ornata resides.
authors.affiliation: Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhao
authors.forename: Vesna
authors.initials: V
authors.lastname: de Serrano
authors.forename: Junjie
authors.initials: J
authors.lastname: Zhao
authors.forename: Peter
authors.initials: P
authors.lastname: Le
authors.forename: Stefan
authors.initials: S
authors.lastname: Franzen
mesh_terms: D019840:2-Propanol / Q000737:chemistry; D000818:Animals; D018360:Crystallography, X-Ray; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D000432:Methanol / Q000737:chemistry; D008958:Models, Molecular; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism; D010636:Phenols / Q000737:chemistry; D011077:Polychaeta / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D011487:Protein Conformation
chemical_list: D006454:Hemoglobins; D010636:Phenols; C098661:DHP I dehaloperoxidase; D010544:Peroxidases; D019840:2-Propanol; D000432:Methanol; D004121:Dimethyl Sulfoxide; C004554:2,4,6-tribromophenol