Substrate Orientation and Catalysis at the Molybdenum Site in Xanthine Oxidase

Abstract

Xanthine oxidoreductase is a ubiquitous cytoplasmic protein that catalyzes the final two steps in purine catabolism. We have previously investigated the catalytic mechanism of the enzyme by rapid reaction kinetics and x-ray crystallography using the poor substrate 2-hydroxy-6-methylpurine, focusing our attention on the orientation of substrate in the active site and the role of Arg-880 in catalysis. Here we report additional crystal structures of as-isolated, functional xanthine oxidase in the course of reaction with the pterin substrate lumazine at 2.2 Å resolution and of the nonfunctional desulfo form of the enzyme in complex with xanthine at 2.6 Å resolution. In both cases the orientation of substrate is such that the pyrimidine subnucleus is oriented opposite to that seen with the slow substrate 2-hydroxy-6-methylpurine. The mechanistic implications as to how the ensemble of active site functional groups in the active site work to accelerate reaction rate are discussed.

Substrate Orientation and Catalytic Specificity in the Action of Xanthine Oxidase

“…The crystal structure of the complex of xanthine oxidase with 6-mercaptopurine was solved at 2.6 Å resolution, with final R factors R cryst of 21.7% and R free of 26.9% (Table 1). The overall structure of the homodimer in both current crystal structures is very similar to that seen previously for the oxidized bovine enzyme (11) and our own previously reported structures of the enzyme in complex with 2-hydroxy-6-methylpurine, xanthine, and lumazine (13,14). As seen previously (13,14), finite electron density for the C-terminal residues of one monomer of the single homodimer in the asymmetric unit was evident, and these residues were added during refinement (residues 1316 -1326 and 1316 -1325 for the structures in complex with hypoxanthine and 6-mercaptopurine, respectively).…”

“…With each substrate, one orientation has C-2 positioned appropriately for hydroxylation by the molybdenum center, and the second has C-8 so positioned instead. Both of the orientations seen here have the exocyclic functional group at the C-6 position adjacent to a conserved arginine residue (Arg 880 in the bovine enzyme), consistent with our previous conclusions regarding the catalytic role of Arg 880 in stabilizing negative charge accumulation on the heterocycle at this position in the course of the reaction (12)(13)(14). In light of these results, we have undertaken further kinetic studies and confirm that xanthine is indeed the sole product of enzyme action on hypoxanthine; to within our detection limits 6,8-dihydroxypurine is not formed, despite the fact that hypoxanthine is able to bind in an orientation that permits such hydroxylation.…”

“…Crystallization, Data Acquisition, and Structure Determination-All enzyme crystals were grown using the batch method according to previously published methods using microbridges to hold batch solutions in sealed wells of a 24-well tray (14). Each batch contained 10 l of 34.5 M (5 mg/ml) enzyme mixed with 5-6 l of 12% polyethylene glycol 8000 precipitant solution.…”

“…We have previously suggested (12) that Arg 880/881 (Arg 310 in the R. capsulatus enzyme) is involved in transition state stabilization by compensation of negative charge accumulation on the C-6ϭO oxygen of xanthine in the course of hydroxylation at C-8, and the orientation we see here indicates that this is also likely to be the case for hydroxylation of hypoxanthine at C-2, because the C-6 carbonyl is again oriented toward the arginine. Similarly, we have suggested (14,34) that Glu 802/803 (Glu 232 in the R. capsulatus enzyme) is involved in proton tautomerization from N-3 to N-9 of xanthine in the course of hydroxylation at C-8, as shown in Fig. 10 …”

Coumarins with Xanthine Oxidase Inhibiting and Radical Scavenging Properties: Tools to Combat Oxidative Stress in Cells

“…Docking of esculetin (1) was performed with the program GOLD (version 5.1; Cambridge Crystallographic Data Centre, UK) [20], [21] and the X-ray crystal structure of the XO/lumazine complex (Protein Data Bank entry 3ETR) [22]. The protein structure was prepared in GOLD for docking by adding hydrogen atoms and deleting all water molecules and lumazine.…”