Iijima H, Fujino T, Minekura H, Suzuki H, Kang M J, Yamamoto T
Tohoku University Gene Research Center, Japan, Sendai, Japan.
Eur J Biochem. 1996 Dec 1;242(2):186-90. doi: 10.1111/j.1432-1033.1996.0186r.x.
Two types of acyl-CoA synthetase (ACS), designated ACS1 and ACS2, are structurally similar isozymes with different tissue distributions. The two enzymes are organized into the following five regions: an NH2 terminus; two luciferase-like regions; a linker connecting the luciferase-like regions; a COOH terminus. Under the control of a lac promoter, rat ACS1 and ACS2 were overproduced in Escherichia coli and purified to homogeneity. The specific activities of the purified ACS1 and ACS2 were 26.2 mumol.min-1.mg-1 and 7.4 mumol.min-1.mg-1, respectively, and the most efficiently utilized saturated fatty acids were those with 10-18 carbon atoms. Among unsaturated fatty acids with 16-22 carbon atoms, the most preferred substrates were palmitoleate, oleate and linoleate for ACS1, and, for ACS2, oleate, arachidonate, eicosapentaenoate and docosahexaenoate. To determine the functionally important regions in the ACS isozymes, we constructed five ACS1 mutants lacking each of the five regions. Introduction of these mutants into E. coli revealed that all five regions in ACS1 are required for functional expression of the enzyme in E. coli; deletion of any one of the five regions almost completely abolished the enzyme activity.
两种酰基辅酶A合成酶（ACS），分别命名为ACS1和ACS2，是结构相似但组织分布不同的同工酶。这两种酶由以下五个区域组成：一个氨基末端；两个类似荧光素酶的区域；连接类似荧光素酶区域的连接子；一个羧基末端。在乳糖启动子的控制下，大鼠ACS1和ACS2在大肠杆菌中过量表达并纯化至同质。纯化后的ACS1和ACS2的比活性分别为26.2 μmol·min⁻¹·mg⁻¹和7.4 μmol·min⁻¹·mg⁻¹，最有效利用的饱和脂肪酸是含10 - 18个碳原子的那些。在含16 - 22个碳原子的不饱和脂肪酸中，ACS1最优选的底物是棕榈油酸、油酸和亚油酸，而对于ACS2，最优选的底物是油酸、花生四烯酸、二十碳五烯酸和二十二碳六烯酸。为了确定ACS同工酶中功能重要的区域，我们构建了五个缺失五个区域中每个区域的ACS1突变体。将这些突变体导入大肠杆菌表明，ACS1中的所有五个区域都是该酶在大肠杆菌中功能表达所必需的；五个区域中任何一个区域的缺失几乎完全消除了酶活性。
