ACTIVE-SITE MUTANTS OF HUMAN CYCLOPHILIN-A SEPARATE PEPTIDYL-PROLYL ISOMERASE ACTIVITY FROM CYCLOSPORINE-A BINDING AND CALCINEURIN INHIBITION
ACTIVE-SITE MUTANTS OF HUMAN CYCLOPHILIN-A SEPARATE PEPTIDYL-PROLYL ISOMERASE ACTIVITY FROM CYCLOSPORINE-A BINDING AND CALCINEURIN INHIBITION
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DOI：
10.1002/pro.5560010903
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发表时间：
1992-09-01
期刊：
影响因子：
8
通讯作者：
WALSH, CT
中科院分区：
文献类型：
--
作者：
ZYDOWSKY, LD;ETZKORN, FA;WALSH, CT
根据最近的 X 射线结构信息，已构建、过表达并从大肠杆菌中纯化出六种人亲环蛋白 A (hCyPA) 定点突变体，这些突变体涉及推定活性位点 H54、R55、F60、Q111、F113 和 H126 中的残基。测定蛋白质 W121A (Liu, J., Chen, C.-M., & Walsh, C.T., 1991a, Biochemistry 30, 2306-23 10)、H54Q、R55A、F60A、Q111A、F113A 和 H126Q 的顺反肽基脯氨酰异构酶 (PPIase) 活性，结合免疫抑制药物环孢菌素 A (CsA) 的能力，以及在 CsA 存在的情况下抑制蛋白磷酸酶 2B（钙调神经磷酸酶）。结果表明，H54Q、Q111A、F113A 和 W121A 保留了野生型重组 hCyPA 的 3-15% 的催化效率 (k(cat)/K(m)）。其余三个突变体（R55A、F60A 和 H126Q）各自保留了不到 1% 的野生型催化效率，表明这些残基参与了 PPIase 催化。每个突变体均与 CsA 亲和基质结合。在 CsA 存在的情况下，突变体 R55A、F60A、F113A 和 H126Q 抑制神经钙蛋白，而 W121A 则不然。尽管 CsA 是 PPIase 活性的竞争性抑制剂，但它可以与酶无活性的亲环蛋白复合并抑制钙调神经磷酸酶的磷酸酶活性。
Based on recent X-ray structural information, six site-directed mutants of human cyclophilin A (hCyPA) involving residues in the putative active site-H54, R55, F60, Q111, F113, and H126-have been constructed, overexpressed, and purified from Escherichia coli to homogeneity. The proteins W121A (Liu, J., Chen, C.-M., & Walsh, C.T., 1991a, Biochemistry 30, 2306-23 10), H54Q, R55A, F60A, Q111A, F113A, and H126Q were assayed for cis-trans peptidyl-prolyl isomerase (PPIase) activity, their ability to bind the immunosuppressive drug cyclosporin A (CsA), and protein phosphatase 2B (calcineurin) inhibition in the presence of CsA. Results indicate that H54Q, Q111A, F113A, and W121A retain 3-15% of the catalytic efficiency (k(cat)/K(m)) of wild-type recombinant hCyPA. The remaining three mutants (R55A, F60A, and H126Q) each retain less than 1% of the wild-type catalytic efficiency, indicating participation by these residues in PPIase catalysis. Each of the mutants bound to a CsA affinity matrix. The mutants R55A, F60A, F113A, and H126Q inhibited calcineurin in the presence of CsA, whereas W121A did not. Although CsA is a competitive inhibitor of PPIase activity, it can complex with enzymatically inactive cyclophilins and inhibit the phosphatase activity of calcineurin.