13C NMR study of the ionizations within a trypsin-chloromethyl ketone inhibitor complex.
13C NMR study of the ionizations within a trypsin-chloromethyl ketone inhibitor complex.
复制标题
胰蛋白酶-氯甲基酮抑制剂复合物内电离的 13C NMR 研究。
DOI：
10.1021/bi00335a014
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Scott,AI
中科院分区：
文献类型：
--
作者：
Malthouse,JP;Primrose,WU;Mackenzie,NE;Scott,AI
13C NMR 用于检测胰蛋白酶-氯甲基酮抑制剂复合物内的电离。将观察到的 pK& 值与自由能关系预测的值进行比较。对于变性/自溶抑制剂复合物，观察到 pKa = 5.26，这归因于组氨酸 57 的咪唑的电离。对于完整的抑制剂复合物，确定 pKa= 7.88。该 pKa 归因于半缩酮羟基的电离 (pKa=7.88-8.1)，并提供了丝氨酸蛋白酶能够稳定四面体加合物的氧阴离子的第一个直接证据。间接证据表明组氨酸57的咪唑pK>8.1。谱线展宽研究表明，可能存在超快的交换谱线展宽，这可能是由于抑制剂复合物内中性和两性离子物质之间的快速互变异构交换所致。讨论了这些结果对于丝氨酸蛋白酶催化机制的意义。
13C NMR is used to detect ionizations within a trypsin-chloromethyl ketone inhibitor complex. The pK& values observed are compared with those predicted by free-energy relationships. For the dena-tured/autolyzed inhibitor complex, a pKa= 5.26 is observed, which is assigned to the ionization of the imidazole of histidine-57. For the intactinhibitor complex a pKa= 7.88 is determined. This pKa is assigned to the ionization of the hemiketal hydroxyl (pKa=7.88-8.1) and provides the first direct evidence that the serine proteases are able to stabilize the oxyanion of tetrahedral adducts. Indirect evidence is adduced that the imidazole pK) of histidine-57 is> 8.1. Line-broadening studies suggest that there may be extra fast exchange line broadening, which could result from rapid tautomeric exchange between neutral and zwitterionic species within the inhibitor complex. The significance of these results for the catalytic mechanism of serine proteases is discussed.