Stafford H A, Baldy R
Biology Department, Reed College, Portland, Oregon 97202.
Plant Physiol. 1970 Feb;45(2):215-22. doi: 10.1104/pp.45.2.215.
A p-hydroxycinnamic acid oxidase activity was present in enzyme preparations from first internodes of Sorghum vulgare variety Wheatland milo when incubated in phosphate buffer at pH 7.5. This preparation had no classical polyphenolase activity but had both peroxidase and catalase activities. Since horseradish preparations catalyzed the same reaction, the oxidation probably is another example of a peroxidase-oxidase reaction. A second substrate was p-hydroxyphenylpyruvic acid. Ferulic acid was slightly active at low concentrations and inhibitory at higher ones. Diphenols such as caffeic and chlorogenic acids were inactive and inhibitory to p-hydroxycinnamic acid oxidation. A variety of monophenols such as tyrosine and cinnamic acid were inactive. An active substrate must have a free monophenolic group and para to this a C(3) side chain with a double bond and probably a free terminal acid group. A sulfhydryl reducing agent at the 5 millimolar level such as mercaptoethanol, reduced glutathione, or dithiothreitol was obligatory. Products were varied and were found in both the ethyl acetate-soluble and insoluble fractions after acidification of the incubation mixtures. With internode extracts, about 1 micromole of O(2) was consumed per micromole of p-hydroxycinnamic acid that disappeared in the presence of mercaptoethanol. Tetrahydrafolic acid plus mercaptoethanol were required for a second step oxidation or a parallel reaction; about 2 micromoles of O(2) were consumed per micromole of p-hydroxycinnamic acid that disappeared. Potassium cyanide, diethyldithiocarbamate, ascorbic acid, and ethylenediaminetetraacetate were inhibitory. A similar mercaptoethanol-dependent monophenol oxidase was present in preparations from green shoots that also contained a classical polyphenolase activity. The activity was present in both soluble and particulate (500 to 100,000 gravity) fractions of internodes. Preliminary studies were made of enzyme complexes in the particulate fractions capable of converting phenylalanine and tyrosine to the level of ferulic acid when the above p-hydroxycinnamic acid oxidase was blocked with ascorbic acid. The ratelimiting step was the hydroxylation of p-hydroxycinnamic acid.
当在pH 7.5的磷酸盐缓冲液中孵育时，来自高粱品种惠特兰 Milo 第一节间的酶制剂中存在对羟基肉桂酸氧化酶活性。该制剂没有经典的多酚酶活性，但具有过氧化物酶和过氧化氢酶活性。由于辣根制剂催化相同的反应，这种氧化可能是过氧化物酶 - 氧化酶反应的另一个例子。第二种底物是对羟基苯丙酮酸。阿魏酸在低浓度时略有活性，在高浓度时具有抑制作用。二酚如咖啡酸和绿原酸无活性且抑制对羟基肉桂酸氧化。多种单酚如酪氨酸和肉桂酸无活性。一种活性底物必须具有一个游离的单酚基团，并且在其对位有一个带有双键且可能带有游离末端酸基团的C(3)侧链。5毫摩尔水平的巯基还原剂如巯基乙醇、还原型谷胱甘肽或二硫苏糖醇是必需的。产物多种多样，在孵育混合物酸化后，在乙酸乙酯可溶和不溶部分中均有发现。对于节间提取物，在巯基乙醇存在的情况下，每消失1微摩尔对羟基肉桂酸大约消耗1微摩尔O(2)。第二步氧化或平行反应需要四氢叶酸加巯基乙醇；每消失1微摩尔对羟基肉桂酸大约消耗2微摩尔O(2)。氰化钾、二乙基二硫代氨基甲酸盐、抗坏血酸和乙二胺四乙酸具有抑制作用。在绿芽的制剂中也存在一种类似的依赖巯基乙醇的单酚氧化酶，该制剂还含有经典的多酚酶活性。该活性存在于节间的可溶性和颗粒（500至100,000重力）部分中。当上述对羟基肉桂酸氧化酶被抗坏血酸阻断时，对颗粒部分中能够将苯丙氨酸和酪氨酸转化为阿魏酸水平的酶复合物进行了初步研究。限速步骤是对羟基肉桂酸的羟基化。
