Interaction Mode of H397A Mutant Carboxypeptidase Y with Protein Substrates Analyzed by the Surface Plasmon Resonance
Interaction Mode of H397A Mutant Carboxypeptidase Y with Protein Substrates Analyzed by the Surface Plasmon Resonance
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通过表面等离子共振分析 H397A 突变体羧肽酶 Y 与蛋白质底物的相互作用模式
DOI：
10.1246/bcsj.73.2587
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发表时间：
2000
影响因子：
4
通讯作者：
Y. Harada
中科院分区：
文献类型：
--
作者：
H. Nakase;G. Jung;H. Ueno;R. Hayashi;Y. Harada
为了研究羧肽酶Y（CPY）的底物结合模式和亚位点性质，用表面等离子体共振法测定了其失活突变体H397 A的解离常数和结合常数，α-酪蛋白、RCM-RNase A、RCM-溶菌酶和RCM-BSA。四种底物获得的KD值在10-8至10-7 M的范围内，等于相同蛋白底物的催化活性野生型CPY的Km值。这些结果表明，酰化步骤是在蛋白质底物的CPY催化水解的限速步骤。N-酰化二肽等小底物的Km值在10-4至10-2 M范围内，远小于蛋白质底物的Km值，表明CPY的底物结合口袋中除了S1′和S1外还存在其他亚位点。
In order to study the substrate binding mode and subsite properties of carboxypeptidase Y (CPY), the dissociation and association constants, KD and KA, of its catalytically inactive mutant, H397A, were determined by surface plasmon resonance with protein substrates, i.e., α-casein, RCM-RNase A, RCM-lysozyme, and RCM-BSA. KD values obtained for four substrates were in a range of 10-8 to 10-7 M, being equal to Km values of catalytically active wild-type CPY for the same protein substrates. These results suggest that the acylation step is a rate-limiting step in the CPY-catalyzed hydrolysis of protein substrates. Small substrates like N-acylated dipeptides gave Km values in a range of 10-4 to 10-2 M, considerably smaller than those for protein substrates, suggesting the presence of additional subsites in addition to S1′ and S1 in the substrate binding pocket of CPY.