4-COUMARATE - COA LIGASE FROM CELL-SUSPENSION CULTURES OF PETROSELINUM-HORTENSE-HOFFM - PARTIAL-PURIFICATION, SUBSTRATE-SPECIFICITY, AND FURTHER PROPERTIES
4-COUMARATE - COA LIGASE FROM CELL-SUSPENSION CULTURES OF PETROSELINUM-HORTENSE-HOFFM - PARTIAL-PURIFICATION, SUBSTRATE-SPECIFICITY, AND FURTHER PROPERTIES
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DOI：
10.1016/0003-9861(77)90347-2
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发表时间：
1977-01-01
影响因子：
3.9
通讯作者：
HAHLBROCK, K
中科院分区：
文献类型：
--
作者：
KNOBLOCH, KH;HAHLBROCK, K
4-香豆酸：CoA连接酶（EC 6.2.1.12）分离自欧芹（P.霍尔滕斯Hoffm.）用紫外光照射15小时。用MnCl_2和（NH_4）_2SO_4分级分离，用二乙氨基乙基纤维素、羟基磷灰石和氨基己基-琼脂糖柱层析，部分纯化了该酶。比活性提高了90倍，总产率为20%。分析型凝胶电泳表明，在最终的酶制剂中仅存在一种4-香豆素酸：CoA连接酶。该酶对4-香豆酸和肉桂酸的其他衍生物具有很强的特异性。4-香豆酸酯具有最低的表观Km和最高的V/Km值（1.4 × 1.5）。10-5 M和14.7 ×. 105 pkatal. M-1）。只有4-香豆酸的反式异构体被激活。2个共底物，ATP和CoA，表现出S形饱和动力学，这被解释为表明同向，变构效应。估计4-香豆酸：CoA连接酶的MW约为67，000。该酶的底物特异性与其在类黄酮生物合成中的功能一致。
4-Coumarate:CoA ligase (EC 6.2.1.12) was isolated from 8 day old cell suspension cultures of parsley (P. hortense Hoffm.) which were irradiated with UV light for 15 h. The enzyme was partially purified by fractionation with MnCl2 and (NH4)2SO4 and by column chromatography on diethylaminoethyl cellulose, hydroxyapatite and aminohexyl-Sepharose. A 90-fold increase in specific activity with an overall yield of 20% was achieved. Analytical gel electrophoresis indicated the occurrence of only one 4-courmarate:CoA ligase species in the final enzyme preparation. The enzyme was largely specific for 4-coumarate and other derivatives of cinnamic acid. 4-Coumarate had the lowest apparent Km and the highest V/Km values (1.4 .times. 10-5 M and 14.7 .times. 105 pkatal .times. M-1, respectively) of all substrates tested. Only the trans isomer of 4-coumarate was activated. The 2 cosubstrates, ATP and CoA, exhibited sigmoidal saturation kinetics, which were interpreted as indicating homotropic, allosteric effects. A MW of about 67,000 was estimated for 4-coumarate:CoA ligase. The substrate specificity of the enzyme was in agreement with its proposed function in flavonoid biosynthesis.