Department of Chemistry and Institute for Biomolecular Science, University of South Florida, 4202 E. Fowler Avenue, CHE205, Tampa, FL 33620-5250, USA.
J Inorg Biochem. 2010 Jan;104(1):19-29. doi: 10.1016/j.jinorgbio.2009.09.019. Epub 2009 Sep 29.
The dinuclear aminopeptidase from Streptomyces griseus (SgAP) and its metal derivatives catalyze the hydrolysis of the phosphoester bis(p-nitrophenyl) phosphate (BNPP) and the phosphonate ester p-nitrophenyl phenylphosphonate with extraordinary rate enhancements at pH 7.0 and 25 degrees C [A. Ercan, H. I. Park, L.-J. Ming, Biochemistry 45, (2006) 13779-13793.], reaching 6.7 billion-fold in terms of the first-order rate constant of the di-Co(II) derivative with respect to the autohydrolytic rates. Since phosphoesters are transition state-like inhibitors in peptide hydrolysis, their hydrolysis by SgAP is quite novel. Herein, we report the investigation of this proficient alternative catalysis of SgAP and the role of each metal ion in the dinuclear site toward peptide and BNPP hydrolysis. Mn(II) selectively binds to one of the dinuclear metal sites (M1), affording MnE-SgAP with an empty (E) second site for the binding of another metal (M2), including Mn(II), Co(II), Ni(II), Zn(II), and Cd(II). Peptide hydrolysis is controlled by M2, wherein the k(cat) values for the derivatives MnM2-SgAP are different yet similar between MnCo- and CoCo-SgAP and pairs of other metal derivatives. On the other hand, BNPP hydrolysis is affected by metals in both sites. Thus, the two hydrolytic catalyses must follow different mechanisms. Based on crystal structures, docking, and the results presented herein, the M1 site is close to the hydrophobic specific site and the M2 site is next to Tyr246 that is H-bonded to a coordinated nucleophilic water molecule in peptide hydrolysis; whereas a coordinated water molecule on M1 becomes available as the nucleophile in phosphodiester hydrolysis.
来自灰色链霉菌（SgAP）的双核氨肽酶及其金属衍生物在 pH7.0 和 25°C 下以极高的速率催化水解双（对硝基苯）磷酸酯（BNPP）和膦酸酯对硝基苯苯膦酸酯[A. Ercan、H.I. Park、L.-J. Ming，生物化学 45，（2006）13779-13793]。对于二-Co（II）衍生物相对于自动水解速率的一级速率常数，达到 67 亿倍。由于磷酸酯是肽水解的过渡态样抑制剂，因此 SgAP 对其水解是相当新颖的。在此，我们报告了对 SgAP 这种高效替代催化作用的研究，以及双核位点中每个金属离子在肽和 BNPP 水解中的作用。Mn(II)选择性地结合到双核金属位点之一（M1），为 MnE-SgAP 提供了一个空的（E）第二位点，用于结合另一种金属（M2），包括 Mn(II)、Co(II)、Ni(II)、Zn(II)和 Cd(II)。肽水解受 M2 控制，其中 MnM2-SgAP 的衍生物的 k(cat)值对于 MnCo-和 CoCo-SgAP 以及其他金属衍生物的配对是不同的，但又相似。另一方面，BNPP 水解受到两个位点中的金属影响。因此，两种水解催化作用必须遵循不同的机制。基于晶体结构、对接和本文提供的结果，M1 位点靠近疏水区特定位点，M2 位点紧邻 Tyr246，该位点与结合的亲核水分子形成氢键，在肽水解中；而 M1 上的一个配位水分子在磷酸二酯水解中可用作亲核试剂。
