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/bi00108a031
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发表时间：
1991-06
期刊：
影响因子：
2.9
通讯作者：
J. Kofron;P. Kuzmič;V. Kishore;Esther Colon-Bonilla;D. Rich
中科院分区：
文献类型：
--
作者：
J. Kofron;P. Kuzmič;V. Kishore;Esther Colon-Bonilla;D. Rich
两种已被充分表征的肽基丙氨酸顺式反式异构酶（PPIases），亲环蛋白和FK-506结合蛋白（FKBP）的动力学性质和底物特异性先前已被研究过[Fischer， G., Bang， H., Berger， E., & Schellenberger， A. (1984) biochem]。Biophys。journal of chengdu electromechanical college；Harrison, r.k., & Stein， R.L.（1990）生物化学29,1684-1689；陈志强、陈志强、陈志强（1990）。化学学报，1995,19(4):444 - 444。在这些研究中使用的凝乳胰蛋白酶偶联酶分析有两个严重的缺点。由于x -顺- pro - ph - pna异构体（PPIase底物）的平衡种群低，再加上胰凝乳酶水解产生的对硝基苯胺的溶解度低，在饱和区域的底物浓度在实验上是无法达到的。其次，未催化的顺反异构化模糊了对初始速度的解释。由于这些限制，稳态动力学参数（Km,Kcat）尚未确定。在这里，我们介绍了一种改进版的分光光度测定法，并首次报道了两种PPIases在确定底物上的米切里斯常数和周转率。实验条件的改善源于介质诱导的顺式X-Pro构象平衡种群的增加，以及在0℃下进行分析以抑制非催化热异构化。此外，我们提出了一个严格的数学模型的分光光度法进展曲线，说明贡献的剩余背景率。（摘要删节250字）
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 (Km,Kcat) 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.(ABSTRACT TRUNCATED AT 250 WORDS)