Dion-Schultz A, Howell E E
Department of Biochemistry, University of Tennessee, Knoxville 37996-0840, USA.
Protein Eng. 1997 Mar;10(3):263-72. doi: 10.1093/protein/10.3.263.
The role of a beta-bulge in Escherichia coli dihydrofolate reductase (DHFR) has been explored by a series of insertion and deletion mutations. Insertion of a seven amino acid sequence from a structurally equivalent 'beta-blowout' sequence from human DHFR destabilizes E. coli DHFR by 3.6 kcal/mol and decreases catalytic efficiency (kcat/K(m)) 34-fold. Deletion of F137, delta 137, the looped out residue in the bulge, also destabilizes E. coli DHFR by 2.8 kcal/mol but only decreases catalytic efficiency threefold. Concurrent deletion of F137 and mutation of, V136 to proline to try and maintain the strand twist associated with the beta-bulge decreases protein stability by 3.4 kcal/mol and decreases catalytic efficiency 84-fold. These insertion/deletion mutations were also constructed in a D27S DHFR background. The D27S mutation has been described previously and proposed to remove the catalytic acid from the active site. The delta 137 mutation partially suppresses the effect of the D27S mutation as it decreases the K(m) for substrate, dihydrofolate, twofold. Non-additive effects are observed for the insertion/deletion mutations in wild-type versus D27S DHFR backgrounds, consistent with structural changes.
通过一系列插入和缺失突变，对大肠杆菌二氢叶酸还原酶（DHFR）中β-凸起的作用进行了探究。从人DHFR结构等效的“β-爆裂”序列插入七个氨基酸序列，使大肠杆菌DHFR的稳定性降低3.6千卡/摩尔，并使催化效率（kcat/K(m)）降低34倍。缺失凸起中伸出的残基F137（Δ137），也使大肠杆菌DHFR的稳定性降低2.8千卡/摩尔，但仅使催化效率降低三倍。同时缺失F137并将V136突变为脯氨酸以试图维持与β-凸起相关的链扭曲，使蛋白质稳定性降低3.4千卡/摩尔，并使催化效率降低84倍。这些插入/缺失突变也构建在D27S DHFR背景中。D27S突变先前已有描述，并被认为可从活性位点去除催化酸。Δ137突变部分抑制了D27S突变的作用，因为它使底物二氢叶酸的K(m)降低了两倍。在野生型与D27S DHFR背景中观察到插入/缺失突变的非加性效应，这与结构变化一致。
