Van den Burg B, Eijsink V G, Vriend G, Veltman O R, Venema G
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.
Biotechnol Appl Biochem. 1998 Apr;27(2):125-32. doi: 10.1111/j.1470-8744.1998.tb01384.x.
Autolytic degradation of the thermolysin-like proteinase of Bacillus subtilis (TLP-sub) is responsible for the irreversible inactivation of the enzyme at elevated temperatures. Previously we have reported five cleavage sites in TLP-sub [Van den Burg et al. (1990) Biochem. J. 272, 93-97]. In an attempt to render the enzyme less susceptible to autolytic breakdown, one of the fission sites, located between Leu-156 and Ile-157, was modified by replacing Ile-157, C-terminally located with respect to the fission site, by an Asp residue. Aspartic acid is less preferred at this position with respect to the substrate preference of TLP-sub. Modelling studies indicated that this mutation was unlikely to cause conformational changes in the enzyme. Although the 156-157 fission was not observed in the mutant enzyme, a new fission site, between Gly-148 and Val-149, was now observed.
枯草芽孢杆菌嗜热菌蛋白酶样蛋白酶（TLP-sub）的自溶降解是该酶在高温下不可逆失活的原因。此前我们已报道了TLP-sub中的五个切割位点[Van den Burg等人（1990年），《生物化学杂志》272卷，93 - 97页]。为使该酶更不易受到自溶分解的影响，位于Leu - 156和Ile - 157之间的一个切割位点被修饰，即将相对于切割位点位于C末端的Ile - 157替换为Asp残基。就TLP-sub的底物偏好而言，天冬氨酸在该位置不太受青睐。建模研究表明，这种突变不太可能导致酶的构象变化。尽管在突变酶中未观察到156 - 157切割，但现在观察到了一个新的切割位点，位于Gly - 148和Val - 149之间。
