Substrate Orientation and Catalytic Specificity in the Action of Xanthine Oxidase

Abstract

Xanthine oxidase is a molybdenum-containing enzyme catalyzing the hydroxylation of a sp 2 -hybridized carbon in a broad range of aromatic heterocycles and aldehydes. Crystal structures of the bovine enzyme in complex with the physiological substrate hypoxanthine at 1.8 Å resolution and the chemotherapeutic agent 6-mercaptopurine at 2.6 Å resolution have been determined, showing in each case two alternate orientations of substrate in the two active sites of the crystallographic asymmetric unit. One orientation is such that it is expected to yield hydroxylation at C-2 of substrate, yielding xanthine. The other suggests hydroxylation at C-8 to give 6,8-dihydroxypurine, a putative product not previously thought to be generated by the enzyme. Kinetic experiments demonstrate that >98% of hypoxanthine is hydroxylated at C-2 rather than C-8, indicating that the second crystallographically observed orientation is significantly less catalytically effective than the former. Theoretical calculations suggest that enzyme selectivity for the C-2 over C-8 of hypoxanthine is largely due to differences in the intrinsic reactivity of the two sites. For the orientation of hypoxanthine with C-2 proximal to the molybdenum center, the disposition of substrate in the active site is such that Arg 880 and Glu 802 , previous shown to be catalytically important for the conversion of xanthine to uric acid, play similar roles in hydroxylation at C-2 as at C-8. Contrary to the literature, we find that 6,8-dihydroxypurine is effectively converted to uric acid by xanthine oxidase.Purine oxidation in nature is catalyzed by three distinct classes of enzymes: the molybdenum-containing hydroxylases such as xanthine oxidoreductase (1, 2), the Fe II -and ␣-ketoglutarate-dependent xanthine hydroxylases (3, 4), and a newly described two-component system, HpxDE, consisting of a [2Fe-2S]/flavin-containing reductase and a Rieske/non-heme iron-containing oxygenase (5, 6). The first class is by far the most broadly distributed, with members found throughout the eubacteria, archaea, and eukaryota. The second class is found principally in fungae, including yeasts (Saccharomyces cerevisiae, for example), and the third is identified to date only in Klebsiella oxytoca and Klebsiella pneumoniae.Xanthine oxidoreductases from eukaryotes are homodimers of ϳ290 kDa, with each monomer containing four redox-active sites: an active site molybdenum center, a pair of spinach ferredoxin-like [2Fe-2S] clusters, and FAD (7). The overall catalytic sequence consists of a reductive half-reaction in which substrate is oxidatively hydroxylated at the molybdenum center (reducing it from Mo(VI) to Mo(IV)) and, after intramolecular electron transfer, an oxidative half-reaction in which reducing equivalents are removed from the enzyme via its FAD. In the reductive half-reaction, purine substrates are hydroxylated at a specific carbon position in a reaction initiated by nucleophilic attack of an equatorial Mo-OH group of the metal center whose deprotonation is thought to be facilitate...

The First Mammalian Aldehyde Oxidase Crystal Structure

“…6, Table 2). It was suggested from the XDH crystal structure that Glu-802 and Arg-880 are essential for the correct positioning/orienting and/or activation of the substrate through the establishment of hydrogen bonds and electrostatic interactions (36), as found in all structures of XOR complexed to inhibitors or substrates (29,(37)(38)(39)(40). Glu-802 in bXDH is replaced by a valine in all AOX1 orthologs and by an alanine (Ala-807) in mAOX3.…”

In Vitro Oxidative Metabolism of 6-Mercaptopurine in Human Liver: Insights into the Role of the Molybdoflavoenzymes Aldehyde Oxidase, Xanthine Oxidase, and Xanthine Dehydrogenase

“…The results are shown in Fig. 2 crystallography study has shown that both 6MP and hypoxanthine bind in two orientations that could lead to either oxidation on the five-membered or six-membered ring of either substrate (Cao et al, 2010). While 6MP metabolism was not explored, this study concluded that xanthine (oxidation on the six-membered ring) was the major intermediate of hypoxanthine oxidation and that oxidation on the five-membered ring was not observed.…”

Reexamining Michaelis-Menten enzyme kinetics for xanthine oxidase

“…In contrast, rabbit myocardial endothelial cells lack XO, so the degradation of purine nucleosides, adenosine and inosine, stops at Hx (18). Cao et al (5) observed that at low pH, a small amount of the closely related 6,8-dihydroxypurine formed as an intermediate in the reaction and that it also degraded to Ua; at pH 8.5, almost none formed, so in our analysis this potentially complicating reaction is not considered.…”

“…They found that in the presence of varied steady-state concentrations of Ua, the half-maximal inhibitory effect was obtained with 178 M Ua and thus used that as K i in Eqs. [5][6][7]. That being the case, the question is how to explain that models 1 and 2, without and with Ua inhibition, fit the data equally well.…”