Reductive Half-reaction Of Xanthine Oxidase Research Articles

Computational exploration of reactive fragment for mechanism-based inhibition of xanthine oxidase

The xanthine oxidase (XO) family is a group of molybdenum-containing enzymes that catalyze the transfer of an oxygen atom from water to a substrate, such as xanthine or aldehyde. The proposed mechanism for the reductive half-reaction of xanthine oxidase involves nucleophilic addition of Mo-bound hydroxide to the substrate and hydride transfer from the substrate to sulfido group (Mo=S) at the molybdenum cofactor (Moco) reaction center. A DFT study of a mechanism involving the near-by glutamic acid (Glu) residue for the oxidation of several model substrates found that the direct involvement of the Glu residue side chain significantly increases the reactivity of the cofactor. The reaction is stepwise with a hydride transfer as the rate determining step. On the basis of mechanistic studies, a QM model was established to search the potential reactive fragments for mechanism-based inhibitors. Two important parameters were designed to evaluate each fragment: the activation enthalpy (ΔH≠) which estimates how effectively an inhibitor competes with xanthine to react with the Moco site, and the enthalpy associated with release of the product (ΔH) which estimates how easily the product in product complex (PRO) could be replaced by a water molecule. The computational findings add to our understanding of the catalytic mechanism of xanthine oxidase, and also assist the screening of mechanism-based inhibitors for this important drug target.

A Combined QM/MM Study on the Reductive Half-Reaction of Xanthine Oxidase: Substrate Orientation and Mechanism

Quantum mechanical/molecular mechanical (QM/MM) methods were used to investigate the conversion of xanthine to uric acid in xanthine oxidase. Seven mechanistic variants were considered with different tautomeric forms of xanthine, different protonation states of the active-site residues, and different substrate orientations. The most favorable pathway (setup G) has a B3LYP/MM barrier of about 14 kcal mol(-1), consistent with the available experimental data. This multistep mechanism starts with Glu1261 deprotonating the xanthine at the N3 position followed by a proton transfer from the cofactor to the N9 atom of xanthine; the thus activated cofactor and substrate then react to form a tetrahedral intermediate, and a subsequent rate-limiting hydride transfer generates the product. The substrate orientation that has commonly been assumed in the literature leads to the most stable reactant complex, but the opposite orientation ("upside down") is computed to be the most favorable one during the reaction (setup G). In the "upside down" conformation, the Arg880 residue can best stabilize the reactive xanthine species with the negatively charged N3 atom, especially the tetrahedral intermediate and the following transition state for hydride transfer which is generally the highest point on the computed energy profiles. QM-only calculations for a minimal gas-phase model and for larger cluster models are performed for comparison, in particular for establishing intrinsic reactivities and a common energy scale. An analysis of the computational results provides detailed insight into the essential mechanistic role of the active-site residues.

A Theoretical Study on the Mechanism of the Reductive Half-Reaction of Xanthine Oxidase

On the basis of the crystal structure of an aldehyde oxidoreductase, Huber et al. proposed a catalytic mechanism for the reductive half-reaction of xanthine oxidase which involves nucleophilic addition of Mo-bound hydroxide (Moco 1) to the substrate and hydride transfer from the substrate to sulfido group (Mo=S). Density functional theory calculations have been carried out for the oxidation of formaldehyde, acetaldehyde, formamide, and formamidine with Moco 2 to understand more detailed catalytic pathways. Our calculation results indicate that the anionic catalyst model acts as a nucleophile and is reactive for the oxidation of aldehyde substrates, which are reactive for nucleophilic addition. In these cases, a concerted mechanism is found to be more favorable than a stepwise mechanism. The concerted mechanism is further shown to be promoted by the presence of a nearby water molecule, in the active site, which serves as a Lewis acid for the nucleophilic addition of hydroxide. For less reactive formamide and formamidine (a model for xanthine) substrates, the calculated activation energies with the above mechanisms are high. These reactions also do not benefit from the presence of the water molecule. The results indicate that different catalyst forms might be responsible for the oxidation of different substrates, which could be regulated by the enzyme active site environment.

