INVESTIGATION OF DIFFUSION-LIMITED RATES OF CHYMOTRYPSIN REACTIONS BY VISCOSITY VARIATION
INVESTIGATION OF DIFFUSION-LIMITED RATES OF CHYMOTRYPSIN REACTIONS BY VISCOSITY VARIATION
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DOI：
10.1021/bi00535a030
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发表时间：
1982-01-01
期刊：
影响因子：
2.9
通讯作者：
KIRSCH, JF
中科院分区：
文献类型：
--
作者：
BROUWER, AC;KIRSCH, JF
胰凝乳蛋白酶的酰化速率由某些高活性底物的方法的扩散控制的限制的可能性进行了研究，通过测量的值的kcat/Km为3个基板作为一个功能的增加粘度与蔗糖和ficoll作为viscosogenic试剂。kcat/Km（pH 8.0，25 ℃）的值为0.001。C）代表酰化速率常数如下：N-（甲氧羰基）-L-色氨酸对硝基苯基酯，3.5 × 104。107 M-1 s-1; N-乙酰基-L-色氨酸甲酯，8 ×105 M-1 s-1; N-乙酰基-L-色氨酸对硝基苯胺，300 M-1 s-1。第一种化合物的速率常数随着粘度的增加而显著降低，第二种化合物的速率常数略有降低，第三种化合物对这种扰动不敏感。对硝基苯胺的结果一起观察到，高浓度的蔗糖或聚蔗糖使用产生的酯底物的kcat的变化微不足道，反对一般的非特异性扰动的酶结构的影响，这些试剂。由这些结果计算的缔合速率常数的值为9 × 10 - 6。107和1 ×107 M-1 s-1的对硝基苯基和甲基酯，分别。kcat/Km值除以缔合速率常数表明，对硝基苯基酯的酰化速率发生在λ。40%和通过在约40%下的甲酯。扩散极限的10%。Possibility涉及重新定位的nonproductively结合底物内的ES复合物或去溶剂化的酶的活性位点的一部分被认为是占较低的缔合速率常数为甲基相比，对-硝基苯酯。
The possibility that the rates of acylation of chymotrypsin by certain highly reactive substrates approach the diffusion-controlled limits was investigated by measuring the values of kcat/Km for 3 substrates as a function of increasing viscosity with sucrose and ficoll as the viscosogenic reagents. The values of kcat/Km (pH 8.0, 25.degree. C) representing the acylation rate constants are the following: N-(methoxycarbonyl)-L-tryptophan p-nitrophenyl ester, 3.5 .times. 107 M-1 s-1; N-acetyl-L-tryptophan methyl ester, 8 .times. 105 M-1 s-1; N-acetyl-L-tryptophan p-nitroanilide, 300 M-1 s-1. The rate constants decrease significantly with increasing viscosity for the first compound, decrease slightly for the second, and are insensitive to this perturbation for the third. The p-nitroanilide results taken together with the observation that the high concentrations of sucrose or ficoll used produce insignificant changes in kcat for the ester substrates argue against a general nonspecific perturbation in the enzyme structure effected by these reagents. The values of the association rate constants calculated from these results are 9 .times. 107 and 1 .times. 107 M-1 s-1 for the p-nitrophenyl and methyl esters, respectively. The values of kcat/Km divided by the association rate constants show that the rates of acylation by the p-nitrophenyl ester occur at .apprx. 40% and by the methyl ester at .apprx. 10% of the diffusion limits. Possibilities involving reorientation of a nonproductively bound substrate within the ES complex or desolvation of part of the active site of the enzyme are considered to account for the lower association rate constant for the methyl as compared to the p-nitrophenyl ester.