X-RAY CRYSTALLOGRAPHIC INVESTIGATION OF SUBSTRATE BINDING TO CARBOXYPEPTIDASE-A AT SUBZERO TEMPERATURE
X-RAY CRYSTALLOGRAPHIC INVESTIGATION OF SUBSTRATE BINDING TO CARBOXYPEPTIDASE-A AT SUBZERO TEMPERATURE
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DOI：
10.1073/pnas.83.20.7568
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发表时间：
1986-10-01
影响因子：
11.1
通讯作者：
LIPSCOMB, WN
中科院分区：
文献类型：
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作者：
CHRISTIANSON, DW;LIPSCOMB, WN
对羧基肽酶A(CPA；肽基-L-氨基酸水解酶，EC 3.4.17.1)与缓慢降解的底物甘氨酰-L-酪氨酸在-9℃下形成的络合物进行了高分辨X射线结晶学研究。虽然这种酶-底物复合体是早期结晶学研究的对象，但人们希望得到底物占有率更高的复合体的更高分辨率的电子密度图。所有的晶体化学(即晶体浸泡和X射线数据收集)都是在安装在衍射仪上的流动池上进行的，晶体被固定在其中。1.6ANG.分辨率的X射线数据得到了一个很好的分辨结构，其中活性中心的锌离子是五配位的：三个酶残基(谷氨酸-72、组氨酸-69和组氨酸-196)和甘氨酸-L-酪氨酸的羰基氧和氨基末端完成了金属的配位多面体。这些结果证实，该底物可能以非生产性的方式结合，因为具有重要水解性的锌结合水被置换并排除在活性中心之外。很可能，所有携带无保护氨基末端的羧基肽酶A的二肽底物都是较差的底物，因为它与活性部位的金属离子具有良好的双齿配位作用。
A high-resolution x-ray crystallographic investigation of the complex between carboxyeptidase A (CPA; peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) and the slowly hydrolyzed substrate glycyl-L-tyrosine was done at -9.degree. C. Although this enzyme-substrate complex has been the subject of earlier crystallographic investigation, a higher resolution electron-density map of the complex with greater occupancy of the substrate was desired. All crystal chemistry (i.e., crystal soaking and x-ray data collection) was performed on a diffractometer-mounted flow cell, in which the crystal was immobilized. The x-ray data to 1.6-.ANG. resolution have yielded a well-resolved structure in which the zinc ion of the active site is five-coordinate: three enzyme residues (glutamate-72, histidine-69, and histidine-196) and the carbonyl oxygen and amino terminus of glycyl-L-tyrosine complete the coordination polyhedron of the metal. These results confirm that this substrate may be bound in a nonproductive manner, because the hydrolytically important zinc-bound water has been displaced and excluded from the active site. It is likely that all dipeptide substrates of carboxypeptidase A that carry an unprotected amino terminus are poor substrates because of such favorable bidentate coordination to the metal ion of the active site.