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Reactions of the oxidized organic cofactor in copper-depleted bovine serum amine oxidase.

机译:缺铜的牛血清胺氧化酶中氧化有机辅因子的反应。

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摘要

A novel copper-depleted bovine serum amine oxidase (BSAO), in which about half the molecules contained the organic cofactor in the oxidized form, was prepared by adding a reductant in anaerobic conditions to the cyanide-reacted protein. The CuI-semiquinone formed in these conditions reoxidizes after the removal of copper. The inactive derivative was reduced by benzylamine at approx. 1/1000 the rate of BSAO. The pseudo-first-order reaction was preceded by the formation of a protein-benzylamine complex with dissociation constant, Kd, of 4.9+/-0.5 mM, similar to the Km of BSAO (2.2 mM). Also the reactions with phenylhydrazine and benzohydrazide were considerably slower than in holo-BSAO, whereas the reactions with p-pyridine-2-ylphenylacetohydrazide, containing a longer aromatic tail, and semicarbazide, lacking an aromatic moiety, were less severely affected. Removal of copper had no effect on the optical spectra of BSAO and of most adducts, containing the cofactor in quinol form, showing that copper is bound to neither the oxidized nor the reduced cofactor. Benzylhydrazine did not produce optical effects but was tightly bound, as inferred from its inhibitory effect on reaction with other molecules. Substrate and inhibitors might bind a hydrophobic pocket at some distance from the quinone, probably near the protein surface, with their affinity depending on the hydrophobic character and pKa. The binding, which is not greatly influenced by copper removal, probably induces a copper-dependent change of conformation, 'opening' a pathway to the active site buried in the protein interior.
机译:通过在厌氧条件下将还原剂添加到氰化物反应的蛋白质上,制备了一种新型的贫铜牛血清胺氧化酶(BSAO),其中约一半的分子含有氧化形式的有机辅因子。在这些条件下形成的CuI-半醌在去除铜后会重新氧化。通过苄胺在约90℃下将无活性衍生物还原。 BSAO的1/1000。在拟一级反应之前,形成蛋白质-苄胺复合物,其解离常数Kd为4.9 +/- 0.5 mM,与BSAO的Km(2.2 mM)相似。而且,与苯肼和苯并肼的反应比在完整的BSAO中要慢得多,而与对苯并吡啶-2-基苯基乙酰肼的反应则较慢,而后者具有较长的芳族尾巴,而氨基脲则缺乏芳香部分。铜的去除对BSAO的光谱没有影响,对大多数加合物(包括以喹诺酚形式的辅因子)的光谱也没有影响,表明铜既不与氧化的辅因子结合,也不与还原的辅因子结合。从对其他分子反应的抑制作用可以推断出苄基肼不会产生光学效应,但却是紧密结合的。底物和抑制剂可能结合离醌较远的疏水口袋(可能靠近蛋白质表面),其亲和力取决于疏水特性和pKa。这种结合不受铜去除的影响很大,可能引起铜依赖性构象变化,从而“打开”一条通往埋在蛋白质内部的活性位点的途径。

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