Kageyama Takashi
Center for Human Evolution Modeling Research, Primate Research Institute, Kyoto University, Inuyama 484-8506, Japan.
Biochemistry. 2006 Dec 5;45(48):14415-26. doi: 10.1021/bi061467a.
Pepsin B is known to be distributed throughout mammalia, including carnivores. In this study, the proteolytic specificity of canine pepsin B was clarified with 2 protein substrates and 37 synthetic octapeptides and compared with that of human pepsin A. Pepsin B efficiently hydrolyzed gelatin but very poorly hydrolized hemoglobin. It was active against only a group of octapeptides with Gly at P2, such as KPAGF/LRL and KPEGF/LRL (arrows indicate cleavage sites). In contrast, pepsin A hydrolyzed hemoglobin but not gelatin and showed high activity against various types of octapeptides, such as KPAEF/FRL and KPAEF/LRL. The specificity of pepsin B is unique among pepsins, and thus, the enzyme provides a suitable model for analyzing the structure and function of pepsins and related aspartic proteinases. Because Tyr13 and Phe219 in/around the S2 subsites (Glu/Ala13 and Ser219 are common in most pepsins) appeared to be involved in the specificity of pepsin B, site-directed mutagenesis was undertaken to replace large aromatic residues with small residues and vice versa. The Tyr13Ala/Phe219Ser double mutant of pepsin B was found to demonstrate broad activity against hemoglobin and various octapeptides, whereas the reverse mutant of pepsin A had significantly decreased activity. According to molecular modeling of pepsin B, Tyr13 OH narrows the substrate-binding space and a peptide with Gly at P2 might be preferentially accommodated because of its high flexibility. The hydroxyl can also make a hydrogen bond with nitrogen of a P3 residue and fix the substrate main chain to the active site, thus restricting the flexibility of the main chain and strengthening preferential accommodation of Gly at P2. The phenyl moiety of Phe219 is bulky and narrows the S2 substrate space, which also leads to a preference for Gly at P2, while lowering the catalytic activity against other peptide types without making a hydrogen-bonding network in the active site.
已知胃蛋白酶B分布于包括食肉动物在内的所有哺乳动物中。在本研究中，用2种蛋白质底物和37种合成八肽阐明了犬胃蛋白酶B的蛋白水解特异性，并与人类胃蛋白酶A进行了比较。胃蛋白酶B能有效水解明胶，但对血红蛋白的水解能力很差。它仅对P2位为甘氨酸的一组八肽有活性，如KPAGF/LRL和KPEGF/LRL（箭头表示切割位点）。相比之下，胃蛋白酶A能水解血红蛋白但不能水解明胶，并且对各种类型的八肽如KPAEF/FRL和KPAEF/LRL表现出高活性。胃蛋白酶B的特异性在胃蛋白酶中是独特的，因此，该酶为分析胃蛋白酶和相关天冬氨酸蛋白酶的结构与功能提供了一个合适的模型。由于S2亚位点（大多数胃蛋白酶中常见的是Glu/Ala13和Ser219）内/周围的Tyr13和Phe219似乎参与了胃蛋白酶B的特异性，因此进行了定点诱变，用小残基取代大的芳香族残基，反之亦然。发现胃蛋白酶B的Tyr13Ala/Phe219Ser双突变体对血红蛋白和各种八肽表现出广泛的活性，而胃蛋白酶A的反向突变体活性显著降低。根据胃蛋白酶B的分子模型，Tyr13的羟基使底物结合空间变窄，P2位为甘氨酸的肽可能因其高柔韧性而被优先容纳。该羟基还可与P3残基的氮形成氢键，并将底物主链固定于活性位点，从而限制主链的柔韧性并增强P2位甘氨酸的优先容纳。Phe219的苯基体积大，使S2底物空间变窄，这也导致对P2位甘氨酸的偏好，同时降低对其他肽类型的催化活性，且不在活性位点形成氢键网络。
