Seidel Antonia, Parker Heather, Turner Rufus, Dickerhof Nina, Khalilova Irada S, Wilbanks Sigurd M, Kettle Anthony J, Jameson Guy N L
From the Department of Chemistry, University of Otago, P. O. Box 56, Dunedin 9054.
the Centre for Free Radical Research, Department of Pathology, University of Otago, Christchurch, Christchurch 8140, New Zealand, and.
J Biol Chem. 2014 Aug 8;289(32):21937-49. doi: 10.1074/jbc.M113.544957. Epub 2014 Jun 13.
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.
尿酸盐的生理功能目前还知之甚少。它可能作为一种危险信号、抗氧化剂或血红素过氧化物酶的底物。尿酸盐能否与乳过氧化物酶（LPO）快速反应以充当生理底物仍不清楚。LPO是一种哺乳动物过氧化物酶，通过将硫氰酸盐氧化为具有杀菌和杀真菌作用的次硫氰酸盐，在先天免疫防御中起关键作用。我们现在证明尿酸盐是牛LPO的良好底物。尿酸盐被LPO氧化生成亲电中间体脱水尿酸盐和5-羟基异尿酸盐，它们随后分解为尿囊素。在超氧化物存在的情况下，LPO和尿酸盐会形成高产率的氢过氧化物。使用停流光谱法，我们测定了尿酸盐与化合物I（k1 = 1.1×10⁷ M⁻¹ s⁻¹）和化合物II（k2 = 8.5×10³ M⁻¹ s⁻¹）反应的速率常数。在尿酸盐氧化过程中，LPO偏离了其过氧化物酶循环，因为过氧化氢与化合物II反应生成了化合物III。在生理相关浓度下，尿酸盐有效地与硫氰酸盐竞争，硫氰酸盐是LPO氧化的主要底物，并抑制了次硫氰酸盐的产生。同样，尿酸盐抑制了铜绿假单胞菌依赖次硫氰酸盐的杀伤作用。尿囊素存在于人类唾液中，并与LPO的浓度相关。当向唾液中加入过氧化氢时，尿酸盐的氧化取决于其浓度和过氧化物酶活性。我们的研究结果表明尿酸盐可能是LPO的生理底物，这将影响宿主防御并产生亲电活性代谢产物。
