31P nuclear magnetic resonance and chemical studies of the phosphorus residues in bovine milk xanthine oxidase.
31P nuclear magnetic resonance and chemical studies of the phosphorus residues in bovine milk xanthine oxidase.
复制标题
牛乳黄嘌呤氧化酶中磷残基的 31P 核磁共振和化学研究。
DOI：
10.1111/j.1432-1033.1984.tb08544.x
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
Müller,F
中科院分区：
文献类型：
--
作者：
Davis,MD;Edmondson,DE;Müller,F
除了酸解离的 FAD 和钼辅因子部分中含有的磷酸盐残基外，牛奶黄嘌呤氧化酶的每个活性中心钼还含有一摩尔共价结合的磷。脱辅基蛋白部分的酸水解和随后的高压薄层电泳分析已鉴定出磷酸化的氨基酸残基为磷酸丝氨酸。31P NMR 数据显示磷酸肽被单取代，与化学分析一致。还观察到钼辅因子部分中磷残基的 pH 依赖性化学位移，这为文献中该辅因子含有单取代磷酸盐的建议提供了明确的支持。对完整酶的 31P NMR 研究显示，磷共振在约 –3 ppm、+1 ppm、+8.8 ppm 和 +13.5 ppm 处。通过与葡萄糖氧化酶上 FAD 的化学位移数据进行类比，+8.8 ppm 和+13.5 ppm 处的共振被指定为 FAD 部分的焦磷酸键的共振[James, T. L., Edmondson, D. E., 和 Husain, M. (1981)Biochemistry 20, 617]，并且在检查 deflavo 制剂时该区域没有任何共振。黄嘌呤氧化酶。约 –3 ppm 共振的强度和分辨率取决于酶的功能程度。相对于 50-60% 功能酶中的 FAD 共振，这种共振的振幅较小，但脱硫酶中的强度显着增加。这种共振是唯一一种暴露于溶剂的共振，因为它是唯一一种在向酶溶液中添加 Mn(II) 时容易受到顺磁线展宽影响的共振。用别嘌呤醇处理酶会导致~‐3-ppm 共振的改变，但不会显着影响其他共振。用乙二醇形成稳定的 Mo(V)“抑制”形式的酶会导致 –3 ppm 和 +1 ppm 处的共振线大幅展宽，但对 FAD 共振没有明显影响。这些数据表明，除了钼辅因子上的磷酸盐外，黄嘌呤氧化酶中的磷酸丝氨酸残基也非常靠近该酶的活性位点钼中心。讨论了这些结果对酶催化机制的可能影响。
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 ∼‐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 active‐site molybdenum center of this enzyme. These results are discussed with respect to possible implications on the catalytic mechanism of the enzyme.