EFFECTS OF POINT MUTATION IN A FLEXIBLE LOOP ON THE STABILITY AND ENZYMATIC FUNCTION OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE
EFFECTS OF POINT MUTATION IN A FLEXIBLE LOOP ON THE STABILITY AND ENZYMATIC FUNCTION OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE
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DOI：
10.1093/oxfordjournals.jbchem.a124007
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发表时间：
1993-01-01
影响因子：
2.7
通讯作者：
IWAKURA, M
中科院分区：
文献类型：
--
作者：
GEKKO, K;YAMAGAMI, K;IWAKURA, M
为了阐明柔性环在大肠杆菌二氢叶酸还原酶的稳定性和功能中的作用，通过定点突变将柔性环（117-131）中的甘氨酸-121替换为缬氨酸和亮氨酸。尽管侧链的疏水性增加，但通过尿素变性在15 ℃下测定的两个突变体（G121 V和G121 L）的解折叠自由能变化分别比野生型降低1.22和0.38 kcal/mol。热变性温度，通过差示扫描量热法监测，分别降低了2.4和5.2度-C的G121 V和G121 L，伴随着变性的焓变的减少。这些发现表明，DHFR的结构是不稳定的突变，主要是由于变性的焓变相对于变性的熵变的大幅下降。酶反应的稳态动力学参数K（m）不受影响，但这些突变使k（cat）大大降低，导致G121 V和G121 L的k（cat）/K（m）分别降低240倍和52倍。突变的主要影响似乎是由于庞大侧链的过度拥挤而改变了环的灵活性，克服了疏水相互作用的增强。
To elucidate the role of a flexible loop in the stability and function of Escherichia coli dihydrofolate reductase, glycine-121 in the flexible loop (117-131) was substituted to valine and leucine by site-directed mutagenesis. Despite the increased hydrophobicity of the side chains, the free energy changes of unfolding of the two mutants (G121V and G121L) determined by urea denaturation at 15-degrees-C were decreased by 1.22 and 0.38 kcal/mol, respectively, compared with that of the wild-type. Thermal denaturation temperature, as monitored by differential scanning calorimetry, was decreased by 2.4 and 5.2-degrees-C for G121V and G121L, respectively, accompanying the decrease in enthalpy change of denaturation. These findings indicate that the structure of DHFR is destabilized by the mutations, predominantly due to the large decrease in enthalpy change of denaturation relative to entropy change of denaturation. The steady-state kinetic parameter in the enzyme reaction, K(m), was not influenced but k(cat) was greatly decreased by these mutations, resulting in 240- and 52-fold decreases in k(cat)/K(m) for G121V and G121L, respectively. The main effect of the mutations appeared to be modification of the flexibility of the loop due to overcrowding of the bulky side chains, overcoming the enhancement of hydrophobic interaction.