Pan S S, Andrews P A, Glover C J, Bachur N R
J Biol Chem. 1984 Jan 25;259(2):959-66.
Under anaerobic conditions and with proper electron donors, NADPH-cytochrome P-450 reductase (EC 1.6.2.4) and xanthine oxidase (EC 1.2.3.2) similarly reductively metabolized mitomycin C. Reversed phase high performance liquid chromatography was used to separate, detect, and isolate several metabolites. Three metabolites were identified by mass spectrometry and thin layer chromatography as 1,2-cis- and trans-2,7-diamino-1-hydroxymitosene and 2,7-diaminomitosene. Three metabolites were phosphate-dependent, and two of them were identified to be 1,2-cis- and trans-2,7-diaminomitosene 1-phosphate. The amounts of the five identified metabolites generated during the reduction of mitomycin C varied with pH and nucleophile concentration. At pH 6.5, 2,7-diaminomitosene was essentially the only metabolite formed, whereas from pH 6.8 to 8.0, trans- and cis-2,7-diamino-1-hydroxymitosene increased in quantity as 2,7-diaminomitosene decreased. The disappearance of mitomycin C and the production of metabolites were enzyme and mitomycin C concentration-dependent. Substrate saturation was not reached for either enzyme up to 5 mM mitomycin C. Electron paramagnetic resonance studies demonstrated the formation of mitomycin C radical anion as an intermediate during enzymatic activation. Our results indicate that either enzyme catalyzed the initial activation of mitomycin C to a radical anion intermediate. Subsequent spontaneous reactions, including the elimination of methanol and the opening of the aziridine ring, generate one active center at C-1 which facilitates nucleophilic attack. Simultaneous generation of two reactive centers was not observed. All five primary metabolites were metabolized further by either flavoenzyme. The secondary metabolites exhibited similar changes in their absorbance spectra and were unlike the primary metabolites, suggesting that a second alkylating center other than C-1 was generated during secondary activation. We propose that secondary activation of monofunctionally bound mitomycin C is probably a main route for the bifunctional binding of mitomycin C to macromolecules and that the cytotoxic actions of mitomycin C result from multiple metabolic activations and reactions.
在厌氧条件下并使用适当的电子供体，NADPH-细胞色素P-450还原酶（EC 1.6.2.4）和黄嘌呤氧化酶（EC 1.2.3.2）同样能将丝裂霉素C进行还原代谢。采用反相高效液相色谱法分离、检测和分离几种代谢产物。通过质谱和薄层色谱法鉴定出三种代谢产物为1,2-顺式和反式-2,7-二氨基-1-羟基丝裂霉素和2,7-二氨基丝裂霉素。三种代谢产物是磷酸依赖性的，其中两种被鉴定为1,2-顺式和反式-2,7-二氨基丝裂霉素1-磷酸。在丝裂霉素C还原过程中生成的五种已鉴定代谢产物的量随pH值和亲核试剂浓度而变化。在pH 6.5时，2,7-二氨基丝裂霉素基本上是唯一形成的代谢产物，而从pH 6.8到8.0，随着2,7-二氨基丝裂霉素的减少，反式和顺式-2,7-二氨基-1-羟基丝裂霉素的量增加。丝裂霉素C的消失和代谢产物的产生是酶和丝裂霉素C浓度依赖性的。在高达5 mM丝裂霉素C的情况下，两种酶均未达到底物饱和。电子顺磁共振研究表明，在酶促活化过程中形成了丝裂霉素C自由基阴离子作为中间体。我们的结果表明，两种酶中的任何一种都催化丝裂霉素C最初活化为自由基阴离子中间体。随后的自发反应，包括甲醇的消除和氮丙啶环的打开，在C-1处产生一个活性中心以促进亲核攻击。未观察到同时产生两个反应中心。所有五种主要代谢产物都被黄素酶进一步代谢。次要代谢产物在其吸收光谱中表现出类似的变化，并且与主要代谢产物不同，这表明在二次活化过程中除了C-1之外还产生了第二个烷基化中心。我们提出，单功能结合的丝裂霉素C的二次活化可能是丝裂霉素C与大分子双功能结合的主要途径，并且丝裂霉素C的细胞毒性作用是由多种代谢活化和反应引起的。
