Analyzes of the mechanisms of thermophilic and alkalophilic properties of aqualysin I,a protease from an extreme thermophile

水溶酶I是一种嗜热和嗜碱的蛋白酶；在钙离子存在下，酶的最佳水解温度为80ºC，酶在80ºC下稳定水解3h。酶活性的最适pH值为10左右，酶在pH值为12、4 -苯甲酸下长期稳定。为了研究使这种酶具有这些特性的结构性质，我们进行了这项研究，得到了以下结果。(1)考察了稀土金属对酶热稳定性的影响。根据离子半径（0.93 ~ 1.06埃），与正电荷无关，一些稀土金属表现出Ca^<2+>（0.98埃）的效应。La^<3+>比Ca^<2+>更有效。La^<3+>与酶的结合常数约为Ca^<2+>的10倍，表明金属的结合常数对酶的稳定很重要。通过PCR随机诱变，在aqualysin I的编码区引入突变，用携带该酶及其突变体基因的重组质粒转化大肠杆菌细胞。选择了在低温或低ph值下似乎能产生具有蛋白水解活性的突变酶的突变克隆。这些克隆的频率为10^<-3>或更低，但从克隆中纯化的酶没有显示出预期的特性。在10 ~ 90 <s:1>温度范围内，丝氨酸替代Asn219的催化缺陷（K_<cat/>K_m）约为野生型的2倍。这种替换导致K_<cat/>而K_<m'>发生显著变化，表明催化残基Ser222附近的侧链影响催化速率常数。

Aqualysin I is a thermophilic and alkalophilic protease ; in the presence of calcium ion, the optimum temperature for the proteolytic activity is 80ﾟC,and the enzyme is stable at 80ﾟC for 3h. The optimum pH for the enzyme activity is about 10, and the enzyme is stable at pH 12 and 4ﾟC for long time. In order to investigate the structural properties which gives these characteristics to the enzyme, we have done this study, and obtained the following resuits.(1) The effects of rare earth metals on the heat-stability of the enzyme were exzmined. Depending on the ionic radii (0.93 to 1.06 angstrom) and independent of the positive charges, some rare earth metals showed the effect like Ca^<2+> (0.98 angstrom). La^<3+> was more effective than Ca^<2+>. The binding constant of La^<3+> with the enzyme was about ten times larger than that of Ca^<2+>, suggesting that the binding constant of metals is important for the stabilization of the enzyme.By random mutagenesis with PCR,mutations were introduced in the coding region of aqualysin I,and Escherichia coli cells were transformed with recombinant plasmids carrying the gene for the enzyme and its mutants. Mutant Clones, which seemed to produce mutant enzymes showing proteolytic activity at low temperatures or at low pHs, were selected. Such clones were obtained at the frequency of 10^<-3> or less, but purified enzymes from the clones did not show the expected properties.Replacement of Asn219 with serine increased the catalytic defficiency (K_<cat/>K_m) about twice as much as that of the wild type at the temperature rage of 10 to 90ﾟC.Such a replacement resulted in a significant change of K_<cat/> rather K_<m'> indicating that the side chain in the vicinity of the catalytic residue Ser222 affects the catalytic rate constant.