胆碱乙酰转移酶与巯基试剂的反应。甲氧基羰基辅酶A二硫化物作为活性位点导向试剂。

The reaction of choline acetyltransferase with sulfhydryl reagents. Methoxycarbonyl-CoA disulfide as an active site-directed reagent.

作者信息

Hersh L B, Nair R V, Smith D J

出版信息

J Biol Chem. 1979 Dec 10;254(23):11988-92.

Abstract

The reaction of choline acetyltransferase with methoxycarbonyl alkyl disulfides leads to a progressive loss in enzyme activity as the size of the alkyl group increases from methyl to n-butyl. Reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or methoxycarbonyl coenzyme A (CoA) disulfide, leads to a total loss of enzyme activity. DTNB inactivation is biphasic (k1 = approximately 9 x 10(2) M-1 s-1, k2 = approximately 6 x 10(1) M-1 s-1) with the slow phase being diminished by acetyl-CoA. Methoxycarbonyl-CoA disulfide inactivation is also biphasic (k1 = approximately 2.1 x 10(3) M-1 s-1, k2 = approximately 6 x 10(1) M-1 s-1), with the rapid phase being diminished in the presence of acetyl-CoA. Inactivation by methoxycarbonyl methyl disulfide, ethyl disulfide, or hydroxyethyl disulfide, or by methyl methanethiosulfonate is not biphasic. Pretreatment of the enzyme with methyl methanethiosulfonate, which leads to a 25% loss in enzyme activity, abolishes the fast phase of DTNB inactivation, the slow phase of methoxycarbonyl-CoA disulfide inactivation, and any further inactivation by methoxycarbonyl ethyl disulfide. These results are interpreted to suggest that choline acetyltransferase contains two classes of reactive sulfhydryl groups, neither of which are required for enzyme activity.

摘要

随着烷基从甲基增长到正丁基，胆碱乙酰转移酶与甲氧基羰基烷基二硫化物的反应会导致酶活性逐渐丧失。与5,5'-二硫代双（2-硝基苯甲酸）（DTNB）或甲氧基羰基辅酶A（CoA）二硫化物反应会导致酶活性完全丧失。DTNB失活是双相的（k1约为9×10² M⁻¹ s⁻¹，k2约为6×10¹ M⁻¹ s⁻¹），慢相被乙酰辅酶A减弱。甲氧基羰基-CoA二硫化物失活也是双相的（k1约为2.1×10³ M⁻¹ s⁻¹，k2约为6×10¹ M⁻¹ s⁻¹），快速相在乙酰辅酶A存在时减弱。甲氧基羰基甲基二硫化物、乙基二硫化物或羟乙基二硫化物，或甲硫基磺酸甲酯导致的失活不是双相的。用甲硫基磺酸甲酯预处理酶会导致酶活性丧失25%，消除DTNB失活的快速相、甲氧基羰基-CoA二硫化物失活的慢相以及甲氧基羰基乙基二硫化物的任何进一步失活。这些结果被解释为表明胆碱乙酰转移酶含有两类反应性巯基，而这两类巯基对于酶活性都不是必需的。