Kinetics and thermodynamics of the molecular mechanism of the reductive half-reaction of xanthine oxidase.

The kinetics and thermodynamics of the reductive half-reaction of xanthine oxidase with xanthine as substrate have been investigated by stopped-flow kinetic measurements. The temperature dependence of the steady-state and transient kinetics of the reductive half-reaction reveals the existence of at least three molecular intermediates during this half-reaction. All the microscopic rate constants and the thermodynamic activation parameters of the elementary steps of the reductive half-reaction have been determined for the first time. The microscopic rate constants and the thermodynamic activation parameters of the individual steps show wide variations in their magnitudes. The present work provides the most detailed and incisive description of the reaction of xanthine oxidase with its physiological substrate xanthine.

Reductive half-reaction of xanthine oxidase: Mechanistic role of the species giving rise to the "rapid type 1" molybdenum(V) electron paramagnetic resonance signal

The reaction of xanthine oxidase with xanthine, 1-methylxanthine, and 2-hydroxy-6-methylpurine has been reinvestigated with the aim of elucidating the mechanistic role of the species giving rise to the "rapid" Mo(V) electron paramagnetic resonance (EPR) signal. It is found that addition of 2.0 mM 1-methylxanthine or 2-hydroxy-6-methylpurine to partially reduced enzyme generates substantial amounts of the Type 1 form of the "rapid" EPR signal, characterized by superhyperfine coupling to one strongly interacting (aav = 13 G) and one weakly interacting (aav = 3 G) proton. The "rapid" signals observed with both substrates are identical to those observed in the course of the anaerobic reaction of enzyme with a stoichiometric excess of substrate. With 2-hydroxy-6-methylpurine at pH 10, a burst phase in the formation of the species giving rise to the "rapid Type 1" signal is observed that is fast relative to the rate of formation of the species giving rise to the "very rapid" EPR signal. At pH 8.5, partial reduction of enzyme prior to reaction with xanthine, 1-methylxanthine, or 2-hydroxy-6-methylpurine reverses the relative amounts of "rapid" and "very rapid" EPR signal observed at the shortest reaction times. The substantial amounts of "rapid Type 1" signal formed by addition of substrates to partially reduced enzyme or by reaction of oxidized enzyme with a stoichiometric excess of substrate contrasts with previous work, which has shown that under single-turnover conditions none of the substrates investigated generates an appreciable amount of "rapid" EPR signal.(ABSTRACT TRUNCATED AT 250 WORDS)

Reductive half-reaction of xanthine oxidase with xanthine. Observation of a spectral intermediate attributable to the molybdenum center in the reaction of enzyme with xanthine.

The reductive half-reaction of xanthine oxidase with substoichiometric concentrations of xanthine and 1-methylxanthine at pH 10 and 8.5 has been examined by UV-visible stopped-flow and rapid-quench electron paramagnetic resonance (EPR) kinetic experiments. A spectral intermediate is observed in stopped-flow experiments with xanthine which exhibits a difference absorbance maximum relative to oxidized enzyme at 480 nm and which decays at the same rate as the decay of the "very rapid" MoV EPR signal observed by freeze-quench EPR experiments both at 5 and 20 degrees C. The intermediate is observed in experiments using enzyme that has had its flavin removed, and most likely arises from the molybdenum center. With 40 microM xanthine oxidase and 10 microM xanthine, rate constants for the appearance and decay of this intermediate at pH 10 are 11 and 1.1 s-1, respectively; at pH 8.5 the corresponding values are 20 and 2.5 s-1. Based on the correlation of the stopped-flow kinetics with the appearance and decay of the MoV EPR signal designated very rapid as monitored in freeze-quench experiments, it is concluded that the spectral intermediate corresponds to the species exhibiting the very rapid EPR signal, the MoIV species that gives rise to it, or a combination of the two. None of the MoV EPR signals designated "rapid" is observed under single turnover conditions with either xanthine or 1-methylxanthine as substrate at pH 8.5, by contrast with the substantial amounts observed in both cases under conditions of excess substrate. These results call into question the prevailing view that the species giving rise to the rapid EPR signal lies downstream in the catalytic cycle from that exhibiting the very rapid signal.

