Aoki M, Uchiumi T, Tsuji E, Hachimori A
Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, 3-15-1, Tokida, Ueda, Nagano 386, Japan.
Biochem J. 1998 Apr 1;331 ( Pt 1)(Pt 1):143-8. doi: 10.1042/bj3310143.
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.
嗜热细菌PS-3无机焦磷酸酶（PPase）中的两个组氨酸残基和一个色氨酸残基分别被丙氨酸取代。H125A和W143A变体的活性降至五分之一，而H118A的活性保持不变。近紫外区域的圆二色光谱表明，前两个变体的构象随取代而改变。与野生型PPase不同，在Mg2+存在下，当酶浓度超过0.1μM时野生型PPase为六聚体，即使在更高的Mg2+浓度下，H118A和H125A变体在酶浓度低于10μM时也无法从三聚体组装成六聚体。特别是，H118A在稀释状态下不可逆地失活。相反，W143A PPase活性的酶浓度依赖性与野生型PPase几乎相同。这些结果表明，His-118和His-125对三聚体-三聚体相互作用和结构完整性都很重要，而Trp-143在结构上很重要。三聚体-三聚体相互作用对于PS-3酶的热稳定性是绝对必要的。
