生物化学和晶体的研究Met144Ala, Asp92Asn His254Phe亚硝酸还原酶的突变体产碱杆菌属xylosoxidans提供洞察酶机制。
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埃利斯•乔丹领域M,多德FE,奇怪的RW, Sawers G,伊迪法官明确指出RR,哈斯南党卫军
生物化学和晶体的研究Met144Ala, Asp92Asn His254Phe亚硝酸还原酶的突变体产碱杆菌属xylosoxidans提供洞察酶机制。
J杂志。2002年2月8日,316 (1):51 - 64。
- PubMed ID
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11829502 (在PubMed]
- 文摘
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异化的亚硝酸还原酶催化作用的减少亚硝酸盐(NO(2)(-))一氧化氮(NO)。铜的亚硝酸盐还原酶同时包含1型和2型铜网站。从氧化还原电子转移合作伙伴认为是介导通过1型铜2型铜催化中心网站和使用以及衬底亚硝酸盐。2型铜网站,Asp92已被确定为一个关键残留在底物利用率,因为它氢键亚硝酸盐的水分子结合位点。我们也建议通过Asp92质子进入催化部位,由His254通过水网络。这些残留的角色一直在调查蓝铜亚硝酸还原酶的产碱杆菌属xylosoxidans (NCIMB 11015)点突变、酶活性测定和结构的决心。此外,有人建议酶通过有序运作机制在一个电子转移到2型铜主要网站当第二衬底亚硝酸盐是束缚,这是通过降低氧化还原电位的控制2型网站时,亚硝酸盐的含量都很高。因此,一个小扰动的1型铜网站应导致显著影响酶的活性。为此Met144的突变,这是最弱的配体1型铜、调查。H254F的结构,D92N和M144A决心1.85,分别为1.9和2.2的决议。 The D92N and H254F mutants have negligible or no activity, while the M144A mutant has 30 % activity of the native enzyme. Structural and spectroscopic data show that the loss of activity in H254F is due to the catalytic site being occupied by Zn while the loss/reduction of activity in D92N/M144A are due to structural reasons. The D92N mutation results in the loss of the Asp92 hydrogen bond to the Cu-ligated water. Therefore, the ligand is no longer able to perform proton abstraction. Even though the loss of activity in H254F is due to lack of catalytic Cu, the mutation does cause the disruption of the water network, confirming its key role in proton channel. The structure of the H254F mutant is the first case where full occupancy Zn at the type 2 Cu site is observed, but despite the previously noted similarity of this site to the carbonic anhydrase catalytic site, no carbonic anhydrase activity is observed. The H254F and D92N mutant structures provide, for the first time, observation of surface Zn sites which may act as a Zn sink and prevent binding of Zn at the catalytic Cu site in the native enzyme.