REFINED ATOMIC MODEL OF WHEAT SERINE CARBOXYPEPTIDASE-II AT 2.2-ANGSTROM RESOLUTION
REFINED ATOMIC MODEL OF WHEAT SERINE CARBOXYPEPTIDASE-II AT 2.2-ANGSTROM RESOLUTION
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DOI：
10.1021/bi00155a037
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发表时间：
1992-10-13
期刊：
影响因子：
2.9
通讯作者：
REMINGTON, SJ
中科院分区：
文献类型：
--
作者：
LIAO, DI;BREDDAM, K;REMINGTON, SJ
来自小麦的同型二聚体丝氨酸羧肽酶II（CPDW-II，M（r）120 K）的晶体结构已被确定，并使用布鲁克海文国家实验室收集的同步加速器数据在2.2埃分辨率下完全精制到16.9%的标准晶体学R因子。该模型与理想键长的均方根偏差为0.018埃，与键角的均方根偏差为2.8度。该模型支持在3.5埃分辨率的早期研究的一般结论，并将形成调查底物结合和机理研究的基础。该酶具有α + β折叠，由中央11链β折叠组成，两侧共有15个螺旋。与其他丝氨酸蛋白酶一样，该酶含有一个“催化三联体”Ser 146-His 397-Asp 338和一个推测的“氧阴离子空穴”，该空穴由Tyr 147和Gly 53的骨架酰胺组成。Asp 338的羧酸和His 397的咪唑与其他丝氨酸蛋白酶不同，它们不共面。三个丝氨酸蛋白酶家族的活性位点特征的比较表明，“催化三联体”实际上应被视为两个二联体，一个组氨酸-天冬氨酸二联体和一个组氨酸-丝氨酸二联体，并且一个二联体相对于另一个二联体的相对取向不是特别重要。四个活性位点残基（52、53、65和146）具有不利的骨架构象，但具有明确的电子密度，表明活性位点区域存在一些应变。游离氨基酸精氨酸的结合已经通过差分傅立叶方法分析，定位离去基团的C-末端羧酸的结合位点。羧酸与Glu 145、Asn 51和Gly 52的酰胺形成氢键。Glu 145的羧酸盐也与Glu 65的羧酸盐形成氢键，这表明其中一个或两个可能被质子化。因此，在pH > 7时肽酶活性的丧失可能部分是由于Glu 145的去质子化。活性位点没有显示出暴露的肽酰胺和羰基氧原子，其可以以扩展的β-折叠方式与底物相互作用。多肽骨架的折叠与胰蛋白酶或枯草杆菌蛋白酶的折叠完全不同，这表明这是趋同分子进化到共同酶活性的第三个例子。此外，活性位点序列基序“G-X-S-X-G/A”，通常被认为是丝氨酸肽酶或酯酶活性的标志，是偶然的，而不是发散进化的结果。然而，折叠的CPDW-II有一个显着的相似性，其他四类水解酶的代表性结构最近已被确定。它们是乙酰胆碱酯酶、几种三酰甘油脂肪酶、卤代烷脱卤酶和二烯内酯水解酶。因此，丝氨酸羧肽酶的建议，以说明收敛和发散分子进化的过程。人丝氨酸羧肽酶carbL或“保护蛋白”和CPDW-II的氨基酸序列的比较表明，CPDW-II的二聚体可能是人酶的二聚体的合理模型，并且二聚体界面在两种蛋白质中是相似的。这可能有助于理解人类遗传性疾病，如半乳糖唾液酸沉积症，其可由该基因座的病变引起[Zhou，X.是的，Galjart，N. J.，威廉森河，Gillemans，N.，Galjaard，H.，& d'Azzo，A. 10，4041-4048]。
The crystal structure of the homodimeric serine carboxypeptidase II from wheat (CPDW-II, M(r) 120K) has been determined and fully refined at 2.2-angstrom resolution to a standard crystallographic R factor of 16.9% using synchrotron data collected at the Brookhaven National Laboratory. The model has an rms deviation from ideal bond lengths of 0.018 angstrom and from bond angles of 2.8-degrees. The model supports the general conclusions of an earlier study at 3.5-angstrom resolution and will form the basis for investigation into substrate binding and mechanistic studies. The enzyme has an alpha+beta fold, consisting of a central 11-stranded beta-sheet with a total of 15 helices on either side. The enzyme, like other serine proteinases, contains a "catalytic triad' Ser146-His397-Asp338 and a presumed "oxyanion hole" consisting of the backbone amides of Tyr147 and Gly53. The carboxylate of Asp338 and imidazole of His397 are not coplanar in contrast to the other serine proteinases. A comparison of the active site features of the three families of serine proteinases suggests that the "catalytic triad" should actually be regarded as two diads, a His-Asp diad and a His-Ser diad, and that the relative orientation of one diad with respect to the other is not particularly important. Four active site residues (52, 53, 65, and 146) have unfavorable backbone conformations but have well-defined electron density, suggesting that there is some strain in the active site region. The binding of the free amino acid arginine has been analyzed by difference Fourier methods, locating the binding site for the C-terminal carboxylate of the leaving group. The carboxylate makes hydrogen bonds to Glu145, Asn51, and the amide of Gly52. The carboxylate of Glu145 also makes a hydrogen bond with that of Glu65, suggesting that one or both may be protonated. Thus, the loss of peptidase activity at pH > 7 may in part be due to deprotonation of Glu145. The active site does not reveal exposed peptide amides and carbonyl oxygen atoms that could interact with substrate in an extended beta-sheet fashion. The fold of the polypeptide backbone is completely different than that of trypsin or subtilisin, suggesting that this is a third example of convergent molecular evolution to a common enzymatic activity, Furthermore, it is suggested that the active site sequence motif "G-X-S-X-G/A", often considered the hallmark of serine peptidase or esterase activity, is fortuitous and not the result of divergent evolution. However, the fold of CPDW-II has a remarkable similarity to four other classes of hydrolytic enzymes for which representative structures have recently been determined. These are the acetylcholine esterase, several triacylglycerol lipases, haloalkane dehalogenase, and dienelactone hydrolase. Thus, serine carboxypeptidases are suggested to illustrate the process of both convergent and divergent molecular evolution. A comparison of the amino acid sequences of human serine carboxypeptidase carbL, or "protective protein", and CPDW-II suggests that the dimer of CPDW-II may be a reasonable model for the dimer of the human enzyme and that the dimer interface is similar in the two proteins. This may be useful toward the understanding of human genetic disorders such as galactosialidosis, which can result from lesions in this locus [Zhou, X. Y., Galjart, N. J., Willemsen, R., Gillemans, N., Galjaard, H., & d'Azzo, A. (1991) EMBO J. 10, 4041-4048].