Zydowsky L D, Etzkorn F A, Chang H Y, Ferguson S B, Stolz L A, Ho S I, Walsh C T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Protein Sci. 1992 Sep;1(9):1092-9. doi: 10.1002/pro.5560010903.
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, 2306-2310), 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 (kcat/Km) 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.
基于最近的X射线结构信息，构建了人亲环素A（hCyPA）的六个定点突变体，这些突变体涉及假定活性位点中的残基——H54、R55、F60、Q111、F113和H126，在大肠杆菌中进行了过表达和纯化，直至达到均一性。对蛋白质W121A（Liu, J., Chen, C.-M., & Walsh, C.T., 1991a, Biochemistry 30, 2306 - 2310）、H54Q、R55A、F60A、Q111A、F113A和H126Q进行了顺反肽基脯氨酰异构酶（PPIase）活性、结合免疫抑制药物环孢素A（CsA）的能力以及在CsA存在下对蛋白磷酸酶2B（钙调神经磷酸酶）抑制作用的检测。结果表明，H54Q、Q111A、F113A和W121A保留了野生型重组hCyPA 3% - 15%的催化效率（kcat/Km）。其余三个突变体（R55A、F60A和H126Q）各自保留的野生型催化效率均低于1%，表明这些残基参与了PPIase催化过程。每个突变体都与CsA亲和基质结合。突变体R55A、F60A、F113A和H126Q在CsA存在下抑制了钙调神经磷酸酶，而W121A则没有。尽管CsA是PPIase活性的竞争性抑制剂，但它可以与无酶活性的亲环素形成复合物，并抑制钙调神经磷酸酶的磷酸酶活性。
