P-31 NUCLEAR MAGNETIC-RESONANCE AND CHEMICAL STUDIES OF THE PHOSPHORUS RESIDUES IN BOVINE-MILK XANTHINE-OXIDASE

In addition to the phosphate residues contained in the acid-dissociable FAD and the Mo cofactor moieties, milk xanthine oxidase contains 1 mol of covalently bound P per active-center Mo. Acid hydrolysis of the apoprotein moiety and subsequent analysis by high-voltage thin-layer electrophoresis identified the phosphorylated amino acid residue to be phosphoserine. 31P NMR data show the phosphopeptide to be monosubstituted, in agreement with the chemical analysis. A pH-dependent chemical shift of the P residue in the Mo cofactor moiety is also observed which provides unequivocal support for suggestions in the literature that this cofactor contains a monosubstituted phosphate. 31P NMR studies on the intact enzyme show P resonances at about -3 ppm, +1 ppm, +8.8 ppm and at +13.5 ppm. The resonances at +8.8 ppm and at +13.5 ppm are assigned to those of the PPi linkage of the FAD moiety by analogy with chemical shift data of the FAD on glucose oxidase and from the absence of any resonances in this region upon examination of preparations of deflavo xanthine oxidase. The intensity and resolution of the resonance at about -3 ppm is dependent on the degree of functionality of the enzyme. This resonance has a small amplitude relative to the FAD resonances in 50-60% functional enzyme, but increases dramatically in intensity in the desulfo enzyme. This resonance is the only one exposed to solvent as it is the only one susceptible to paramagnetic line-broadening on the addition of Mn(II) to the enzyme solution. Treatment of the enzyme with allopurinol leads to alteration of the .apprxeq.-3-ppm resonance, but does not significantly affect the other resonances. Formation of the stable Mo(V) inhibited form of the enzyme with ethylene glycol results in extensive line-broadening of the resonances at -3 ppm and +1 ppm, but has no observable affect on the FAD resonances. In addition to the phosphate or the Mo cofactor, the phosphoserine residue in xanthine oxidase is also in close proximity to the active-site Mo center of this enzyme. These results are discussed with respect to possible implications on the catalytic mechanism of the enzyme.