Li Jing-Ya, Chen Ling-Ling, Cui Yong-Mei, Luo Qun-Li, Li Jia, Nan Fa-Jun, Ye Qi-Zhuang
Chinese National Center for Drug Screening, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 189 Guo-Shou-Jing Road, Shanghai 201203, China.
Biochem Biophys Res Commun. 2003 Jul 18;307(1):172-9. doi: 10.1016/s0006-291x(03)01144-6.
Methionine aminopeptidases (MetAPs) have been studied in vitro as Co(II) enzymes, but their in vivo metal remains to be defined. While activation of Escherichia coli MetAP (EcMetAP1) by Co(II), Mn(II), and Zn(II) was detectable by a colorimetric Met-S-Gly-Phe assay, significant activation by Ni(II) was shown in a fluorescence Met-AMC assay, in addition to Co(II) and Mn(II) activation. When tested on the metal-substituted EcMetAP1s, a few inhibitors that we obtained recently from a random screening on Co-EcMetAP1 either became much weak or lost activity on Mn- or Zn-EcMetAP1, although they kept inhibitory activity on Ni-EcMetAP1. A couple of peptidic inhibitors and the methionine mimetic (3R)-amino-(2S)-hydroxyheptanoic acid (AHHpA, 6) maintained moderate activities on Co-, Mn-, Zn-, and Ni-EcMetAP1s. Our results clearly demonstrate that the metal-substitution has changed the enzyme specificity for substrates and inhibitors. Therapeutic applications call for inhibitors specific for MetAP with a physiologically relevant metal at its active site.
甲硫氨酸氨基肽酶（MetAPs）在体外作为钴（II）酶进行了研究，但其体内金属仍有待确定。虽然通过比色法Met-S-Gly-Phe测定法可检测到钴（II）、锰（II）和锌（II）对大肠杆菌MetAP（EcMetAP1）的激活，但除了钴（II）和锰（II）激活外，在荧光Met-AMC测定法中还显示镍（II）有显著激活作用。当对金属取代的EcMetAP1进行测试时，我们最近通过对钴-EcMetAP1的随机筛选获得的一些抑制剂，对锰-或锌-EcMetAP1要么变得非常弱，要么失去活性，尽管它们对镍-EcMetAP1仍保持抑制活性。一些肽类抑制剂和甲硫氨酸模拟物（3R）-氨基-（2S）-羟基庚酸（AHHpA，6）对钴-、锰-、锌-和镍-EcMetAP1保持中等活性。我们的结果清楚地表明，金属取代改变了酶对底物和抑制剂的特异性。治疗应用需要对活性位点带有生理相关金属的MetAP具有特异性的抑制剂。
