Effects of introducing negative charges into the molecular surface of thermolysin by site-directed mutagenesis on its activity and stability.
Effects of introducing negative charges into the molecular surface of thermolysin by site-directed mutagenesis on its activity and stability.
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DOI：
10.1016/j.bbapap.2007.12.004
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发表时间：
2008-03
期刊：
影响因子：
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通讯作者：
T. Takita;Takahiro Aono;Haruko Sakurama;T. Itoh;Takumi Wada;Masashi Minoda;K. Yasukawa;K. Inouye
中科院分区：
文献类型：
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作者：
T. Takita;Takahiro Aono;Haruko Sakurama;T. Itoh;Takumi Wada;Masashi Minoda;K. Yasukawa;K. Inouye
嗜热菌蛋白酶是显着激活和稳定的中性盐，表面电荷的建议在其活性和稳定性的重要。介绍了在分子表面引入负电荷对其活性和稳定性的影响。选择7个丝氨酸残基，并通过定点诱变在嗜热菌蛋白酶突变体中将它们中的每一个改变为天冬氨酸。在N-[3-（2-呋喃基）丙烯酰基]-甘氨酰-1-亮氨酸酰胺的水解中，所有突变体的kcat/Km值与野生型酶（WT）的kcat/Km值几乎相似。然而，7个突变体中的6个的那些在4 M NaCl下增强17-19倍，略高于WT。S53 D和S65 D突变体（Ser 53和Ser 65分别被替换为Asp）在85 °C下与10 mM CaCl 2孵育30分钟后的剩余酪蛋白水解活性为78%和63%，高于WT（51%）和其它突变体（35-53%）。S53 D随热失活活化焓变的增加而稳定，而S65 D随热失活活化熵变的减小而稳定。CaCl 2可增强WT的稳定性，并在100 mM时达到S53 D和S65 D的水平，表明S53 D和S65 D可能通过加强Ca 2+结合结构而稳定。
Thermolysin is remarkably activated and stabilized by neutral salts, and surface charges are suggested important in its activity and stability. The effects of introducing negative charge into the molecular surface on its activity and stability are described. Seven serine residues were selected, and each of them was changed for aspartate by site-directed mutagenesis in a thermolysin mutant. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide, the kcat/Kmvalues of all mutants were almost similar to that of the wild-type enzyme (WT). However, those of six out of seven mutants were enhanced 17–19 times with 4 M NaCl, being slightly higher than WT. The remaining casein-hydrolyzing activities of the S53D and S65D mutants (Ser53 and Ser65 are replaced with Asp, respectively) after 30-min incubation with 10 mM CaCl2at 85 °C were 78 and 63%, being higher than those of WT (51%) and the other mutants (35–53%). S53D was stabilized with increase in the enthalpy change of activation for thermal inactivation while S65D was with decrease in the entropy change of activation. The stability of WT was enhanced by CaCl2and reached the level of S53D and S65D at 100 mM, suggesting that S53D and S65D might be stabilized by reinforcement of the Ca2+-binding structures.