Mock W L, Liu Y
Department of Chemistry, University of Illinois, Chicago 60607-7061, USA.
J Biol Chem. 1995 Aug 4;270(31):18437-46. doi: 10.1074/jbc.270.31.18437.
The velocity of enzymic cleavage of 4-substituted picolinylprolines by swine kidney prolidase approaches that of physiological dipeptides, but depends substantially upon the nature of the pyridine-ring substituent. The pH dependence of kcat/Km for picolinylproline is sigmoidal, with optimum activity on the acidic limb and a delimiting enzymic pKa of 6.6, unlike glycylproline (bell-shaped pH profile, maximum at pH 7.7). Productive chelation to an active site metal ion by the N terminus of substrates is indicated, with a water molecule ligated to that hyper(Lewis)acidic center prior to substrate binding supplying the pKa of 6.6. The rate-governing catalytic step differs according to the 4-substituent on the picolinyl residue; productive binding is slow in the case of electron-withdrawing groups, but subsequent nucleophilic addition to the metal ion-activated scissile linkage becomes controlling with more basic pyridine rings. Rate constants yield a Brønsted-type correlation with substrate pKa, providing a gauge of active-site Lewis acidity. A mechanism is suggested involving the cooperative participation of two especially acidic metal ions positioned adjacently within the active site (situated as in an homologous and structurally characterized aminopeptidase), with both serving to stabilize a bridging carboxamide-hydrate intermediate.
猪肾脯氨酰二肽酶对4-取代吡啶甲酰基脯氨酸的酶促裂解速度接近生理二肽的裂解速度，但很大程度上取决于吡啶环取代基的性质。吡啶甲酰基脯氨酸的kcat/Km对pH的依赖性呈S形，在酸性区间具有最佳活性，酶的pKa为6.6，这与甘氨酰脯氨酸不同（pH曲线呈钟形，在pH 7.7时达到最大值）。表明底物的N端与活性位点金属离子发生有效螯合，在底物结合之前，与该超（路易斯）酸性中心相连的水分子提供了6.6的pKa。根据吡啶甲酰基残基上的4-取代基，限速催化步骤有所不同；对于吸电子基团，有效结合较慢，但随后对金属离子活化的可裂解键的亲核加成在吡啶环碱性更强时起控制作用。速率常数与底物pKa呈现布朗斯特型相关性，为活性位点的路易斯酸性提供了一个衡量标准。提出了一种机制，涉及活性位点内相邻定位的两个特别酸性的金属离子的协同参与（其位置与同源且结构已表征的氨肽酶中的情况相同），两者都用于稳定桥连的羧酰胺-水合物中间体。
