Rendina Alan R, Cheng Dong
Pharmaceutical Research Institute, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
Biochem J. 2005 Jun 15;388(Pt 3):895-903. doi: 10.1042/BJ20041963.
C75, a synthetic inhibitor of FAS (fatty acid synthase), has both anti-tumour and anti-obesity properties. In this study we provide a detailed kinetic characterization of the mechanism of in vitro inhibition of rat liver FAS. At room temperature, C75 is a competitive irreversible inhibitor of the overall reaction with regard to all three substrates, i.e. acetyl-CoA, malonyl-CoA and NADPH, exhibiting pseudo-first-order kinetics of the complexing type, i.e. a weak non-covalent enzyme-inhibitor complex is formed before irreversible enzyme modification. C75 is a relatively inefficient inactivator of FAS, with a maximal rate of inactivation of 1 min(-1) and an extrapolated K(I) (dissociation constant for the initial complex) of approx. 16 mM. The apparent second-order rate constants calculated from these values are 0.06 mM(-1).min(-1) at room temperature and 0.21 mM(-1).min(-1) at 37 degrees C. We also provide experimental evidence that C75 inactivates the beta-ketoacyl synthase (3-oxoacyl synthase) partial activity of FAS. Unexpectedly, C75 also inactivates the enoyl reductase and thioesterase partial activities of FAS with about the same rates as for inactivation of the beta-ketoacyl synthase. In contrast with the overall reaction, the beta-ketoacyl synthase activity and the enoyl reductase activity, substrates do not protect the thioesterase activity of rat liver FAS from inactivation by C75. These results differentiate inactivation by C75 from that by cerulenin, which only inactivates the beta-ketoacyl synthase activity of FAS, by forming an adduct with an active-site cysteine. Interference by dithiothreitol and protection by the substrates, acetyl-CoA, malonyl-CoA and NADPH, further distinguish the mechanism of C75-mediated inactivation from that of cerulenin. The most likely explanation for the multiple effects observed with C75 on rat liver FAS and its partial reactions is that there are multiple sites of interaction between C75 and FAS.
C75是脂肪酸合酶（FAS）的一种合成抑制剂，具有抗肿瘤和抗肥胖特性。在本研究中，我们对大鼠肝脏FAS体外抑制机制进行了详细的动力学表征。在室温下，C75是整个反应针对所有三种底物（即乙酰辅酶A、丙二酰辅酶A和NADPH）的竞争性不可逆抑制剂，呈现复合类型的假一级动力学，即在不可逆的酶修饰之前形成弱的非共价酶-抑制剂复合物。C75是一种相对低效的FAS失活剂，最大失活速率为1分钟⁻¹，外推的K（I）（初始复合物的解离常数）约为16 mM。根据这些值计算出的表观二级速率常数在室温下为0.06 mM⁻¹·分钟⁻¹，在37℃下为0.21 mM⁻¹·分钟⁻¹。我们还提供了实验证据表明C75使FAS的β-酮酰基合酶（3-氧代酰基合酶）部分活性失活。出乎意料的是，C75还使FAS的烯酰还原酶和硫酯酶部分活性失活，失活速率与β-酮酰基合酶的失活速率大致相同。与整个反应不同，对于β-酮酰基合酶活性和烯酰还原酶活性，底物不能保护大鼠肝脏FAS的硫酯酶活性不被C75失活。这些结果将C75的失活作用与浅蓝菌素的失活作用区分开来，浅蓝菌素仅通过与活性位点半胱氨酸形成加合物使FAS的β-酮酰基合酶活性失活。二硫苏糖醇的干扰以及底物乙酰辅酶A、丙二酰辅酶A和NADPH的保护作用进一步区分了C75介导的失活机制与浅蓝菌素的失活机制。对于C75对大鼠肝脏FAS及其部分反应所观察到的多种效应，最可能的解释是C75与FAS之间存在多个相互作用位点。
