Urate Oxidase Reaction Research Articles

Urate to allantoin, specifically (S)-allantoin

Enzymes that catalyze the formation of (S)-allantoin from the product of the urate oxidase reaction have been identified. This finding answers the longstanding question of how living organisms produce a single enantiomer of allantoin.

EPR Spin Trapping of a Radical Intermediate in the Urate Oxidase Reaction

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EPR Spin Trapping of a Radical Intermediate in the Urate Oxidase Reaction

Urate oxidase, or uricase (EC 1.7.3.3), is a peroxisomal enzyme that catalyses the oxidation of uric acid to allantoin. The chemical mechanism of the urate oxidase reaction has not been clearly established, but the involvement of radical intermediates was hypothesised. In this study EPR spectroscopy by spin trapping of radical intermediates has been used in order to demonstrate the eventual presence of radical transient urate species. The oxidation reaction of uric acid by several uricases (Porcine Liver, Bacillus Fastidiosus, Candida Utilitis) was performed in the presence of 5‐diethoxyphosphoryl‐5‐methyl‐pyrroline‐N‐oxide (DEPMPO) as spin trap. DEPMPO was added to reaction mixture and a radical adduct was observed in all cases. Therefore, for the first time, the presence of a radical intermediate in the uricase reaction was experimentally proved.

A familiar motif in a new context: the catalytic mechanism of hydroxyisourate hydrolase.

Hydroxyisourate hydrolase is a recently discovered enzyme that participates in the ureide pathway in soybeans. Its role is to catalyze the hydrolysis of 5-hydroxyisourate, the product of the urate oxidase reaction. There is extensive sequence homology between hydroxyisourate hydrolase and retaining glycosidases; in particular, the conserved active site glutamate residues found in retaining glycosidases are present in hydroxyisourate hydrolase as Glu 199 and Glu 408. However, experimental investigation of their roles, as well as the catalytic mechanism of the enzyme, have been precluded by the instability of 5-hydroxyisourate. Here, we report that diaminouracil serves as a slow, alternative substrate and can be used to investigate catalysis by hydroxyisourate hydrolase. The activity of the E199A protein was reduced 400-fold relative to wild-type, and no activity could be detected with the E408A mutant. Steady-state kinetic studies of the wild-type protein revealed that the pH-dependence of V(max) and V/K describe bell-shaped curves, consistent with the hypothesis that catalysis requires two ionizable groups in opposite protonation states. Addition of 100 mM azide accelerated the reaction catalyzed by the wild-type enzyme 8-fold and the E199A mutant 20-fold but had no effect on the E408A mutant. These data suggest that Glu 408 acts as a nucleophile toward the substrate forming a covalent anhydride intermediate, and Glu 199 facilitates formation of the intermediate by serving as a general acid and then activates water for hydrolysis of the intermediate. Thus, the mechanism of hydroxyisourate hydrolase is strikingly similar to that of retaining glycosidases, even though it catalyzes hydrolysis of an amide bond.

General base catalysis in the urate oxidase reaction: evidence for a novel Thr-Lys catalytic diad.

Urate oxidase catalyzes the oxidation of urate without the involvement of any cofactors. The gene encoding urate oxidase from Bacillus subtilis has been cloned and expressed, and the enzyme was purified and characterized. Formation of the urate dianion is believed to be a key step in the oxidative reaction. Rapid-mixing chemical quench studies provide evidence that the dianion is indeed an intermediate; at 15 degrees C the dianion forms within the mixing time of the rapid-quench instrument, and it disappears with a rate constant of 8 s(-)(1). Steady-state kinetic studies indicate that an ionizable group on the enzyme with a pK of 6.4 must be unprotonated for catalysis, and it is presumed that the role of this group is to abstract a proton from the substrate. Surprisingly, examination of the active site provided by the previously reported crystal structure does not reveal any obvious candidates to act as the general base. However, Thr 69 is hydrogen-bonded to the ligand at the active site, and Lys 9, which does not contact the ligand, is hydrogen-bonded to Thr 69. The T69A mutant enzyme has a V(max) that is 3% of wild type, and the K9M mutant enzyme has a V(max) that is 0.4% of wild type. The ionization at pH 6.4 that is observed with wild-type enzyme is absent in both of these mutants. It is proposed that these residues form a catalytic diad in which K9 deprotonates T69 to allow it to abstract the proton from the N9 position of the substrate to generate the dianion.

