PURIFICATION AND CHARACTERIZATION OF (2S)-FLAVANONE 3-HYDROXYLASE FROM PETUNIA-HYBRIDA
PURIFICATION AND CHARACTERIZATION OF (2S)-FLAVANONE 3-HYDROXYLASE FROM PETUNIA-HYBRIDA
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DOI：
10.1111/j.1432-1033.1986.tb09616.x
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发表时间：
1986-05-02
期刊：
影响因子：
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通讯作者：
GRISEBACH, H
中科院分区：
文献类型：
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作者：
BRITSCH, L;GRISEBACH, H
矮牵牛花中的(2S)-黄烷酮3-羟基酶催化(2S)-柚皮素转化为(2R，3R)-二氢山奈酚。在有抗坏血酸存在的厌氧条件下，该酶可以部分稳定。为了提纯，必须向缓冲液中加入2-羟基戊二酸和Fe2+。羟基酶经六步法纯化约200倍，回收率较低。经凝胶过滤，该酶的相对分子质量约为74,000。羟基酶反应的最适pH为8.5，需要氧、2-羟基戊二酸、Fe2+和抗坏血酸作为辅酶。与2-氧代[1-14C]戊二酸反应生成二氢山奈酚和14CO2的摩尔比为1：1，过氧化氢酶对反应有促进作用。该产物被明确鉴定为(+)-(2R，3R)-二氢山奈酚。羟基酶的底物是(2S)-柚皮苷元，而不是(2R)-对映体。(2S)-Eriodictyol转化为(2R，3R)-二氢槲皮素。相反，5，7，3‘，4’，5‘-五羟基黄烷酮不是底物。测定了(2S)-柚皮苷和2-氧代戊二酸的表观米氏常数分别为5.6微克分子倍。L-1和20微克分子倍。L-1，pH 8.5。(2S)-芥子醇的Km值为12微克分子倍。L-1，pH 8.0。2，4-二羧酸吡啶和2，5-二羧酸对2-羟基戊二酸具有较强的竞争性抑制作用，其KI值为1.2×10~(-6)。L-1和40微克分子倍。L-1号。
(2S)-Flavanone 3-hydroxylase from flowers of Petunia hybrida catalyses the conversion of (2S)-naringenin to (2R,3R)-dihydrokaempferol. The enzyme could be partially stabilized under anaerobic conditions in the presence of ascorbate. For purification, 2-oxoglutarate and Fe2+ had to be added to the buffers. The hydroxylase was purified about 200-fold by a six-step procedure with low recovery. The Mr of the enzyme was estimated by gel filtration to be about 74,000. The hydroxylase reaction has a pH optimum at pH 8.5 and requires as cofactors oxygen, 2-oxoglutarate, Fe2+ and ascorbate. With 2-oxo[1-14C]glutarate in the enzymes assay dihydrokaempferol and 14CO2 are formed in a molar ratio of 1:1. Catalase stimulates the reaction. The product was unequivocally identified as (+)-(2R,3R)-dihydrokaempferol. (2S)-Naringenin, but not the (2R)-enantiomer is a substrate of the hydroxylase. (2S)-Eriodictyol is converted to (2R,3R)-dihydroquercetin. In contrast, 5,7,3'',4'',5''-pentahydroxyflavanone is not a substrate. Apparent Michaelis constants for (2S)-naringenin and 2-oxoglutarate were determined to be respectively 5.6 .mu.mol .times. l-1 and 20 .mu.mol .times. l-1 at pH 8.5. The Km for (2S)-eriodictyol is 12 .mu.mol .times. l-1 at pH 8.0. Pyridine 2,4-dicarboxylate and 2 5-dicarboxylate are strong competitive inhibitors with respect to 2-oxoglutarate with Ki values of 1.2 .mu.mol .times. l-1 and 40 .mu.mol .times. l-1, respectively.