Escherichia coli methionine aminopeptidase:: Implications of crystallographic analyses of the native, mutant, and inhibited enzymes for the mechanism of catalysis
Escherichia coli methionine aminopeptidase:: Implications of crystallographic analyses of the native, mutant, and inhibited enzymes for the mechanism of catalysis
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DOI：
10.1021/bi990684r
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发表时间：
1999-06-15
期刊：
影响因子：
2.9
通讯作者：
Matthews, BW
中科院分区：
文献类型：
--
作者：
Lowther, WT;Orville, AM;Matthews, BW
通过改进大肠杆菌蛋氨酸氨基肽酶（eMetAP）的表达和纯化，并采用略有不同的结晶条件，将亲本结构的分辨率从2.4埃提高到1.9埃。这使得活性位双核金属中心的配位几何和溶剂结构的可视化成为可能。一个溶剂分子（很可能是氢氧化物）连接着三角双锥体（Co1）和八面体（Co2 -钴离子）。第二种溶剂（可能是氢氧化物离子）与Co2末端结合，在距离金属中心约13埃的地方，在蛋白质两个亚结构域之间的界面处，还发现了一个单价阳离子结合位点。类底物抑制剂的第一个结构(3R)-氨基-(2S)-羟基heptanyl -l - ala -l与甲硫氨酸氨基肽酶结合，也已被确定。该抑制剂通过以下四种相互作用来协调金属中心：(i) n端(3R)-氮与Co2的连接，（ii, iii） (2S)-羟基的桥接配合，（iv）假肽链的酮氧与Col的末端连接。除了四齿、双螯合的金属配位外，底物类似物还与所有MetAPs活性位点内的两个保守残基His79和His178形成氢键。为了评估它们在催化中的重要性，将His79和His178替换为丙氨酸，这两种取代，尤其是His79的取代，都降低了活性。His79Ala脱酶的结构及其电子吸收光谱与其他变体的比较表明，活性的丧失不是由于构象变化或金属中心缺陷所致。提出了两种不同的反应机理，并与相关酶的反应机理进行了比较。这些结果还表明，类似于本文报道的抑制剂可能在预防癌症血管生成和治疗微生物和真菌感染方面有用。
By improving the expression and purification of Escherichia coli methionine aminopeptidase (eMetAP) and using slightly different crystallization conditions, the resolution of the parent structure was extended from 2.4 to 1.9 Angstrom resolution. This has permitted visualization of the coordination geometry and solvent structure of the active-site dinuclear metal center. One solvent molecule (likely a mu-hydroxide) bridges the trigonal bipyramidal (Co1) and octahedral (Co2 cobalt ions. A second solvent (possibly a hydroxide ion) is bound terminally to Co2, A monovalent cation binding site was also identified about 13 Angstrom away from the metal center at an interface between the two subdomains of the protein. The first structure of a substrate-like inhibitor, (3R)-amino-(2S)-hydroxyheptanoyl-L-Ala-L bound to a methionine aminopeptidase, has also been determined, This inhibitor coordinates the metal center through four interactions as follows: (i) ligation of the N-terminal (3R)-nitrogen to Co2, (ii, iii) bridging coordination of the (2S)-hydroxyl group, and (iv) terminal ligation to Col by the keto oxygen of the pseudo-peptide linkage. Inhibitor binding occurs with the displacement of two solvent ligands and the expansion of the coordination sphere of Col, In addition to the tetradentate, bis-chelate metal coordination, the substrate analogue forms hydrogen bonds with His79 and His178, two conserved residues within the active site of all MetAPs, To evaluate their importance in catalysis His79 and His178 were replaced with alanine, Both substitutions, but especially that of His79, reduce activity. The structure of the His79Ala apoenzyme and the comparison of its electronic absorption spectra with other variants suggest that the loss in activity is not due to a conformational change or a defective metal center. Two different reaction mechanisms are proposed and are compared to those of related enzymes. These results also suggest that inhibitors analogous to that reported here may be useful in preventing angiogenesis in cancer and in the treatment of microbial and fungal infections.