Klein J, Post K, Thomas H, Wörner W, Setiabudi F, Frank H, Oesch F, Platt K L
Institute of Toxicology, University of Mainz, F.R.G.
Chem Biol Interact. 1990;76(2):211-26. doi: 10.1016/0009-2797(90)90089-6.
Rat liver dihydrodiol dehydrogenase (DDH, EC 1.3.1.20) has been shown to reduce the mutagenicity of benz[a]anthracene (BA) in the bacterial Ames test. BA-3,4-dihydrodiol is a highly mutagenic and tumorigenic metabolite of BA. In order to test the hypothesis that this dihydrodiol may be a substrate of DDH, we established two novel assay systems for the NADP(+)-dependent oxidation of BA-3,4-dihydrodiol by rat liver DDH, an HPLC-based assay procedure and a radiometric assay with specifically labelled [3,4-3H]-BA-3,4-dihydrodiol as substrate. With the HPLC-based assay, the kinetic constants of the enzymatic catalysis were as follows: Km(app) = 21 microM for BA-3,4-dihydrodiol and Vmax = 20.0 nmol/min.mg enzyme. The reaction product was identified by cochromatography, fluorimetry and mass spectroscopy as BA-3,4-catechol, but interconversions between the catechol and the corresponding o-quinone during the analytical procedures were detected. With the radiolabelled substrate, a linear relationship between substrate concentration and reaction velocity was found. The V/K value for labelled substrate was 0.155 ml/min.mg enzyme and a (V/K)H/(V/K)T kinetic isotope effect of 6.7 was observed. The non-labelled substrate acted as a competitive inhibitor of the enzymatic oxidation of tritiated BA-3,4-dihydrodiol with a Ki value of 56.4 microM. The reaction rates determined in this study suggest an important role of DDH activity in the metabolism of BA.
大鼠肝脏二氢二醇脱氢酶（DDH，EC 1.3.1.20）已被证明在细菌艾姆斯试验中可降低苯并[a]蒽（BA）的致突变性。BA - 3,4 - 二氢二醇是BA的一种高致突变性和致癌性代谢产物。为了验证这种二氢二醇可能是DDH的底物这一假设，我们建立了两种新的检测系统，用于检测大鼠肝脏DDH对BA - 3,4 - 二氢二醇的NADP(+)依赖性氧化反应，一种是基于高效液相色谱的检测方法，另一种是以特异性标记的[3,4 - 3H] - BA - 3,4 - 二氢二醇为底物的放射性检测方法。在基于高效液相色谱的检测中，酶催化的动力学常数如下：BA - 3,4 - 二氢二醇的表观米氏常数Km(app) = 21 μM，最大反应速率Vmax = 20.0 nmol/min·mg酶。通过共色谱法、荧光法和质谱法鉴定反应产物为BA - 3,4 - 儿茶酚，但在分析过程中检测到儿茶酚与相应邻醌之间的相互转化。使用放射性标记底物时，发现底物浓度与反应速度之间呈线性关系。标记底物的V/K值为0.155 ml/min·mg酶，观察到(V/K)H/(V/K)T动力学同位素效应为6.7。未标记底物对氚标记的BA - 3,4 - 二氢二醇的酶促氧化反应起竞争性抑制作用，抑制常数Ki值为56.4 μM。本研究测定的反应速率表明DDH活性在BA代谢中起重要作用。
