Davis M D, Edmondson D E, Müller F
Eur J Biochem. 1984 Dec 3;145(2):237-43. doi: 10.1111/j.1432-1033.1984.tb08544.x.
In addition to the phosphate residues contained in the acid-dissociable FAD and the molybdenum cofactor moieties, milk xanthine oxidase contains one mole of covalently bound phosphorus per active-center molybdenum. Acid hydrolysis of the apoprotein moiety and subsequent analysis by high-voltage thin-layer electrophoresis has 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 phosphorus residue in the molybdenum 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 phosphorus 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 pyrophosphate linkage of the FAD moiety by analogy with chemical shift data of the FAD on glucose oxidase [James, T.L., Edmondson, D.E., and Husain, M. (1981) Biochemistry 20, 617] 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 desulpho 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 approximately equal to -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. These data suggest that in addition to the phosphate on the molybdenum cofactor, the phosphoserine residue in xanthine oxidase is also in close proximity to the activesite molybdenum center of this enzyme. These results are discussed with respect to possible implications on the catalytic mechanism of the enzyme.
除了存在于酸可解离的黄素腺嘌呤二核苷酸（FAD）和钼辅因子部分中的磷酸残基外，乳黄嘌呤氧化酶每个活性中心钼还含有一摩尔共价结合的磷。对脱辅基蛋白部分进行酸水解，随后通过高压薄层电泳分析，已确定磷酸化氨基酸残基为磷酸丝氨酸。磷-31核磁共振（³¹P NMR）数据表明该磷酸肽为单取代，这与化学分析结果一致。还观察到钼辅因子部分中磷残基的pH依赖性化学位移，这为文献中关于该辅因子含有单取代磷酸的观点提供了明确支持。对完整酶的³¹P NMR研究显示，磷共振峰出现在约-3 ppm、+1 ppm、+8.8 ppm和+13.5 ppm处。通过与葡萄糖氧化酶上FAD的化学位移数据[詹姆斯，T.L.，埃德蒙森，D.E.，和侯赛因，M.（1981年）《生物化学》20卷，617页]进行类比，并通过检查脱黄素黄嘌呤氧化酶制剂时该区域没有任何共振峰，将+8.8 ppm和+13.5 ppm处的共振峰归属于FAD部分的焦磷酸键。约-3 ppm处共振峰的强度和分辨率取决于酶的功能程度。在50 - 60%功能的酶中，该共振峰的幅度相对于FAD共振峰较小，但在脱硫酶中强度显著增加。该共振峰是唯一暴露于溶剂中的峰，因为它是唯一在向酶溶液中添加锰（II）时易受顺磁线宽展影响的峰。用别嘌呤醇处理该酶会导致约-3 ppm共振峰发生改变，但对其他共振峰没有显著影响。用乙二醇形成酶的稳定钼（V）“抑制”形式会导致-3 ppm和+1 ppm处的共振峰出现广泛的线宽展，但对FAD共振峰没有可观察到的影响。这些数据表明，除了钼辅因子上的磷酸外，黄嘌呤氧化酶中的磷酸丝氨酸残基也紧邻该酶的活性位点钼中心。针对这些结果对酶催化机制可能产生的影响进行了讨论。
