Flavin substrate specificity of the vitamin B2-aldehyde-forming enzyme from Schizophyllum commune.
Flavin substrate specificity of the vitamin B2-aldehyde-forming enzyme from Schizophyllum commune.
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来自裂褶藻的维生素 B2-醛形成酶的黄素底物特异性。
DOI：
10.1006/abbi.1994.1476
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发表时间：
1994
影响因子：
3.9
通讯作者：
McCormick,DB
中科院分区：
文献类型：
--
作者：
Kekelidze,TN;Edmondson,DE;McCormick,DB
裂殖酵母的维生素B2-醛形成酶催化核黄素的5‘-羟甲基氧化成甲酰基。我们以2，6-二氯苯酚-吲哚苯酚为电子受体，用分光光度法检测了35个核黄素类似物作为潜在底物或竞争性抑制剂的活性，以确定底物结合部位的结构要求。侧链长度为2-6个碳的类似物不被氧化，这些侧链通过删除仲羟基或改变其表观构型而被修饰。ω-羟基烷基黄素(n=2-6)是核黄素氧化的竞争性抑制剂(Ki=7-16μM)，与侧链上的L-仲羟基类似。异四氮杂环上具有大体积取代基的类似物也不是底物。该酶不能显著结合黄素：8α-N-咪唑；8位二乙氨基、甲乙氨基、二甲氨基、乙氨基或乙氧基；6位甲基；2位β-羟乙氨基。此外，1-脱氮胞黄素中的CH取代N也不允许底物反应。在第8位具有氟、氯、甲基、氨基或甲氨基的类似物；在第7位有氯；在第3位有甲基或羧甲基；在第2位有硫代，在第3或5位用C取代N是底物，其相对Vmax值在核黄素的27%至110%之间。被氧化的类似物的Km值都在微摩尔范围内(22-176μM)。核黄素酶的总体专一性被发现相当狭窄和空间限制，这表明维生素是天然的底物。
Vitamin B2-aldehyde-forming enzyme fromSchizophyllum communecatalyzes oxidation of the 5′-hydroxymethyl of riboflavin to the formyl group. We have monitored enzyme activity by spectrophotometrically measuring the reduction of 2,6-dichlorophenol-indolphenol as electron acceptor to assess 35 riboflavin analogs as potential substrates or competitive inhibitors with the purpose of delimiting structural requirements of the substrate binding site. Analogs with side chains of two-to six-carbon length modified by deletion of secondary hydroxyls or by changes in their epimeric configuration are not oxidized. The ω-hydroxyalkyl-flavins (n= 2-6) are competitive inhibitors (Ki= 7-16 μM) of riboflavin oxidation, as are some analogs with L-secondary hydroxyls in the side chain. Analogs with bulky substituents on the isoalloxazine ring are also not substrates. The enzyme does not significantly bind flavins with an 8α-N-imidazole; diethylamino, methylethylamino, dimethylamino, ethylamino, or ethoxy groups at position 8; methyl at 6; and β-hydroxyethylamino at position 2. Also the replacement of N with CH in 1-deazariboflavin disallows substrate reaction. Analogs with fluoro, chloro, methyl, amino, or methylamino at position 8; chloro at 7; methyl or carboxymethyl at 3; thio at 2, and C replacing N at positions 3 or 5 are substrates with relativeVmaxvalues ranging from 27 to 110% that of riboflavin. TheKmvalues for the analogs oxidized are all found to be in the micromolar range (22-176 μM). Overall specificity of the enzyme for riboflavin is found to be rather narrow and sterically limited, which suggests that the vitamin is the natural substrate.