Purification and characterization of hydroxycinnamoyl D-glucose. Quinate hydroxycinnamoyl transferase in the root of sweet potato, Ipomoea batatas Lam.
Purification and characterization of hydroxycinnamoyl D-glucose. Quinate hydroxycinnamoyl transferase in the root of sweet potato, Ipomoea batatas Lam.
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发表时间：
1986-07
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通讯作者：
R. Villegas;M. Kojima
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作者：
R. Villegas;M. Kojima
我们以前提出了一个绿原酸的生物合成途径，其中包括羟基肉桂酰D-葡萄糖和D-奎尼酸之间的酯交换反应。所提出的途径基于示踪剂实验结果（Kojima，M.，和Uritani，I.（1972）Plant Cell Physiol.13，311-319）。催化上述反应的酶已从甘薯根（Ipomoea batatas Lam.）和表征了纯化的酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上产生一条26，000道尔顿的条带，通过凝胶过滤色谱法估计其分子量为25，000。因此，该酶似乎由25，000 - 26，000道尔顿的单一多肽组成。该酶的等电点为8.6。酶反应的最适pH为6.0。该酶不需要任何金属活性，并显示出广泛的底物特异性对羟基肉桂酰D-葡萄糖作为供体。对于t-肉桂酰D-葡萄糖，Km和Vmax值为3.7 mM和8.5单位/mg蛋白质，对于p-香豆酰D-葡萄糖，Km和Vmax值为3.9 mM和15.1单位/mg蛋白质，对于咖啡酰D-葡萄糖，Km和Vmax值为14.3 mM和38.1单位/mg蛋白质。该酶表现出严格的底物特异性对D-奎尼酸相关化合物作为受体，Km和Vmax值分别为16.7 mM和15.1单位/毫克蛋白质的D-奎尼酸，250 mM和19.0单位/毫克蛋白质的莽草酸，并没有与L-苹果酸或内消旋酒石酸的活动。酶的活性变化的方式表明其参与绿原酸的生物合成在孵育切片甘薯根组织。
We have previously proposed a chlorogenic acid biosynthetic pathway which involves a transesterification reaction between hydroxycinnamoyl D-glucose and D-quinic acid. The proposed pathway was based on tracer experimental results (Kojima, M., and Uritani, I. (1972) Plant Cell Physiol. 13, 311-319). The enzyme that catalyzes the above reaction has been purified 160-fold from sweet potato root (Ipomoea batatas Lam.) and characterized. The purified enzyme yielded one band of 26,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 25,000 by gel filtration chromatography. Therefore, the enzyme seems to consist of a single polypeptide of 25,000-26,000 daltons. The isoelectric point of the enzyme was 8.6. The optimum pH of the enzyme reaction was 6.0. The enzyme did not require any metal for activity and showed a broad substrate specificity toward hydroxycinnamoyl D-glucose as donors. The Km and Vmax values were 3.7 mM and 8.5 units/mg of protein for t-cinnamoyl D-glucose, 3.9 mM and 15.1 units/mg of protein for p-coumaroyl D-glucose, and 14.3 mM and 38.1 units/mg of protein for caffeoyl D-glucose. The enzyme showed a strict substrate specificity toward D-quinic acid-related compounds as acceptors; the Km and Vmax values were 16.7 mM and 15.1 units/mg of protein for D-quinic acid, 250 mM and 19.0 units/mg of protein for shikimic acid, and there was no activity with either L-malic acid or meso-tartaric acid. The enzyme activity changed in a manner suggesting its involvement in chlorogenic acid biosynthesis during incubation of sliced sweet potato root tissues.