A colorimetric 96-well microtiter plate assay for the determination of urate oxidase activity and its kinetic parameters

Urate oxidase (E.C.1.7.3.3; uricase, urate oxygen oxidoreductase) is an enzyme of the purine breakdown pathway that catalyzes the oxidation of uric acid in the presence of oxygen to allantoin and hydrogen peroxide. A 96-well plate assay measurement of urate oxidase activity based on hydrogen peroxide quantitation was developed. The 96-well plate method included two steps: an incubation step for the urate oxidase reaction followed by a step in which the urate oxidase activity is stopped in the presence of 8-azaxanthine, a competitive inhibitor. Hydrogen peroxide is quantified during the second step by a horseradish peroxidase-dependent system. Under the defined conditions, uric acid, known as a radical scavenger, did not interfere with hydrogen peroxide quantification. The general advantages of such a colorimetric assay performed in microtiter plates, compared to other methods and in particular the classical UV method performed with cuvettes, are easy handling of large amounts of samples at the same time, the possibility of automation, and the need for less material. The method has been applied to the determination of the kinetic parameters of rasburicase, a recombinant therapeutic enzyme.

Urate oxidase: Single-turnover stopped-flow techniques for detecting two discrete enzyme-bound intermediates

Urate oxidase is a true oxidase; although the product is hydroxylated, the oxygen in the hydroxyl group derives from solvent, and one equivalent of dioxygen is reduced to H 2 0 2 concomitant with urate oxidation. In leguminous plants, urate oxidase is a constituent of the ureide pathway by which fixed nitrogen is converted to allantoin and allantoate, the metabolites that serve to transport nitrogen from the roots to the stems, leaves, and seed pods of the plant. In mammals urate oxidase facilitates the excretion of excess nitrogen by virtue of the greater solubility of allantoin relative to urate. Urate oxidase is also found in a wide variety of microorganisms, and some bacteria can grow on urate as their sole carbon and nitrogen source. The goals of these studies were both qualitative and quantitative. In particular, the mechanism illustrated in the chapter proposes the formation of three discrete intermediates in the course of the catalytic reaction, and stopped-flow studies were conducted to determine whether evidence could be adduced for their existence. An alternative mechanism for the urate oxidase reaction has been proposed that suggests that oxidation of urate occurs without formation of any discrete intermediates.

Evidence for Urate Hydroperoxide as an Intermediate in the Urate Oxidase Reaction

Evidence for Urate Hydroperoxide as an Intermediate in the Urate Oxidase Reaction

Identification and Purification of Hydroxyisourate Hydrolase, a Novel Ureide-metabolizing Enzyme

We report the identification and purification of a novel enzyme from soybean root nodules that catalyzes the hydrolysis of 5-hydroxyisourate, which is the true product of the urate oxidase reaction. The product of this reaction is 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, and the new enzyme is designated 5-hydroxyisourate hydrolase. The enzyme was purified from crude extracts of soybean root nodules approximately 100-fold to apparent homogeneity with a final specific activity of 10 micromol/min/mg. The enzyme exhibited a native molecular mass of approximately 68 kDa by gel filtration chromatography and migrated as a single band on SDS-polyacrylamide gel electrophoresis with a subunit molecular mass of 68 +/- 2 kDa. The purified enzyme obeyed normal Michaelis-Menten kinetics, and the K(m) for 5-hydroxyisourate was determined to be 15 microM. The amino-terminal end of the purified protein was sequenced, and the resulting sequence was not found in any available data bases, confirming the novelty of the protein. These data suggest the existence of a hitherto unrecognized enzymatic pathway for the formation of allantoin.

