Gekko K, Kunori Y, Takeuchi H, Ichihara S, Kodama M
Department of Applied Biological Science, Faculty of Agriculture, Nagoya University.
J Biochem. 1994 Jul;116(1):34-41. doi: 10.1093/oxfordjournals.jbchem.a124499.
To elucidate the role of a flexible loop in the stability and function of Escherichia coli dihydrofolate reductase, glycine-121 in a flexible loop (residues 117-131), separated by 19 A from active site Asp27, was substituted by site-directed mutagenesis with eight amino acids (Ala, Val, Leu, Asp, Ser, Cys, Tyr, and His). The free energy change of unfolding decreased in the order of G121A > G121D > G121C > G121S, wild-type > G121H > G121Y > G121L > G121V. The thermal denaturation temperature decreased with all mutations, accompanied by a decrease in the calorimetric enthalpy of denaturation. The steady-state kinetic parameter for the enzyme reaction, Km, was only slightly influenced, but kcat was significantly decreased by the mutations, there being 3- (G121C) to 42-fold (G121L) decreases in kcat/Km compared to that of the wild-type enzyme. The effects of mutations on the stability and enzyme activity were statistically examined as a function of the hydrophobicity and volume of amino acids introduced. The diminished stability and activity with increases in the hydrophobicity and volume of amino acids suggest that the main effect of the mutations would be modification of the flexibility of the loop due to overcrowding of the bulky side chains, overcoming the enhancement of the hydrophobic interaction.
为阐明柔性环在大肠杆菌二氢叶酸还原酶稳定性和功能中的作用，通过定点诱变将柔性环（残基117 - 131）中与活性位点Asp27相隔19 Å的甘氨酸-121替换为8种氨基酸（丙氨酸、缬氨酸、亮氨酸、天冬氨酸、丝氨酸、半胱氨酸、酪氨酸和组氨酸）。去折叠的自由能变化按以下顺序降低：G121A > G121D > G121C > G121S、野生型 > G121H > G121Y > G121L > G121V。所有突变均导致热变性温度降低，同时变性的量热焓也降低。酶反应的稳态动力学参数Km仅受到轻微影响，但kcat因突变而显著降低，与野生型酶相比，kcat/Km降低了3倍（G121C）至42倍（G121L）。作为引入氨基酸的疏水性和体积的函数，对突变对稳定性和酶活性的影响进行了统计学检验。随着氨基酸疏水性和体积的增加，稳定性和活性降低，这表明突变的主要影响可能是由于庞大侧链过度拥挤导致环的柔性发生改变，从而克服了疏水相互作用的增强。
