title: Uric acid and thiocyanate as competing substrates of lactoperoxidase.
abstract: The physiological function of urate is poorly understood. It may act as a danger signal, an antioxidant, or a substrate for heme peroxidases. Whether it reacts sufficiently rapidly with lactoperoxidase (LPO) to act as a physiological substrate remains unknown. LPO is a mammalian peroxidase that plays a key role in the innate immune defense by oxidizing thiocyanate to the bactericidal and fungicidal agent hypothiocyanite. We now demonstrate that urate is a good substrate for bovine LPO. Urate was oxidized by LPO to produce the electrophilic intermediates dehydrourate and 5-hydroxyisourate, which decayed to allantoin. In the presence of superoxide, high yields of hydroperoxides were formed by LPO and urate. Using stopped-flow spectroscopy, we determined rate constants for the reaction of urate with compound I (k1 = 1.1 × 10(7) M(-1) s(-1)) and compound II (k2 = 8.5 × 10(3) M(-1) s(-1)). During urate oxidation, LPO was diverted from its peroxidase cycle because hydrogen peroxide reacted with compound II to give compound III. At physiologically relevant concentrations, urate competed effectively with thiocyanate, the main substrate of LPO for oxidation, and inhibited production of hypothiocyanite. Similarly, hypothiocyanite-dependent killing of Pseudomonas aeruginosa was inhibited by urate. Allantoin was present in human saliva and associated with the concentration of LPO. When hydrogen peroxide was added to saliva, oxidation of urate was dependent on its concentration and peroxidase activity. Our findings establish urate as a likely physiological substrate for LPO that will influence host defense and give rise to reactive electrophilic metabolites.
authors.affiliation: From the Department of Chemistry, University of Otago, P. O. Box 56, Dunedin 9054.
authors.forename: Antonia
authors.initials: A
authors.lastname: Seidel
authors.affiliation: the Centre for Free Radical Research, Department of Pathology, University of Otago, Christchurch, Christchurch 8140, New Zealand, and.
authors.forename: Heather
authors.initials: H
authors.lastname: Parker
authors.affiliation: the Centre for Free Radical Research, Department of Pathology, University of Otago, Christchurch, Christchurch 8140, New Zealand, and.
authors.forename: Rufus
authors.initials: R
authors.lastname: Turner
authors.affiliation: the Centre for Free Radical Research, Department of Pathology, University of Otago, Christchurch, Christchurch 8140, New Zealand, and.
authors.forename: Nina
authors.initials: N
authors.lastname: Dickerhof
authors.affiliation: the Centre for Free Radical Research, Department of Pathology, University of Otago, Christchurch, Christchurch 8140, New Zealand, and.
authors.forename: Irada S
authors.initials: IS
authors.lastname: Khalilova
authors.affiliation: the Department of Biochemistry, University of Otago, P. O. Box 56, Dunedin 9054, New Zealand.
authors.forename: Sigurd M
authors.initials: SM
authors.lastname: Wilbanks
authors.affiliation: the Centre for Free Radical Research, Department of Pathology, University of Otago, Christchurch, Christchurch 8140, New Zealand, and.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Kettle
authors.affiliation: From the Department of Chemistry, University of Otago, P. O. Box 56, Dunedin 9054, gjameson@chemistry.otago.ac.nz.
authors.forename: Guy N L
authors.initials: GN
authors.lastname: Jameson
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism; D001667:Binding, Competitive; D002417:Cattle; D006801:Humans; D007113:Immunity, Innate; D007700:Kinetics; D007784:Lactoperoxidase / Q000276:immunology / Q000378:metabolism*; D008954:Models, Biological; D010084:Oxidation-Reduction; D011550:Pseudomonas aeruginosa / Q000276:immunology; D012463:Saliva / Q000276:immunology / Q000378:metabolism; D013379:Substrate Specificity; D013861:Thiocyanates / Q000378:metabolism*; D014527:Uric Acid / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D013861:Thiocyanates; D014527:Uric Acid; C014607:hypothiocyanite ion; D007784:Lactoperoxidase; C031760:thiocyanate
keywords: Host Defense; Kinetics; Peroxidase; Pre-steady-state Kinetics; Uric Acid