表征metal-substituted克雷伯氏菌aerogenes脲酶。
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山口K, Cosper新泽西,Stalhandske C,斯科特•拉皮尔森马,Karplus PA, Hausinger RP
表征metal-substituted克雷伯氏菌aerogenes脲酶。
J Inorg化学杂志。1999年8月,4 (4):468 - 77。
- PubMed ID
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10555581 (在PubMed]
- 文摘
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脲酶具有双核的Ni活性位点的蛋白质提供桥接carbamylated赖氨酸残留物以及天门冬氨酰和四个histidyl配体。脱辅基蛋白可以在体外激活的孵化与碳酸氢/二氧化碳和镍(II);然而,只有大约15%的形式活跃酶(Ni-CO2-ureaseA),其余形成活性carbamylated Ni-containing蛋白质(Ni-CO2-ureaseB)。没有二氧化碳,脱辅基蛋白+镍(II)形成一个独特的活性Ni-containing物种(Ni-urease)。这里描述的研究进行了更好的为活动定义metal-binding网站Ni-urease Ni-CO2-ureaseB物种,并检查各种形式的公司的性质,Mn和Cu-substituted脲酶。x光吸收光谱(xa)表示,两个镍原子中Ni-urease metallocenter协调平均两个组氨酸和3 - 4 N / O配体,符合绑定通常与赖氨酸氨基甲酸酯酶配体取代溶剂。xa和电子光谱提供了证据不活跃的硫醇盐结扎Ni-containing物种。相比之下,比较研究Co-CO2-urease及其C319A变体的电子光谱符合部分两个公司被Cys319协调。而不活跃Co-CO2-urease具有单个histidyl每金属配位,形成的物种使用C319A脱辅基蛋白更类似于本机metallocenter和展品低水平的活动。活动也涉及两种Mn-CO2-urease之一。 A crystal structure of the inactive Mn-CO2-urease species shows a metallocenter very similar in structure to that of native urease, but with a disordering of the Asp360 ligand and movement in the Mn-coordinated solvent molecules. Cu(II) was bound to many sites on the protein in addition to the usual metallocenter, but most of the adventitious metal was removed by treatment with EDTA. Cu-treated urease was irreversibly inactivated, even in the C319A variant, and was not further characterized. Metal speciation between Ni, Co, and Mn most affected the higher of two pKa values for urease activity, consistent with this pKa being associated with the metal-bound hydrolytic water molecule. Our results highlight the importance of precisely positioned protein ligands and solvent structure for urease activity.