Radicals in anaerobic aromatic metabolism

本文以细菌Thauera aromatica的两种厌氧芳香代谢酶为研究对象，研究了自由基在酶催化中的作用。(1)苯甲酰辅酶a还原酶通过将电子转移偶联到atp水解来还原芳环。这种反应被认为类似于桦树还原。该模型表明单电子转移和质子化交替进行，第一个电子转移的E'o为-1.9 V。“Birch”假说将通过改变底物的电子特性、使用抑制剂和同位素以及检测动力学同位素效应来探测。中间底物和/或酶自由基将通过EPF-， ENDOR-和FT-IR光谱技术检测。为了进行比较，将研究厌氧细菌中的环还原酶，它们被认为可以将电子不耦合地转移到ATP水解中。(2)苯磷酸酯羧化酶催化苯磷酸酯对羧基化生成4-羟基苯甲酸酯和14CO2/羧酸盐同位素交换。将对氧和自由基捕获剂敏感的酶进行纯化，并采用与苯甲酰辅酶a还原酶类似的方法研究潜在的自由基反应机理。

The role of radicals in enzyme catalysis will be studied with two enzymes of anaerobic aromatic metabolism in the bacterium Thauera aromatica. (1) Benzoyl-CoA reductase reduces the aromatic ring by coupling electron transfer to ATP-hydrolysis. This reaction is thought to be similar to a Birch reduction. This model implies alternate one-electron transfer and protonation steps, with E'o of the first electron transfer of -1.9 V. The 'Birch' hypothesis will be probed by altering the electronic characteristics of the substrate, by the use of of inhibitors and isotopes and by the detection of kinetic isotope effects. Intermediate substrates and/or enzyme radicals will be detected by EPF-, ENDOR- and FT-IR spectroscopic techniques. For comparison, ring reducing enzymes will be studied from anaerobic bacteria which are thought to transfer electrons without coupling to ATP hydrolysis. (2) Phenylphosphate carboxylase catalyzes phenylphosphate para-carboxylation to 4-hydroxybenzoate and 14CO2/carboxylate isotope exchange. The enzyme which is sensitive towards oxygen and radical trapping agents will be purified and the potential radical reaction mechanism will be investigated by similar methods as described for benzoyl-CoA reductase.