Effect of replacement of His-118, His-125 and Trp-143 by alanine on the catalytic activity and subunit assembly of inorganic pyrophosphatase from thermophilic bacterium PS-3.
Effect of replacement of His-118, His-125 and Trp-143 by alanine on the catalytic activity and subunit assembly of inorganic pyrophosphatase from thermophilic bacterium PS-3.
复制标题
丙氨酸取代His-118、His-125和Trp-143对嗜热菌PS-3无机焦磷酸酶催化活性和亚基组装的影响。
DOI：
10.1042/bj3310143
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
A. Hachimori
中科院分区：
文献类型：
--
作者：
M. Aoki;T. Uchiumi;E. Tsuji;A. Hachimori
嗜热菌PS-3无机焦磷酸酶(PPase)的2个组氨酸残基和1个色氨酸残基分别被丙氨酸取代。H125A和W143A变异体的活性下降到五分之一，而H118A的活性保持不变。近紫外区的圆二色谱表明，前两个变异体的构象随取代度的变化而变化。与野生型PPase相比，野生型PPase在存在镁离子的情况下超过0.1微米的酶浓度，而H118A和H125A变体不能在低于10微米的酶浓度下从三聚体组装成六聚体，即使在较高的镁离子浓度下也是如此。特别是，H118A在稀释状态下被不可逆灭活。相反，W143A PPase的活性与野生型PPase的活性几乎相同。这些结果表明，His-118和His-125对于三聚体-三聚体相互作用和结构完整性都是重要的，而Trp-143在结构上是重要的。三聚体-三聚体相互作用对于PS-3酶的热稳定性是必不可少的。
Each of two histidine residues and one tryptophan residue in thermophilic bacterium PS-3 inorganic pyrophosphatase (PPase) was replaced by alanine. The activities of the H125A and W143A variants decreased to one-fifth, whereas the activity of H118A remained unaltered. CD spectra in the near-UV region indicated that the conformations of the first two variants changed with the substitution. In contrast with wild-type PPase, which is hexameric beyond an enzyme concentration of 0.1 microM in the presence of Mg2+, the H118A and H125A variants cannot be assembled from trimers into hexamers at less than an enzyme concentration of 10 microM even at a higher concentration of Mg2+. In particular, H118A was irreversibly inactivated in a diluted state. In contrast, the enzyme concentration dependence of W143A PPase activity was almost the same as that of wild-type PPase. These results indicated that His-118 and His-125 are important for both trimer-trimer interaction and structural integrity, whereas Trp-143 is important structurally. The trimer-trimer interaction is absolutely necessary for the thermostability of the PS-3 enzyme.