Liu J, Albers M W, Chen C M, Schreiber S L, Walsh C T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
Proc Natl Acad Sci U S A. 1990 Mar;87(6):2304-8. doi: 10.1073/pnas.87.6.2304.
The cDNA encoding human cyclophilin from the Jurkat T-cell lymphoma line has been cloned by the expression cassette polymerase chain reaction and sequenced, and an expression vector has been constructed under control of the tac promoter for efficient expression in Escherichia coli. Active cyclophilin is produced at up to 40% of soluble cell protein, facilitating a one-column purification to homogeneity. Wild-type cyclophilin was characterized for binding of the potent immunosuppressant agent cyclosporin A (Kd = 46 nM) by tryptophan fluorescence enhancement and for inhibition (IC50 = 19 nM) of cyclophilin's peptidyl-prolyl cis-trans isomerase (rotamase) activity. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as the substrate, recombinant human cyclophilin has a high catalytic efficiency; kcat/Km is 1.4 X 10(7) M-1.S-1 at 10 degrees C. To test the prior suggestion that a cysteine residue may be essential for catalysis and immunosuppressant binding, the four cysteines at positions 52, 62, 115, and 161 were mutated individually to alanine and the purified mutant proteins were shown to retain full affinity for cyclosporin A and equivalent catalytic efficiency as a rotamase. Clearly the cysteines play no essential role in catalysis or cyclosporin A binding. These results rule out the recently proposed mechanism [Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T. & Schmid, F. X. (1989) Nature (London) 337, 476-478)] involving the formation of tetrahedral hemithioorthoamide. Whereas mechanisms that embody other tetrahedral intermediates may be operative, an alternative mechanism is considered that involves distortion of bound substrate with a twisted (90 degrees) peptidyl-prolyl amide bond.
通过表达盒聚合酶链反应克隆并测序了来自Jurkat T细胞淋巴瘤系的编码人亲环蛋白的cDNA，并构建了在tac启动子控制下用于在大肠杆菌中高效表达的表达载体。活性亲环蛋白的产量高达可溶性细胞蛋白的40%，便于通过单柱纯化达到均一性。通过色氨酸荧光增强对野生型亲环蛋白与强效免疫抑制剂环孢菌素A的结合（解离常数Kd = 46 nM）进行了表征，并对亲环蛋白的肽基脯氨酰顺反异构酶（旋转异构酶）活性的抑制作用（半数抑制浓度IC50 = 19 nM）进行了测定。以N - 琥珀酰 - 丙氨酸 - 丙氨酸 - 脯氨酸 - 苯丙氨酸 - 对硝基苯胺为底物，重组人亲环蛋白具有较高的催化效率；在10℃时，催化常数与米氏常数的比值kcat/Km为1.4×10⁷ M⁻¹·s⁻¹。为了验证先前提出的半胱氨酸残基可能对催化和免疫抑制剂结合至关重要的观点，将第52、62、115和161位的四个半胱氨酸分别突变为丙氨酸，结果显示纯化的突变蛋白对环孢菌素A仍保留完全亲和力，并且作为旋转异构酶具有同等的催化效率。显然，半胱氨酸在催化或环孢菌素A结合中不起关键作用。这些结果排除了最近提出的涉及四面体半硫代原酰胺形成的机制[菲舍尔，G.，维特曼 - 利博尔德，B.，朗，K.，基费哈伯，T. & 施密德，F. X.（1989年）《自然》（伦敦）337, 476 - 478]。虽然体现其他四面体中间体的机制可能起作用，但考虑了一种替代机制，该机制涉及结合底物的扭曲，其中肽基脯氨酰酰胺键呈扭曲（90度）状态。
