Tsai I H, Lu P J, Chuang J L
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, Republic of China.
Biochim Biophys Acta. 1991 Oct 11;1080(1):59-67. doi: 10.1016/0167-4838(91)90112-d.
The midgut chymotrypsins (EC 3.4.4.5) of three species of shrimps, Penaeus monodon, Penaeus japonicus and Penaeus penicillatus were purified and studied in detail to clarify previous ambiguity in their identification. In each of the species there are two major forms of chymotrypsin, both single-chained with three disulfide bonds. One has a pI of 3.2 and Mr 27,000 or 28,000, while the other has a pI of 3.0 and Mr 25,000 or 26,000. The N-terminal amino acid sequences of the P. monodon enzymes are homologous to those of the crab (Uca pugilator) collagenase and to the other chymotrypsins. However, the active sites of the shrimp chymotrypsins are different from that of the well studied bovine alpha-chymotrypsin in some respects: (1) in spite of showing the typical specificity of chymotrypsin, the shrimp enzymes are more stringently selective for substrates with extended polypeptide chain; (2) some titration agents of alpha-chymotrypsin, including t-cinnamoylimidazole, 4-nitrophenyl guanidinobenzoate and its fluorescent derivative, do not react with the shrimp enzymes, neither do some of the alpha-chymotrypsin inhibitors: Tosyl-PheCH2Cl, methyl-4-nitrobenzenesulfonate and benzeneboronic acid; (3) the shrimp chymotrypsins are more reactive than the bovine enzyme toward native protein substrates including collagen; (4) the kinetic-salt-effects of the shrimp enzyme toward N-succinyl- and acetyl-Ala-Ala-Pro-Phe-4-nitroanilide mainly reflect electrostatic rather than hydrophobic interactions between the substrates and the enzyme. The shrimp enzymes are acid-labile but resistent to autolysis. Our results suggest that most Crustacea decapods contain chymotrypsins as one of the major digestive endopeptidases.
对斑节对虾、日本对虾和长毛对虾这三种虾的中肠胰凝乳蛋白酶（EC 3.4.4.5）进行了纯化和详细研究，以澄清此前在其鉴定方面的模糊之处。在每个物种中都有两种主要形式的胰凝乳蛋白酶，均为单链且含有三个二硫键。一种的pI为3.2，Mr为27,000或28,000，而另一种的pI为3.0，Mr为25,000或26,000。斑节对虾酶的N端氨基酸序列与蟹（凶猛招潮蟹）胶原酶及其他胰凝乳蛋白酶的序列同源。然而，虾类胰凝乳蛋白酶的活性位点在某些方面与已深入研究的牛α-胰凝乳蛋白酶不同：（1）尽管虾类酶表现出典型的胰凝乳蛋白酶特异性，但它们对具有延伸多肽链的底物具有更严格的选择性；（2）一些α-胰凝乳蛋白酶的滴定剂，包括反式肉桂酰咪唑、4-硝基苯基胍基苯甲酸及其荧光衍生物，不与虾类酶反应，一些α-胰凝乳蛋白酶抑制剂也不反应：甲苯磺酰苯丙氨酸氯甲基酮、甲基-4-硝基苯磺酸盐和苯硼酸；（3）虾类胰凝乳蛋白酶对包括胶原蛋白在内的天然蛋白质底物的反应性比牛酶更高；（4）虾类酶对N-琥珀酰-和乙酰-丙氨酰-丙氨酰-脯氨酰-苯丙氨酸-4-硝基苯胺的动力学盐效应主要反映底物与酶之间的静电相互作用而非疏水相互作用。虾类酶对酸不稳定但抗自溶。我们的结果表明，大多数十足目甲壳动物含有胰凝乳蛋白酶作为主要的消化内肽酶之一。
