Wang T T, Hofmann T
Biochem J. 1976 Mar 1;153(3):691-9. doi: 10.1042/bj1530691.
The action of pig pepsin on a variety of small peptides including Leu-Trp-Met-Arg, Leu-Trp-Met, Leu-Leu-NH2, benzyloxycarbonyl-Phe-Leu and Gly-Leu-Tyr was studied. Leu-Leu-Leu was found to be the major product from the substrates Leu-Trp-Met-Arg and Leu-Trp-Met, indicating that the predominant reaction at pH 3.4 was a transpeptidation of the acyl-transfer type. Leu-Leu-Leu was also formed in high yield by amino transfer from benzyloxycarbonyl-Phe-Leu. Like the amino-transfer reactions the acyl transfer proceeded via a covalent intermediate, since [14C]leucine was not incorporated into transpeptidation products and did not exchange with enzyme-bound leucine in the presence of acceptors. With Leu-Trp-Met both acyl and amino transpeptidation products, namely Leu-Leu, Leu-Leu-Leu, Met-Met and Met-Met-Met, were formed in addition to methionine and leucine. With Leu-Trp-Met-Arg (1 mM) the pH optimum for the rates of hydrolysis and acyl transfer is about pH 3.4. At this pH the rate of acyl transfer exceeds that of hydrolysis; at pH 2, however, hydrolysis was faster than transfer. A comparison of the effect of the length of substrates and products on the reaction rates allows the conclusion that the binding site can extend over eight to nine amino acid residues. Although the experiments provide no conclusive evidence for or against the involvement of amino and/or acyl intermediates in the hydrolysis of long peptides and proteins, the high yield of transpeptidation reactions of both types observed with some substrates suggests a major role for the intermediates in pepsin-catalysed reactions. The results also show that when pig pepsin is used for the digestion of proteins for sequence work, the likelihood of the formation of transpeptidation products is considerable. In this way peptides not present in the original sequence could easily form in a reasonably good yield.
研究了猪胃蛋白酶对多种小肽的作用，这些小肽包括亮氨酸-色氨酸-甲硫氨酸-精氨酸、亮氨酸-色氨酸-甲硫氨酸、亮氨酸-亮氨酸-氨基、苄氧羰基-苯丙氨酸-亮氨酸和甘氨酸-亮氨酸-酪氨酸。发现亮氨酸-亮氨酸-亮氨酸是底物亮氨酸-色氨酸-甲硫氨酸-精氨酸和亮氨酸-色氨酸-甲硫氨酸的主要产物，这表明在pH 3.4时主要反应是酰基转移型的转肽反应。通过苄氧羰基-苯丙氨酸-亮氨酸的氨基转移也能高产率地生成亮氨酸-亮氨酸-亮氨酸。与氨基转移反应一样，酰基转移也是通过共价中间体进行的，因为在有受体存在的情况下，[14C]亮氨酸没有掺入转肽产物中，也没有与酶结合的亮氨酸发生交换。对于亮氨酸-色氨酸-甲硫氨酸，除了甲硫氨酸和亮氨酸外，还形成了酰基和氨基转肽产物，即亮氨酸-亮氨酸、亮氨酸-亮氨酸-亮氨酸、甲硫氨酸-甲硫氨酸和甲硫氨酸-甲硫氨酸-甲硫氨酸。对于亮氨酸-色氨酸-甲硫氨酸-精氨酸（1 mM），水解速率和酰基转移的最适pH约为3.4。在此pH下，酰基转移速率超过水解速率；然而，在pH 2时，水解比转移快。比较底物和产物长度对反应速率的影响可以得出结论，结合位点可以延伸到八个到九个氨基酸残基。尽管实验没有提供确凿证据支持或反对氨基和/或酰基中间体参与长肽和蛋白质的水解，但在一些底物上观察到的两种类型转肽反应的高产率表明中间体在胃蛋白酶催化反应中起主要作用。结果还表明，当使用猪胃蛋白酶消化蛋白质进行序列分析时，形成转肽产物的可能性相当大。通过这种方式，原始序列中不存在的肽很容易以相当高的产率形成。
