DETERMINATION OF KINETIC CONSTANTS FOR PEPTIDYL PROLYL CIS TRANS ISOMERASES BY AN IMPROVED SPECTROPHOTOMETRIC ASSAY
DETERMINATION OF KINETIC CONSTANTS FOR PEPTIDYL PROLYL CIS TRANS ISOMERASES BY AN IMPROVED SPECTROPHOTOMETRIC ASSAY
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DOI：
10.1021/bi00239a007
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发表时间：
1991-06-25
期刊：
影响因子：
2.9
通讯作者：
RICH, DH
中科院分区：
文献类型：
--
作者：
KOFRON, JL;KUZMIC, P;RICH, DH
先前已经检查了两种充分表征的肽基脯氨酰顺反异构酶（PPI酶）亲环蛋白和FK-506结合蛋白（FKBP）的动力学性质和底物特异性[Fischer，G.，邦，H.，Berger，E.，& Schellenberger，A.（1984）Biochim. Biophys. Acta 791，87-97; Harrison，R. K.，和斯泰因河L.（1990）生物化学29，1684 -1689;阿尔伯斯，M. W.，沃尔什，C. T.，& Schreiber，S. L.（1990）J. Org. Chem. 55，4984-4986]。在这些研究中采用的胰凝乳蛋白酶偶联酶法有两个严重的缺点。由于X-顺式-Pro-Phe-pNA异构体（PPI酶底物）的平衡群体较低，以及胰凝乳蛋白酶水解产生的对硝基苯胺的溶解度较低，因此在实验中无法达到饱和区域的底物浓度。其次，未催化的顺反异构化模糊了对初速度的解释。由于这些限制，稳态动力学参数（K（m），k（cat））尚未确定。在这里，我们介绍了一个改进的版本的分光光度测定和报告的第一次米氏常数和营业额为两个PPIases与既定的基板。在实验条件的改进起源于介质诱导的增加的平衡人口的顺式X-Pro构象，并在进行测定在0 ℃，以抑制未催化的热异构化。此外，我们提出了一个严格的数学模型的分光光度计的进展曲线，占剩余背景率的贡献。对于具有牛亲环蛋白的Suc-Ala-Ala-cis-Pro-Phe-pNA，K（m）= 0.98 +/- 0.14 mM和k（cat）= 13200 +/- 880 s-1;对于重组人亲环蛋白，K（m）= 0.87 +/- 0.084 mM和k（cat）= 12700 +/- 550 s-1。Suc-Ala-Leu-cis-Pro-Phe-pNA与FKBP的动力学参数为K（m）= 0.520 4- 0.08 mM和k（cat）= 344 +/- 26 s-1。我们还表明，[（Boc）Dab]8-CsA，环孢菌素A类似物，是一个紧密结合，缓慢结合的亲环素抑制剂，另一个环孢菌素A类似物，[Me 5 Bth]1-CsA，是相同的酶的竞争性抑制剂。
The kinetic properties and substrate specificity of two well-characterized peptidyl prolyl cis-trans isomerases (PPIases), cyclophilin and the FK-506 binding protein (FKBP), have been previously examined [Fischer, G., Bang, H., Berger, E., & Schellenberger, A. (1984) Biochim. Biophys. Acta 791, 87-97; Harrison, R. K., & Stein, R. L. (1990) Biochemistry 29,1684-1689; Albers, M. W., Walsh, C. T., & Schreiber, S. L. (1990) J. Org. Chem. 55, 4984-4986]. The chymotrypsin-coupled enzymatic assay employed in these studies suffers from two serious shortcomings. Due to the low equilibrium population of the X-cis-Pro-Phe-pNA isomer (the PPIase substrate), in conjunction with the low solubility of p-nitroaniline generated by chymotrypsin hydrolysis, substrate concentrations in the saturating region are not experimentally attainable. Secondly, the uncatalyzed cis-trans isomerization obscures the interpretation of the initial velocity. As a result of these limitations, the steady-state kinetic parameters (K(m), k(cat)) have not been determined. Here we introduce an improved version of the spectrophotometric assay and report for the first time the Michaelis constants and turnover numbers for both PPIases with established substrates. The improvements in the experimental conditions originate in a medium-induced increase in the equilibrium population of the cis X-Pro conformer and in conducting the assay at 0-degrees-C to suppress the uncatalyzed thermal isomerization. In addition, we present a rigorous mathematical model of the spectrophotometric progress curves that accounts for the contributions of the residual background rate. For Suc-Ala-Ala-cis-Pro-Phe-pNA with bovine cyclophilin, K(m) = 0.98 +/- 0.14 mM and k(cat) = 13200 +/- 880 s-1; for recombinant human cyclophilin, K(m) = 0.87 +/- 0.084 mM and k(cat) = 12700 +/- 550 s-1. The kinetic parameters for Suc-Ala-Leu-cis-Pro-Phe-pNA with FKBP are K(m) = 0.520 4- 0.08 mM and k(cat) = 344 +/- 26 s-1. We also demonstrate that [(Boc)Dab]8-CsA, a cyclosporin A analogue, is a tight-binding, slow-binding inhibitor of cyclophilin and that another cyclosporin A analogue, [Me5Bth]1-CsA, is a competitive inhibitor of the same enzyme.