Two inhibitor molecules bound in the active site of Pseudomonas sedolisin: a model for the bi-product complex following cleavage of a peptide substrate.
Two inhibitor molecules bound in the active site of Pseudomonas sedolisin: a model for the bi-product complex following cleavage of a peptide substrate.
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两个抑制剂分子结合在假单胞菌 sedolisin 的活性位点：肽底物裂解后的副产物复合物模型。
DOI：
10.1016/j.bbrc.2003.12.130
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发表时间：
2004
影响因子：
3.1
通讯作者：
Dunn,BenM
中科院分区：
文献类型：
--
作者：
Wlodawer,Alexander;Li,Mi;Gustchina,Alla;Oyama,Hiroshi;Oda,Kohei;Beyer,BretB;Clemente,Jose;Dunn,BenM
丝氨酸羧基蛋白酶sedolisin与假碘酪氨酸抑制素复合物的高分辨率晶体学分析显示，该抑制剂的两个分子结合在酶的活性位点，标记S3至S3′的亚位点。结合模式代表蛋白水解反应的两种产物。利用肽库研究了sedolisin的底物特异性，并基于酶促和晶体学研究的结果合成了sedolisin的新肽底物MCA-Lys-Pro-Pro-Leu-Glu#Tyr-Arg-Leu-Gly-Lys（DNP）-Gly，并显示其被酶有效切割。底物的动力学参数，通过猝灭解除后荧光的增加来测量，为：kcat=73±5s−1，Km=0.12±0.011μM，kcat/Km=608± 85 s −1μM−1。
High-resolution crystallographic analysis of a complex of the serine-carboxyl proteinase sedolisin with pseudo-iodotyrostatin revealed two molecules of this inhibitor bound in the active site of the enzyme, marking subsites from S3 to S3′. The mode of binding represents two products of the proteolytic reaction. Substrate specificity of sedolisin was investigated using peptide libraries and a new peptide substrate for sedolisin, MCA–Lys–Pro–Pro–Leu–Glu#Tyr–Arg–Leu–Gly–Lys(DNP)–Gly, was synthesized based on the results of the enzymatic and crystallographic studies and was shown to be efficiently cleaved by the enzyme. The kinetic parameters for the substrate, measured by the increase in fluorescence upon relief of quenching, were: kcat=73±5s−1, Km=0.12±0.011μM, and kcat/Km=608±85s−1μM−1.