Theoretical Study of Intermediates in the Urate Oxidase Reaction

The energetics of the urate oxidase reaction has been studied employing HF, MP2, and DFT calculations. Gas-phase energies of possible urate oxidase reaction intermediates were calculated; radical species and dehydrourate had high relative energies while 5-hydroperoxy isourate was stable in the gas phase. Significant stabilization of the initial radical pair relative to reactants occurred in an aqueous environment, suggesting that oxidation of urate in water proceeds via a single-electron transfer pathway. The possibility of enzyme catalysis through proton abstraction from urate has been investigated by comparing the calculated properties of uric acid, urate monoanion, and urate dianion. Results indicate that ionization of uric acid is accompanied by polarization of the central carbon–carbon double bond and weaker binding of valence electrons.

Spectroscopic Characterization of Intermediates in the Urate Oxidase Reaction

The oxidation of urate catalyzed by soybean urate oxidase was studied under single-turnover conditions using stopped-flow absorbance and fluorescence spectrophotometry. Two discrete enzyme-bound intermediates were observed; the first intermediate to form had an absorbance maximum at 295 nm and was assigned to a urate dianion species; the second intermediate had an absorbance maximum at 298 nm and is believed to be urate hydroperoxide. These data are consistent with a catalytic mechanism that involves formation of urate hydroperoxide from O2 and the urate dianion, collapse of the peroxide to form dehydrourate, and hydration of dehydrourate to form the observed product, 5-hydroxyisourate. The rate of formation of the first intermediate was too fast to measure accurately at 20 degreesC; the second intermediate formed with a rate constant of 32 s-1 and decayed with a rate constant of 6.6 s-1. The product of the reaction, 5-hydroxyisourate, is fluorescent, and its release from the active site occurred with a rate constant of 31 s-1.

Identification of the True Product of the Urate Oxidase Reaction

The O2-dependent oxidation of urate catalyzed by urate oxidase has been examined in order to identify the immediate product of the enzymatic reaction. Specifically labeled [13C]urates were utilized as substrates, and the time courses were monitored by 13C NMR. On the basis of chemical shift values and 18O labeling, the product of the reaction was identified as 5-hydroxyisourate. This identification was substantiated by calculation of the 13C NMR spectrum of 5-hydroxyisourate using ab initio density functional theory methods. The predominant tautomers of urate and allantoin in aqueous solution were identified from 13C NMR titration data; the ionization behavior of urate and 5-hydroxyisourate were also examined by computational methods. The nonenzymatic pathway for production of allantoin from 5-hydroxyisourate was delineated; the reaction proceeds by the hydrolysis of the N1−C6 bond, followed by an unusual 1,2-carboxylate shift and decarboxylation to form allantoin.

Purification and properties of urate oxidase from Streptomyces cyanogenus.

Urate oxidase [EC 1.7.3.3] was purified to homogeneity from cell-free extracts of a strain of Streptomyces cyanogenus. The enzyme had a molecular weight of 100,000 and consisted of three subunits each with a molecular weight of 32,000. The isoelectric point was at pH 4.0. No evidence was found for the involvement of copper, iron or coenzymes in the urate oxidase reaction. The enzyme was most active at pH 8 and at 35 degrees C, and was stable between pH 6 and 11 (35 degrees C, 1 h) and below 50 degrees C (pH 7.8, 10 min). The enzyme was inhibited by cyanide and sulfhydryl reagents, but only slightly by heavy metal ions and chelating agents. The activity was inhibited by xanthine and 2-hydroxypurine. The enzyme was found not to be inhibited by high concentrations of uric acid when the activity was assayed in terms of hydrogen peroxide formation. Urea and racemic allantoin were formed from uric acid by the enzyme reaction in phosphate buffer, and urea and other ninhydrin-positive materials in borate buffer.