Spectroscopically distinct cobalt(II) sites in heterodimetallic forms of the aminopeptidase from Aeromonas proteolytica: Characterization of substrate binding
Spectroscopically distinct cobalt(II) sites in heterodimetallic forms of the aminopeptidase from Aeromonas proteolytica: Characterization of substrate binding
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DOI：
10.1021/bi970735p
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发表时间：
1997-08-12
期刊：
影响因子：
2.9
通讯作者：
Holz, RC
中科院分区：
文献类型：
--
作者：
Bennett, B;Holz, RC
本文报道了溶蛋白气单胞菌（Aeromonasproteolytica，AAP）氨肽酶的Co（Ⅱ）Zn（Ⅱ）和Zn（Ⅱ）Co（Ⅱ）取代衍生物的EPR谱。在[CoZn（AAP）]和[ZnCo（AAP）]中的高自旋S = 3/2 Co（II）离子的EPR谱表明，每个金属结合位点提供光谱上不同的特征。对于[CoZn（AAP）]，在pH 7.5和10下记录的EPR光谱的减法显示存在两种物质，并且对每个实验光谱的相对贡献是pH依赖性的。在较低pH值下占优势的第一EPR物质被模拟为相对无特征的轴向信号，g（eff）值为2.20、3.92和5.23，其对应于具有2.29的g（真实的）和0.1的E/D的M-s = 1 +/-1/2]基态跃迁。第二个物种，主要在高pH值，模拟与g（eff）值为1.80，2.75，和6.88，并表现出一个特征的八线Co-59超精细图案与A（z）（Co-59）的7.0 mT。这些参数对应于g（真实的）为2.54的M-s = \+/-1/2]基态跃迁;然而，信号表现出明显的菱形性（E/D = 0.32），指示不对称四面体或五配位Co（II）离子。这两个物种的总和提供了一个很好的模拟所观察到的[CoZn（AAP）] EPR谱。[ZnCo（AAP）]的EPR谱也包含两个物种，其中至少一个物种也表现出Co-59超精细特征。然而，该信号表现出很小的pH依赖性，并且不能分离出单个物质。加入竞争性抑制剂1-丁烷硼酸（BuBA）[CoZn（AAP）]导致EPR谱的明显变化;然而，加入BuBA [ZnCo（AAP）]使EPR谱完全不受干扰。这些数据表明BuBA仅结合AAP中的第一个金属结合位点，不与第二个位点相互作用。基于AAP和来自牛透镜的氨肽酶的过渡态类似物抑制复合物的X射线晶体学数据，BuBA被重新分类为底物类似物抑制剂，而不是如先前所建议的过渡态类似物抑制剂[Baker，J.O.，& Prescott，J. M.（1983）Biochemistry 22，5322-5331]。从差谱和从[CoZn（AAP）] EPR谱的模拟中，分离出BuBA结合后出现的第三个信号。该信号用2.08、3.15和6.15的g（eff）值模拟，其对应于具有2.41的g（真实的）和0.22的E/D的M-s = 1 +/-1/2]基态跃迁。该模拟还调用了A（z）（Co-59）值为4.0 mT的八线未分辨Co-59超精细图案。这三种物质的总和提供了在两种pH值下观察到的[CoZn（AAP）] + BuBA EPR谱的极好模拟。这项工作确立了底物仅与AAP中的第一个金属结合位点结合，从而证实了最近提出的AAP作用机制中催化的第一步。
The Co(II)Zn(II)- and Zn(II)Co(II)-substituted derivatives of the aminopeptidase from Aeromonas proteolytica (AAP) were probed by EPR spectroscopy. EPR spectra of the high-spin S = 3/2 Co(II) ions in [CoZn(AAP)] and [ZnCo(AAP)] indicated that each metal binding site provides a spectroscopically distinct signature. For [CoZn(AAP)], subtraction of EPR spectra recorded at pH 7.5 and 10 revealed that two species were present and that the relative contributions to each of the experimental spectra were pH-dependent. The first EPR species, predominant at lower pH values, was simulated as a relatively featureless axial signal with g(eff) values of 2.20, 3.92, and 5.23 which correspond to an M-s = \+/-1/2] ground state transition with a g(real) of 2.29 and an E/D of 0.1. The second species, predominant at high pH, was simulated with g(eff) values of 1.80, 2.75, and 6.88 and exhibited a characteristic eight-line Co-59 hyperfine pattern with an A(z)(Co-59) of 7.0 mT. These parameters correspond to an M-s = \+/-1/2] ground state transition with a g(real) of 2.54; however, the signal exhibited marked rhombicity (E/D = 0.32) indicative of an asymmetric tetrahedral or five-coordinate Co(II) ion. Summation of these two species provided an excellent simulation of the observed [CoZn(AAP)] EPR spectrum. The EPR spectrum of [ZnCo(AAP)] also contained two species, at least one of which also exhibited Co-59 hyperfine features. However, this signal exhibited little pH dependence, and individual species could not be isolated. The addition of the competitive inhibitor 1-butaneboronic acid (BuBA) to [CoZn(AAP)] resulted in a distinct change in the EPR spectrum; however, addition of BuBA to [ZnCo(AAP)] left the EPR spectrum completely unperturbed. These data indicate that BuBA binds only to the first metal binding site in AAP and does not interact with the second site. On the basis of the X-ray crystallographic data for the transition state analog-inhibited complexes of AAP and the aminopeptidase from bovine lens, BuBA was reclassified as a substrate analog inhibitor rather than a transition state analog inhibitor as previously suggested [Baker, J. O., & Prescott, J. M. (1983) Biochemistry 22, 5322-5331]. From difference spectroscopy and from the simulation of the [CoZn(AAP)] EPR spectrum, a third signal appearing upon BuBA binding was isolated. This signal was simulated with g(eff) values of 2.08, 3.15, and 6.15 which correspond to an M-s = \+/-1/2] ground state transition with a g(real) of 2.41 and an E/D of 0.22. This simulation also invoked an eight-line unresolved Co-59 hyperfine pattern with an A(z)(Co-59) value of 4.0 mT. Summation of the these three species provided an excellent simulation of the observed [CoZn(AAP)] + BuBA EPR spectrum at both pH values. This work establishes that substrate binds only to the first metal binding site in AAP and thus substantiates the first step in catalysis in the recently proposed mechanism of action for AAP.