Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
J Biochem. 2012 Sep;152(3):231-9. doi: 10.1093/jb/mvs064. Epub 2012 May 29.
In the N-terminal domain of thermolysin, two anti-parallel β-strands, Asn112-Ala113-Phe114-Trp115 and Ser118-Gln119-Met120-Val121-Tyr122 are connected by an Asn116-Gly117 turn to form a β-hairpin structure. In this study, we examined the role of Asn116 in the activity and stability of thermolysin by site-directed mutagenesis. Of the 19 Asn116 variants, four (N116A, N116D, N116T and N116Q) were produced in Escherichia coli, by co-expressing the mature and pro domains separately, while the other 15 were not. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide (FAGLA) at 25°C, the intrinsic k(cat)/K(m) value of N116D was 320% of that of the wild-type thermolysin (WT), and in the hydrolysis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (ZDFM) at pH 7.5 at 25°C, the k(cat)/K(m) value of N116D was 140% of that of WT, indicating that N116D exhibited higher activity than WT. N116Q exhibited similar activity as WT, and N116A and N116T exhibited reduced activities. The first-order rate constants, k(obs), of the thermal inactivation at 80°C were in the order N116A, N116D, N116T > N116Q > WT at all CaCl(2) concentrations examined (1-100 mM), indicating that all variants exhibited reduced stabilities. These results suggest that Asn116 plays an important role in the activity and stability of thermolysin presumably by stabilizing this β-hairpin structure.
在嗜热菌蛋白酶的 N 端结构域中，两个反平行的β-折叠，Asn112-Ala113-Phe114-Trp115 和 Ser118-Gln119-Met120-Val121-Tyr122 通过一个 Asn116-Gly117 转角连接，形成一个β-发夹结构。在这项研究中，我们通过定点突变研究了 Asn116 在嗜热菌蛋白酶活性和稳定性中的作用。在 19 种 Asn116 突变体中，有 4 种（N116A、N116D、N116T 和 N116Q）是通过分别共表达成熟酶和前导肽在大肠杆菌中产生的，而其余 15 种则不能。在 25°C 下，N-[3-(2-呋喃基)丙烯酰基]-甘氨酰-L-亮氨酸酰胺（FAGLA）的水解反应中，N116D 的固有 kcat/Km 值比野生型嗜热菌蛋白酶（WT）高 320%，在 25°C 下，pH7.5 时，N-碳苄氧羰基-L-天冬氨酰-L-苯丙氨酸甲酯（ZDFM）的水解反应中，N116D 的 kcat/Km 值比 WT 高 140%，表明 N116D 的活性比 WT 高。N116Q 的活性与 WT 相似，而 N116A 和 N116T 的活性降低。在所有研究的 CaCl2浓度（1-100mM）下，80°C 时的热失活动力学的一级速率常数 kobs 的顺序为 N116A、N116D、N116T>N116Q>WT，表明所有变体的稳定性都降低了。这些结果表明，Asn116 可能通过稳定这个β-发夹结构，在嗜热菌蛋白酶的活性和稳定性中发挥重要作用。
