Initial-rate kinetics of the flavin reductase reaction catalysed by human biliverdin-IXβ reductase (BVR-B)
Initial-rate kinetics of the flavin reductase reaction catalysed by human biliverdin-IXβ reductase (BVR-B)
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DOI：
10.1042/0264-6021:3450393
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发表时间：
2000-01-15
影响因子：
4.1
通讯作者：
Mantle, TJ
中科院分区：
文献类型：
--
作者：
Cunningham, O;Gore, MG;Mantle, TJ
在pH 7.5时，胆绿素-IX β还原酶催化的黄素还原酶反应的初始速率动力学与快速平衡有序机制一致，首先与吡啶核苷酸结合。NADPH结合到游离酶的特征在于使用停流荧光猝灭，并计算出15.8 μ M的Kd。平衡荧光猝灭实验表明，0.55 μ M的K-D，这表明酶-NADPH遇到复杂的（K-D 15.8 μ M）异构化到一个更稳定的“核苷酸诱导的”构象。该酶催化FMN、FAD和核黄素的还原，K-m值分别为52 μ M、125 μ M和53 μ M。Lumichrome被证明是FMN的竞争性抑制剂，具有76 μ M的Ki，表明与异咯嗪环的相互作用可能足以结合。在最初的实验中，它被观察到，黄素还原酶和胆绿素还原酶活性的酶表现出尖锐的最佳pH值为5的柠檬酸盐缓冲液。初始速率研究表明，该酶在该缓冲液中服从稳态有序机制。在pH 5的乙酸钠中的初始速率动力学与快速平衡有序机制一致，表明柠檬酸盐可能直接影响酶在pH 5的行为。中胆绿素XIII α是一种合成胆绿素，与黄素还原酶结合，但不作为酶的底物，与FMN（Ki 0.59 μ M）表现出竞争动力学，与NADPH表现出混合抑制动力学。这与单个吡啶核苷酸位点以及FMN和胆绿素对第二个位点的竞争一致。有趣的是，黄素还原酶/胆绿素-IX β还原酶也显示出三价铁还原酶活性，三价铁的表观Km为2.5 μ M。铁还原酶反应需要NAD（P）H和FMN。这种活性是有趣的，因为胎儿中的血红素分裂产生非α。胆绿素和三价铁的异构体，二者都是黄素还原酶/胆绿素-IX β还原酶的底物。
The initial-rate kinetics of the flavin reductase reaction catalysed by biliverdin-IX beta reductase at pH 7.5 are consistent with a rapid-equilibrium ordered mechanism, with the pyridine nucleotide binding first. NADPH binding to the free enzyme was characterized using stopped-flow fluorescence quenching, and a K-d of 15.8 mu M was calculated. Equilibrium fluorescence quenching experiments indicated a K-d of 0.55 mu M, suggesting that an enzyme-NADPH encounter complex (K-d 15.8 mu M) isomerizes to a more stable 'nucleotide-induced' conformation. The enzyme was shown to catalyse the reduction of FMN, FAD and riboflavin, with K-m values of 52 mu M, 125 mu M and 53 mu M, respectively. Lumichrome was shown to be a competitive inhibitor against FMN, with a K-i of 76 mu M, indicating that interactions with the isoalloxazine ring are probably sufficient for binding. During initial experiments it was observed that both the flavin reductase and biliverdin reductase activities of the enzyme exhibit a sharp optimum at pH 5 in citrate buffer. An initial-rate study indicated that the enzyme obeys a steady-state ordered mechanism in this buffer. The initial-rate kinetics in sodium acetate at pH 5 are consistent with a rapid-equilibrium ordered mechanism, indicating that citrate may directly affect the enzyme's behaviour at pH 5. Mesobiliverdin XIII alpha, a synthetic biliverdin which binds to flavin reductase but does not act as a substrate for the enzyme, exhibits competitive kinetics with FMN (K-i 0.59 mu M) and mixed-inhibition kinetics with NADPH. This is consistent with a single pyridine nucleotide site and competition by FMN and biliverdin for a second site. Interestingly, flavin reductase/biliverdin-IX beta reductase has also been shown to exhibit ferric reductase activity, with an apparent K-m of 2.5 mu M for the ferric iron. The ferric reductase reaction requires NAD(P)H and FMN. This activity is intriguing, as haem cleavage in the foetus produces non-alpha. isomers of biliverdin and ferric iron, both of which are substrates for flavin reductase/biliverdin-IX beta reductase.