Wilson E J, Massoulié J, Bon S, Rosenberry T L
Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106-4965, USA.
FEBS Lett. 1996 Jan 29;379(2):161-4. doi: 10.1016/0014-5793(95)01504-3.
The rate of thermal inactivation of Torpedo AChE at pH 8.5 was increased by the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). At 30 degrees C or 37 degrees C, inactivation rates with 0.3 mM DTNB increased about 5-fold for the wild-type enzyme and for two site-specific mutants, D72S and V129R. The reversible active site inhibitor, ambenonium, completely stabilized the wild type enzyme and partially stabilized the D72S mutant. However, ambenonium did not protect against the destabilization introduced by DTNB, which still accelerated inactivation of D72S 5-fold. When the only free sulfhydryl group in AChE was removed by replacing cysteine 231 with serine, increased rates of thermal inactivation were observed. The inactivation rate increased by a factor of 2 to 3 for the single mutant (C231S) and by a factor of 5 for the double mutant V129R/C231S. Even in the C231S mutants, DTNB still had an additional effect. It increased the inactivation rate for C231S and V129R/C231 by a factor of about 1.5 to 3 beyond the rates seen in the absence of DTNB. Therefore, at least part of the destabilization seen with DTNB in enzymes that retain C231 does not involve reaction of DTNB with C231.
在pH 8.5条件下，巯基试剂5,5'-二硫代双（2-硝基苯甲酸）（DTNB）可提高电鳐乙酰胆碱酯酶（Torpedo AChE）的热失活速率。在30℃或37℃时，对于野生型酶以及两个位点特异性突变体D72S和V129R，0.3 mM DTNB存在时的失活速率增加了约5倍。可逆性活性位点抑制剂安贝氯铵可完全稳定野生型酶，并部分稳定D72S突变体。然而，安贝氯铵不能防止DTNB引入的去稳定化作用，DTNB仍能使D72S的失活加速5倍。当用丝氨酸取代半胱氨酸231从而去除AChE中唯一的游离巯基时，观察到热失活速率增加。单突变体（C231S）的失活速率增加了2至3倍，双突变体V129R/C231S的失活速率增加了5倍。即使在C231S突变体中，DTNB仍有额外作用。它使C231S和V129R/C231S的失活速率比在无DTNB时增加了约1.5至3倍。因此，在保留C231的酶中，DTNB导致的至少部分去稳定化作用并不涉及DTNB与C231的反应。
