Kim Y S, Byun H S
Department of Biochemistry, College of Science, Yonsei University, Seoul, Korea.
J Biol Chem. 1994 Nov 25;269(47):29636-41.
A novel type of malonate decarboxylase which is induced and located in periplasm of Acinetobacter calcoaceticus grown on malonate as sole carbon and energy source was purified to homogeneity. The purified 185-kDa decarboxylase was found to be an oligomer composed of three different polypeptides in a ratio of 2:1:1, designated alpha (65 kDa), beta (32 kDa), and gamma (25 kDa). Optimum pH was 6.8, while pI was 6.39. The enzyme was highly specific for malonate with Km 1.4 mM. This enzyme contained a biotin prosthetic group on alpha subunit which does not seem to be directly related to malonate decarboxylation. The purified enzyme showed almost no activity; activity was restored, however, by treatment with acetic anhydride or malonyl-CoA, which formed an acetyl-enzyme. The 14C-acylated enzyme was isolated by a gel filtration from the reaction mixtures containing [2-14C]malonyl-CoA and the enzyme or [2-14C]malonate, malonyl-CoA, and the enzyme. When treated by thiol-specific reagents, such as N-ethylmaleimide, 5,5'-dinitro-bis(2-nitrobenzoic acid), and 2-nitro-5-thiocyanobenzoic acid, the purified enzyme showed no increase in activity after the acetic anhydride treatment. The thiol-specific reagents failed to inhibit, however, the catalytically active acetyl-enzyme, suggesting that the site of acylation is the thiol group. Further evidence was provided when sodium borohydride inactivated the acetyl-enzyme. These results suggest that malonate decarboxylation by this enzyme may proceed by a catalytic cycle in which the acetyl group on the active enzyme is displaced by malonate, which binds covalently to a thiol group on the enzyme and is subsequently decarboxylated.
一种新型丙二酸脱羧酶被纯化至同质，该酶是在以丙二酸作为唯一碳源和能源生长的醋酸钙不动杆菌的周质中被诱导产生并定位的。纯化后的185 kDa脱羧酶是一种由三种不同多肽组成的寡聚体，其比例为2:1:1，分别命名为α（65 kDa）、β（32 kDa）和γ（25 kDa）。最适pH为6.8，而等电点为6.39。该酶对丙二酸具有高度特异性，Km为1.4 mM。这种酶在α亚基上含有一个生物素辅基，这似乎与丙二酸脱羧没有直接关系。纯化后的酶几乎没有活性；然而，用乙酸酐或丙二酰辅酶A处理可恢复其活性，二者会形成乙酰化酶。通过凝胶过滤从含有[2-14C]丙二酰辅酶A和该酶或[2-14C]丙二酸、丙二酰辅酶A和该酶的反应混合物中分离出14C-酰化酶。当用硫醇特异性试剂如N-乙基马来酰亚胺、5,5'-二硝基-双（2-硝基苯甲酸）和2-硝基-5-硫氰基苯甲酸处理时，纯化后的酶在乙酸酐处理后活性没有增加。然而，硫醇特异性试剂未能抑制具有催化活性的乙酰化酶，这表明酰化位点是硫醇基团。硼氢化钠使乙酰化酶失活时提供了进一步的证据。这些结果表明，该酶催化的丙二酸脱羧可能通过一个催化循环进行，其中活性酶上的乙酰基团被丙二酸取代，丙二酸与酶上的硫醇基团共价结合，随后脱羧。
