The importance of Asn47 for structure and reactivity of azurin from Alcaligenes denitrificans as studied by site-directed mutagenesis and spectroscopy.
The importance of Asn47 for structure and reactivity of azurin from Alcaligenes denitrificans as studied by site-directed mutagenesis and spectroscopy.
复制标题
通过定点诱变和光谱学研究 Asn47 对脱氮产碱菌天青蛋白的结构和反应性的重要性。
DOI：
10.1016/s0021-9258(19)49644-0
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
G. Canters
中科院分区：
文献类型：
--
作者：
C. Hoitink;G. Canters
为了研究刚性铜位点对天青蛋白结构和功能的重要性，制备并表征了铜周围内部氢键数量减少的突变体。为此，先前从产碱杆菌脱氮菌中克隆的 azu 基因（Hoitink, C. W. G.、Woudt, L. P.、Turenhout, J. C. M.、Van de Kamp, M. 和 Canters, G. W. (1990) Gene (Amst.) 90, 15-20）在大肠杆菌中表达，并开发了天青蛋白的分离和纯化程序。在大肠杆菌中异源表达后获得的天青蛋白在光谱上与源自脱氮菌的天青蛋白无法区分。通过定点诱变用亮氨酸取代 Asn47，改变了铜位点周围的氢键网络。 Asn47 是所有蓝铜蛋白中的保守残基，其一级结构已被报道。通过紫外-可见光、电子自旋共振和核磁共振波谱对突变蛋白进行表征，并与野生型天青蛋白进行比较，结果表明铜位点的结构以及蛋白的整体结构在很大程度上得到了保留。通过电子自交换率测量的氧化还原活性似乎也没有改变。然而，突变体在稳定性和中点电位方面与野生型天青蛋白不同。突变型和野生型天青蛋白的中点电位分别为 396 和 286 mV。差异是由于相当大的熵和焓贡献在很大程度上抵消了。讨论了这些实验结果的可能解释。
To study the importance of a rigid copper site for the structure and function of azurin, a mutant with a reduced number of internal hydrogen bonds around the copper has been prepared and characterized. To this purpose, the previously cloned azu gene from Alcaligenes denitrificans (Hoitink, C. W. G., Woudt, L. P., Turenhout, J. C. M., Van de Kamp, M., and Canters, G. W. (1990) Gene (Amst.) 90, 15-20) was expressed in Escherichia coli and an isolation and purification procedure for the azurin was developed. The azurin obtained after heterologous expression in E. coli appears spectroscopically indistinguishable from azurin derived from A. denitrificans. The hydrogen bonding network around the copper site was altered by replacing Asn47 by a leucine by means of site-directed mutagenesis. Asn47 is a conserved residue in all blue copper proteins of which the primary structure has been reported. Characterization of the mutant protein with UV-visible, electron spin resonance, and NMR spectroscopy, and comparison with the wild type azurin revealed that the structure of the copper site as well as the overall structure of the protein have been largely retained. The redox activity as measured by the electron self-exchange rate appears not to have changed either. However, the mutant differs from the wild type azurin with respect to stability and midpoint potential. Midpoint potentials of mutant and wild type azurin amount to 396 and 286 mV, respectively. The difference is due to sizable entropic and enthalpic contributions which to a large extent cancel. Possible explanations for the outcome of these experiments are discussed.