KINETIC STUDIES ON A 4-METHOXYBENZOATE O-DEMETHYLASE FROM PSEUDOMONAS-PUTIDA
KINETIC STUDIES ON A 4-METHOXYBENZOATE O-DEMETHYLASE FROM PSEUDOMONAS-PUTIDA
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DOI：
10.1111/j.1432-1033.1977.tb11230.x
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发表时间：
1977-01-01
期刊：
影响因子：
--
通讯作者：
SEYDEWITZ, V
中科院分区：
文献类型：
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作者：
BERNHARDT, FH;NASTAINCZYK, W;SEYDEWITZ, V
本文介绍了一种直接、灵敏、可靠的测定微生物非特异性4-甲氧基苯甲酸O-脱甲基酶活性的光度法。该测定基于3-硝基-4-甲氧基苯甲酸酯O-脱甲基化生成黄色产物3-硝基-4-羟基苯甲酸酯。使用该测定并通过监测还原的吡啶核苷酸的氧化速率，研究了由4-甲氧基苯甲酸单加氧酶[EC 1.14.99.15]（O-脱甲基化）和来自恶臭假单胞菌的还原酶组成的纯化的重构酶系统的动力学性质。该酶系统的单加氧酶对不同底物的Km值（即，紧耦合器、解耦合器和部分解耦合器）从紧耦合器的0.07 μ M的低值上升到约0.07 μ M。55 μ M用于解偶联剂。研究了可能的抑制剂和金属离子对重组酶系统的影响。的抑制模式是几乎相同的，发现为纯化的还原酶，只有红菲咯啉二磺酸盐显示出更大的抑制重构酶系统。还原酶对NADH的亲和力为约。200-比对NADPH的反应大一倍。该还原酶对NADH的亲和力取决于电子受体的性质，当使用单加氧酶底物复合物作为电子受体时，比使用细胞色素c或2，6-二氯靛酚时高10倍以上。这些差异进行了讨论的基础上的酶之间的相互作用的还原酶和单加氧酶。
A direct, sensitive and reliable photometric assay procedure for monitoring the activity of non-specific 4-methoxybenzoate O-demethylases of microorganisms is described. The assay is based on the O-demethylation of 3-nitro-4-methoxybenzoate to the yellow-colored product 3-nitro-4-hydroxybenzoate. Using this assay and by monitoring the oxidation rate of reduced pyridine nucleotides, the kinetic properties of a purified, reconstituted enzyme system composed of 4-methoxybenzoate monooxygenase [EC 1.14.99.15] (O-demethylating) and a reductase from P. putida were investigated. The Km value of the monooxygenase of this enzyme system towards different substrates (i.e., tight couplers, uncouplers and partial uncouplers) rises from the low value of 0.07 .mu.M for the tight couplers to .apprx. 55 .mu.M for the uncouplers. The effect of possible inhibitors and metal ions on the reconstituted enzyme system was investigated. The inhibition pattern was almost identical to that found for the purified reductase, only bathophenanthrolinedisulfonate showing a greater inhibition of the reconstituted enzyme system. The affinity of the reductase towards NADH was .apprx. 200-fold greater than that towards NADPH. The affinity of this reductase to NADH depended on the nature of the electron acceptor and was > 10 times higher when the monooxygenase.cntdot.substrate complex was used as the electron acceptor, than when cytochrome c or 2,6-dichloroindophenol was used. These differences are discussed on the basis of enzyme-enzyme interactions between the reductase and the monooxygenase.