Science. 1986 Aug 8;233(4764):659-63. doi: 10.1126/science.233.4764.659.
Steric and hydrophobic effects on substrate specificity were probed by protein engineering of subtilisin. Subtilisin has broad peptidase specificity and contains a large hydrophobic substrate binding cleft. A conserved glycine (Gly(166)), located at the bottom of the substrate binding left, was replaced by 12 nonionic amino acids by the cassette mutagenesis method. Mutant enzymes showed large changes in specificity toward substrates of increasing size and hydrophobicity. In general, the catalytic efficiency (k(cat)/K(m)) toward small hydrophobic substrates was increased (up to 16 times) by hydrophobic substitutions at position 166 in the binding cleft. Exceeding the optimal binding volume of the cleft ( approximately 160 A(3)), by enlarging either the substrate side chain or the side chain at position 166, evoked precipitous drops in catalytic efficiency (k(cat)/K(m)) (up to 5000 times) as a result of steric hindrance.
通过枯草杆菌蛋白酶的蛋白质工程研究了立体和疏水性效应对底物特异性的影响。枯草杆菌蛋白酶具有广泛的肽酶特异性，并包含一个大的疏水性底物结合裂隙。位于底物结合左侧底部的保守甘氨酸（Gly（166））通过盒式诱变方法被 12 个非离子氨基酸取代。突变酶对大小和疏水性增加的底物的特异性发生了很大变化。一般来说，在结合裂隙中位置 166 处的疏水性取代增加了对小疏水性底物的催化效率（kcat/Km）（高达 16 倍）。通过增大裂隙的最佳结合体积（约 160A3），无论是增大底物侧链还是位置 166 的侧链，都会由于空间位阻导致催化效率（kcat/Km）急剧下降（高达 5000 倍）。
