KINETIC-ANALYSIS OF CYCLOPHILIN-CATALYZED PROLYL CIS/TRANS ISOMERIZATION BY DYNAMIC NMR-SPECTROSCOPY
KINETIC-ANALYSIS OF CYCLOPHILIN-CATALYZED PROLYL CIS/TRANS ISOMERIZATION BY DYNAMIC NMR-SPECTROSCOPY
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DOI：
10.1021/bi00041a039
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发表时间：
1995-10-17
期刊：
影响因子：
2.9
通讯作者：
DRAKENBERG, T
中科院分区：
文献类型：
--
作者：
KERN, D;KERN, G;DRAKENBERG, T
采用一维动态H-1 NMR技术研究了肽基脯氨酰顺反异构酶（PPIases）催化N-琥珀酰-Ala-Phe-Pro-Phe-（4-）硝基苯胺脯氨酰肽键顺反异构化反应的动力学.为此，在细胞溶质猪肾亲环蛋白（Cyp 18）和肽底物的各种浓度下进行质子信号的线形分析。Cyp 18的顺式/反式异构化的催化作用最好由四个位点交换模型描述，其中四个位点代表溶液中游离的顺式和反式异构体并与酶结合。结合动态NMR光谱与经典的蛋白酶偶联PPIase测定允许确定一套完整的微观速率常数描述的四个网站交换模型。比较溶液中游离肽与亲环蛋白结合肽的顺式-->反式异构化速率常数，得出加速因子为3.5 x 10（5）。米氏络合物的解离与酶的异构化速率具有相同的数量级。因此，所有微观速率常数有助于稳态参数。首次在pH6.0和10 ℃的可逆条件下测定了反式异构体的k（cat）（620 s（-1））和K-M（220 μ M）值以及顺式异构体的值（k（cat）= 680 s（-1），K-M = 80 μ M）。Cyp 18对顺式异构体的亲和力比对反式异构体的亲和力高4倍。这导致顺式/反式平衡向顺式异构体移动。Phe（5）底物酰胺质子顺式信号的1.8ppm低场化学位移可以通过顺式肽单元与酶的氢键结合来解释。这可能是顺式异构体优先结合的原因。最后，结果表明，在催化过程中的残基N-末端脯氨酸保持固定Cyp 18，而C-末端部分旋转。
To investigate the kinetics of the prolyl peptide bond cis/trans isomerization of N-succinyl-Ala-Phe-Pro-Phe-(4-)nitroanilide catalyzed by peptidyl prolyl cis/trans isomerases (PPIases), one-dimensional dynamic H-1 NMR spectroscopy was employed. To this end line shape analyses of proton signals were performed at various concentrations of both cytosolic porcine kidney cyclophilin (Cyp18) and peptide substrate. Catalysis of the cis/trans isomerization by Cyp18 is best described by a four-site exchange model, where the four sites represent the cis and trans isomers free in solution and bound to the enzyme. Combination of dynamic NMR spectroscopy with the classical protease-coupled PPIase assay allowed determination of the complete set of the microscopic rate constants describing the four site exchange model. The comparison of the rate constants of cis-->trans isomerization of the peptide free in solution and bound to cyclophilin yields an acceleration factor of 3.5 x 10(5). Dissociation of the Michaelis complexes are of the same order of magnitude as the isomerization rates on the enzyme. Therefore, all microscopic rate constants contribute to the steady state parameters. For the first time, the k(cat) (620 s(-1)) and K-M (220 mu M) value for the trans isomer in addition to the values of the cis isomer (k(cat) = 680 s(-1), K-M = 80 mu M) could be determined under reversible conditions at pH 6.0 and 10 degrees C. The affinity of Cyp18 for the cis isomer is 4 times higher than for the trans isomer. This results in a shift of the cis/trans equilibrium toward the cis isomer. The 1.8 ppm downfield chemical shift of the cis signal of the substrate amide proton of Phe(5) could be explained by hydrogen bonding of the cis peptide unit to the enzyme. This might be the reason for the preferred binding of the cis isomer. Finally, the results suggest that during catalysis the residues N-terminal to proline remain fixed to Cyp18 while the C-terminal part is rotated.