Azide inhibition of urate oxidase

Abstract

The inhibition of urate oxidase (UOX) by azide was investigated by X-ray diffraction techniques and compared with cyanide inhibition. Two well characterized sites for reagents are present in the enzyme: the dioxygen site and the substrate-binding site. To examine the selectivity of these sites towards azide inhibition, several crystallization conditions were developed. UOX was co-crystallized with azide (N3) in the presence or absence of either uric acid (UA, the natural substrate) or 8-azaxanthine (8AZA, a competitive inhibitor). In a second set of experiments, previously grown orthorhombic crystals of the UOX-UA or UOX-8AZA complexes were soaked in sodium azide solutions. In a third set of experiments, orthorhombic crystals of UOX with the exchangeable ligand 8-nitroxanthine (8NXN) were soaked in a solution containing uric acid and azide simultaneously (competitive soaking). In all assays, the soaking periods were either short (a few hours) or long (one or two months). These different experimental conditions showed that one or other of the sites, or the two sites together, could be inhibited. This also demonstrated that azide not only competes with dioxygen as cyanide does but also competes with the substrate for its enzymatic site. A model in agreement with experimental data would be an azide in equilibrium between two sites, kinetically in favour of the dioxygen site and thermodynamically in favour of the substrate-binding site.

Keywords: 8-azaxanthine; Aspergillus flavus; azide; cyanide; urate oxidase; uric acid; uricase.

Figure 1

(a) The catalyzed reaction leading to 5-hydroxyisourate (5-HIU). In solution, 5-HIU spontaneously evolves to (±)-allantoin with a lifetime of about 20 min. In vivo, 5-HIU is rapidly degraded to (+)-allantoin by another enzymatic system. (b) A schematic view of the substrate-binding site. In red, the minimal consensus for a chemical compound to be a ‘good’ ligand of UOX. The binding site at the interface of two subunits A and B is built by residues from two chains: A176 and A227–228 from the first subunit and B57–58 from the second. In addition (not shown), Phe159 from subunit A closely stacks with the six-membered ring of uric acid.

Figure 2

Co-crystallizations. (a) Contents of the substrate active site when UOX is crystallized in the presence of 0.3 M sodium azide. The dioxygen site (peroxo hole; Thr57* and Asn254) contains a water molecule, while an azide is bound in the substrate-binding site, filling the cavity with two structural water molecules (W2 and W3). A third water (W4) completes the hydrogen bonding but with weaker interactions. (b) The same result from competitive co-crystallizations of UOX with UA (saturated) and 0.3 M sodium azide. Omit maps are at the 3σ level in both cases.

Figure 3

Soaking of the UOX–UA and UOX–8AZA complexes with N3. (a) 8AZA, (b) UA. The substrate is locked in the active site whilst azide fills the dioxygen site. No change was observed when analyzing ‘aged’ crystals (harvested after one or two months).

Figure 4

‘Aged’ crystals from co-crystallizations: the evolution of the contents of the active sites in the case of the UOX–N3 crystals depicted in Fig. 2 ▶. Two azide anions fill both the dioxygen and the substrate-binding sites.