Zarnt T, Lang K, Burtscher H, Fischer G
Max-Planck-Gesellschaft, Arbeitsgruppe Enzymologie der Peptidbindung, Halle, Germany.
Biochem J. 1995 Jan 1;305 ( Pt 1)(Pt 1):159-64. doi: 10.1042/bj3050159.
Free in solution, the immunosuppressive compounds cyclosporin A (CsA), FK506, ascomycin and rapamycin are present in many solvents in various slowly interconverting conformations. Together with their cellular receptor proteins, cyclophilin (CyP) and FK506-binding protein (FKBP), however, these inhibitors have been shown to have a homogeneous conformation. The existence of a slow cis-trans interconversion of an imidic bond in the inhibitor molecule during the course of the formation of the CsA-CyP18cy complex (where CyP18cy is human 18 kDa cytosolic CyP) prompted us to investigate the reaction of the peptidomacrolides FK506, ascomycin and rapamycin with two specific binding-proteins in more detail. Since formation of the FK506-FKBP complex results in the inhibition of the peptidylprolyl cis-trans-isomerase activity of the binding protein, we used the enzyme's decrease in enzymic activity to monitor binding of the inhibitors to their enzyme targets. For FK506, the kinetics of inhibition of human 12 kDa cytosolic FKBP (FKBP12cy) were clearly dependent on time. Subsequent to a rapid inactivation reaction, not resolved in its kinetics due to manual mixing, a slow dominant first-order inactivation process with a relaxation time of 1163 s at 10 degrees C was observed. Concomitantly the Ki value of the slow phase dropped 2.6-fold within the first 60 min of incubation. Using the FKBP12cy homologue 25 kDa membrane FKBP (FKBP25mem), a bacterial peptidylprolyl cis-trans-isomerase, the rate and amplitudes of the inhibition reactions were very similar to FKBP12cy. On the other hand, the kinetics and amplitudes of the inhibition of FKBP12cy varied significantly if rapamycin was used as an inhibitor instead of FK 506. Owing to reduced conformation transition in rapamycin upon binding to FKBP12cy, the slow phase during inhibition was significantly decreased in amplitude. A likely reason for this became apparent when the activation-enthalpy and the pH-dependence of the rate constants of the slow phase were determined. We conclude that the cis to trans interconversion of the pipecolinyl bond of the three peptidomacrolides may be responsible for the slow process. There was no indication of a suicide catalysis of this process by FKBPs.
在溶液中自由存在时，免疫抑制化合物环孢菌素A（CsA）、FK506、子囊霉素和雷帕霉素以多种缓慢相互转化的构象存在于许多溶剂中。然而，与它们的细胞受体蛋白亲环蛋白（CyP）和FK506结合蛋白（FKBP）一起，这些抑制剂已被证明具有均一的构象。在CsA-CyP18cy复合物（其中CyP18cy是人18 kDa胞质CyP）形成过程中，抑制剂分子中一个亚胺键存在缓慢的顺反相互转化，这促使我们更详细地研究肽基大环内酯类FK506、子囊霉素和雷帕霉素与两种特异性结合蛋白的反应。由于FK506-FKBP复合物的形成导致结合蛋白的肽基脯氨酰顺反异构酶活性受到抑制，我们利用该酶活性的降低来监测抑制剂与其酶靶点的结合。对于FK506，抑制人12 kDa胞质FKBP（FKBP12cy）的动力学明显依赖于时间。在一个快速失活反应之后（由于手动混合，其动力学未得到解析），观察到一个缓慢的主导一级失活过程，在10℃时弛豫时间为1163 s。同时，慢相的Ki值在孵育的前60分钟内下降了2.6倍。使用FKBP12cy的同源物25 kDa膜FKBP（FKBP25mem），一种细菌肽基脯氨酰顺反异构酶，抑制反应的速率和幅度与FKBP12cy非常相似。另一方面，如果使用雷帕霉素代替FK506作为抑制剂，FKBP12cy抑制的动力学和幅度会有显著变化。由于雷帕霉素与FKBP12cy结合后构象转变减少，抑制过程中的慢相幅度显著降低。当测定慢相速率常数的活化焓和pH依赖性时，其可能的原因变得明显。我们得出结论，三种肽基大环内酯类的哌啶基键的顺反相互转化可能是导致这个缓慢过程的原因。没有迹象表明FKBPs对这个过程有自杀催化作用。
