Margolis H C, Nakagawa Y, Douglas K T, Kaiser E T
J Biol Chem. 1978 Nov 10;253(21):7891-7.
In the course of our further investigation of the active site titration of carboxypeptidase Y, using 4-nitrophenyl trimethylacetate, we have found that carboxypeptidase Y can be isolated in different molecular forms. Carboxypeptidase Y obtained from Fleischmann baker's yeast has a molecular weight of 53,000, as compared to 64,000 for an enzyme species isolated from Anheuser-Busch baker's yeast. The amino acid analyses of both enzymes were essentially identical and very similar to those reported by others. However, we have found that the molecular weight difference is due to a variation in carbohydrate content as determined by gas chromatography. When carboxypeptidase Y was isolated from a single source, Anheuser-Busch baker's yeast, we observed a smaller variation in carbohydrate content. In all cases, sugar analyses revealed only mannose and N-acetylglucosamine to be present. The effect of the enzyme's carbohydrate content on the "burst kinetics" of the 4-nitrophenyl trimethylacetate reaction has been examined. In general, the Anheuser-Busch enzyme, containing more carbohydrate than the Fleischmann enzyme, reacts with a larger apparent bimolecular rate constant, kcat/Km. On the other hand, the deacylation rate constant, k3, is affected only slightly.
在我们使用4-硝基苯基三甲基乙酸酯对羧肽酶Y活性位点滴定进行的进一步研究过程中，我们发现羧肽酶Y可以以不同的分子形式分离出来。从弗莱施曼面包酵母中获得的羧肽酶Y分子量为53,000，而从安海斯-布希面包酵母中分离出的一种酶分子量为64,000。两种酶的氨基酸分析基本相同，且与其他人报道的结果非常相似。然而，我们发现分子量差异是由于气相色谱法测定的碳水化合物含量不同所致。当从单一来源安海斯-布希面包酵母中分离羧肽酶Y时，我们观察到碳水化合物含量的变化较小。在所有情况下，糖类分析表明仅存在甘露糖和N-乙酰葡糖胺。已经研究了该酶的碳水化合物含量对4-硝基苯基三甲基乙酸酯反应“爆发动力学”的影响。一般来说，安海斯-布希酶比弗莱施曼酶含有更多的碳水化合物，它以更大的表观双分子速率常数kcat/Km进行反应。另一方面，脱酰基速率常数k3仅受到轻微影响。
