Both nucleophile and substrate bind to the catalytic Fe(II)-center in the type-II methionyl aminopeptidase from Pyrococcus furiosus
Both nucleophile and substrate bind to the catalytic Fe(II)-center in the type-II methionyl aminopeptidase from Pyrococcus furiosus
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DOI：
10.1021/ic0487934
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发表时间：
2005-03-07
影响因子：
4.6
通讯作者：
Holz, RC
中科院分区：
文献类型：
--
作者：
Copik, AJ;Waterson, S;Holz, RC
金属蛋白酶利用其活性位点二价金属离子生成亲核水/氢氧化物。对于蛋氨酸氨基肽酶（MetAPs），这种亲核试剂以及底物相对于活性位点金属离子的确切位置是未知的。为了解决这个问题，我们通过动力学和光谱（EPR, UV-vis）方法研究了在没有和存在一氧化氮（NO）和底物类似抑制剂丁硼酸（BuBA）的情况下，PfMetAP-II （[Fe(PfMetAP-II)]）负载Fe（II）的催化能力。NO与[Fe(PfMetAP-II)]结合，K-d为200mum，形成{FeNO}(7)配合物。得到的[Fe(PfMetAP-II)]-NO配合物的紫外-可见光谱表明，Fe（II）离子为六坐标。这些数据表明，NO结合发生时不会取代[Fe(PfMetAP-II)]中结合的水/羟基部分。根据EPR谱，得到的Fe-NO配合物最好描述为NO- (S = 1)与高自旋Fe（III）离子（S = 5/2）的反铁磁耦合。BuBA加入[Fe(PfMetAP-II)]-NO取代配位水分子形成六坐标加合物。EPR数据还表明，结合的NO-和BuBA之间发生相互作用，形成一个复合物，模拟Michaelis复合物和四面体过渡态之间的中间步骤。
Metalloproteases utilize their active site divalent metal ions to generate a nucleophilic water/hydroxide. For methionine aminopeptidases (MetAPs), the exact location of this nucleophile, as well as of the substrate, with respect to the active site metal ion is unknown. In order to address this issue, we have examined the catalytically competent Fe(II)-loaded form of PfMetAP-II ([Fe(PfMetAP-II)]) in the absence and presence of both nitric oxide (NO) and the substrate-analogue inhibitor butaneboronic acid (BuBA) by kinetic and spectroscopic (EPR, UV-vis) methods. NO binds to [Fe(PfMetAP-II)] with a K-d of 200 muM forming an {FeNO}(7) complex. UV-vis spectra of the resulting [Fe(PfMetAP-II)]-NO complex indicate that the Fe(II) ion is six coordinate. These data suggest that NO binding occurs without displacing the bound aquo/ hydroxo moiety in [Fe(PfMetAP-II)]. On the basis of EPR spectra, the resulting Fe-NO complex is best described as NO- (S = 1) antiferromagnetically coupled to a high-spin Fe(III) ion (S = 5/2). The addition of BuBA to [Fe(PfMetAP-II)]-NO displaces the coordinated water molecule forming a six-coordinate adduct. EPR data also indicate that an interaction between the bound NO- and BuBA occurs forming a complex that mimics an intermediate step between the Michaelis complex and the tetrahedral transition-state.