Kekelidze T N, Edmondson D E, McCormick D B
Department of Biochemistry, Emory University, Atlanta, Georgia 30322-3050.
Arch Biochem Biophys. 1994 Nov 15;315(1):100-3. doi: 10.1006/abbi.1994.1476.
Vitamin B2-aldehyde-forming enzyme from Schizophyllum commune catalyzes 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 omega-hydroxyalkyl-flavins (n = 2-6) are competitive inhibitors (Ki = 7-16 microM) 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 8 alpha-N-imidazole; diethylamino, methylethylamino, dimethylamino, ethylamino, or ethoxy groups at position 8; methyl at 6; and beta-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 carboxylmethyl at 3; thio at 2, and C replacing N at positions 3 or 5 are substrates with relative Vmax values ranging from 27 to 110% that of riboflavin. The Km values for the analogs oxidized are all found to be in the micromolar range (22-176 microM). 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.
裂褶菌中的维生素B2醛形成酶催化核黄素5'-羟甲基氧化为甲酰基。我们通过分光光度法测量作为电子受体的2,6-二氯酚靛酚的还原反应来监测酶活性，以此评估35种核黄素类似物作为潜在底物或竞争性抑制剂，目的是确定底物结合位点的结构要求。具有两到六个碳原子长度侧链且仲羟基缺失或差向异构构型改变的类似物不会被氧化。ω-羟烷基黄素（n = 2 - 6）是核黄素氧化的竞争性抑制剂（Ki = 7 - 16 microM），一些在侧链带有L-仲羟基的类似物也是如此。在异咯嗪环上带有庞大取代基的类似物也不是底物。该酶与在8位带有α-N-咪唑、二乙氨基、甲基乙基氨基、二甲氨基、乙氨基或乙氧基；在6位带有甲基；在2位带有β-羟乙氨基的黄素没有明显结合。同样，1-去氮核黄素中N被CH取代也不允许底物反应。在8位带有氟、氯、甲基、氨基或甲基氨基；在7位带有氯；在3位带有甲基或羧甲基；在2位带有硫，以及在3或5位C取代N的类似物是底物，其相对Vmax值为核黄素的27%至110%。所有被氧化的类似物的Km值都在微摩尔范围内（22 - 176 microM）。发现该酶对核黄素的总体特异性相当狭窄且受空间限制，这表明该维生素是天然底物。
