Taddei N, Magherini F, Chiti F, Bucciantini M, Raugei G, Stefani M, Ramponi G
Department of Biochemical Sciences, University of Florence, Italy.
FEBS Lett. 1996 Apr 15;384(2):172-6. doi: 10.1016/0014-5793(96)00292-x.
Ser-Ala and Ser-Ala-Ser-Ala C-terminus elongated (delta+2 and delta+4, respectively) and two C-terminus deleted (delta-2 and delta-3) muscle acylphosphatase mutants were investigated to assess the catalytic and structural roles of the C-terminal region. The kinetic analysis of these mutants shows that the removal of two or three C-terminal residues reduces the catalytic activity to 7% and 4% of the value measured for the wild-type enzyme, respectively; instead, the elongation of the C-terminus does not significantly change the enzyme behaviour. 1H Nuclear magnetic resonance spectroscopy indicates that all mutants display a native-like fold though they appear less stable, particularly delta-2 and delta-3 mutants, as compared to the wild-type enzyme. Such destabilisation of the C-terminal modified mutants is further confirmed by urea inactivation experiments. The results here presented account for an involvement of the C-terminal region in the stabilisation of the three-dimensional structure of acylphosphatase, particularly at the active-site level. Moreover, a participation of the C-terminal carboxyl group to the catalytic mechanism can be excluded.
研究了丝氨酸 - 丙氨酸（Ser - Ala）和丝氨酸 - 丙氨酸 - 丝氨酸 - 丙氨酸C末端延长（分别为δ + 2和δ + 4）以及两个C末端缺失（δ - 2和δ - 3）的肌肉酰基磷酸酶突变体，以评估C末端区域的催化和结构作用。对这些突变体的动力学分析表明，去除两个或三个C末端残基会使催化活性分别降至野生型酶测量值的7%和4%；相反，C末端的延长并未显著改变酶的行为。1H核磁共振光谱表明，所有突变体均呈现出类似天然的折叠结构，尽管与野生型酶相比，它们显得不太稳定，尤其是δ - 2和δ - 3突变体。尿素失活实验进一步证实了C末端修饰突变体的这种不稳定。此处呈现的结果表明，C末端区域参与了酰基磷酸酶三维结构的稳定，特别是在活性位点水平。此外，可以排除C末端羧基参与催化机制的可能性。