The reductive half-reaction of xanthine oxidase. Identification of spectral intermediates in the hydroxylation of 2-hydroxy-6-methylpurine.

The reaction of xanthine oxidase with 2-hydroxy-6-methylpurine (also called 2-oxo-6-methylpurine) has been studied under both anaerobic and aerobic conditions. Reaction of enzyme with substoichiometric concentrations of hydroxymethylpurine in aerobic 0.1 M 3-(cyclohexylamino)propanesulfonic acid, 0.1 N KCl, 0.3 mM EDTA, pH 10.0, exhibits two reaction intermediates detectable by UV-visible spectrophotometry. The rate constants for formation of the first intermediate, conversion of the first to the second, and the decay of the second to give oxidized enzyme are 18, 1.2, and 0.13 s-1, respectively. The difference spectra of these two intermediates relative to oxidized enzyme are characterized by absorbance maxima at 470 and 540 nm, respectively, with extinction changes (relative to oxidized enzyme) of approximately 410 M-1 cm-1. The 0.13 s-1 decay of the second intermediate agrees well with kcat of 0.11 s-1 determined under the same conditions. Based on a comparison of the kinetics of the reaction as monitored by UV-visible absorption and electron paramagnetic resonance spectrometry, it is concluded that these spectral intermediates arise from the molybdenum center of the enzyme in the MoIV and MoV valence states, respectively, the latter corresponding to the species exhibiting the "very rapid" MoV EPR signal known to be formed in the course of the reaction. This conclusion is supported by the results of experiments using cytochrome c reduction to follow the formation of superoxide production in the course of the aerobic reaction of xanthine oxidase with substoichiometric hydroxymethylpurine, which demonstrate unequivocally that the species exhibiting the very rapid EPR signal is formed by one-electron oxidation of a MoIV species rather than direct one-electron reduction of MoVI by substrate. No evidence is found for the formation of any of the MoV EPR signals designated "rapid" in the present studies, and it is concluded that this species is not a bona fide catalytic intermediate in the reductive half-reaction of xanthine oxidase.

The reductive half-reaction of xanthine oxidase: Spectral intermediates in the hydroxylation of 2-hydroxy-6-methylpurine

The reductive half-reaction of xanthine oxidase: Spectral intermediates in the hydroxylation of 2-hydroxy-6-methylpurine

Influence and role of metal ions in enzymatic catalysis with e.c. 1.2.3.2. xanthine oxidase: Application to trace analysis

The effect of metal ions on the reductive half-reaction of xanthine oxidase (XOD) in the catalytic conversion of xanthine into uric acid has been studied spectrophotometrically in Tris-HCl buffer at pH 7.4, 37 ± 0.1° and ionic strength 0.04 M. Some metal ions display inhibitor properties, the sequence of inhibiting efficiency being Ag(I) > Hg(II) > Cu(II) > Cr(VI) > V(V) > Au(III) > Tl(I) and for these the I 50 values were determined. Only Tl(I), V(V) and Cu(II) showed reversible inhibition and therefore for these the mechanisms were assessed [competitive for V(V) and Tl(I); uncompetitive for Cu(II)]. The conditional inhibition constants ( K i) were also determined. The effect of EDTA for protection of the enzyme against metal inhibition, and for its reactivation after inhibition, was also investigated. Utilization of the linear relationship between relative enzyme activity and inhibitor concentration allowed sensitive and selective (though not specific) determination of Ag(I) and Hg(II) (10 −9−10 −8 M), and of Cu(II) and Cr(VI) (10 −7-10 −6 M), the maximum relative error being ± 4%. For a few metal ions, e.g., Ag(I) and Cr(VI), in the presence of EDTA, a certain specificity is observed.