St Leger R J, Charnley A K, Cooper R M
Arch Biochem Biophys. 1987 Feb 15;253(1):221-32. doi: 10.1016/0003-9861(87)90655-2.
Two chymoelastases and three trypsinlike proteases were separated from culture filtrates of the entomopathogen Metarhizium anisopliae. A chymoelastase (Pr1) (pI 10.3 Mr 25,000) and trypsin (Pr2) (pI 4.42, Mr 28,500) were purified to homogeneity by ammonium sulphate precipitation, isoelectric focusing, and affinity chromatography. Inhibition studies showed that both enzymes possessed essential serine and histidine residues in the active site. Pr1 shows greater activity than Pr2 or mammalian enzymes against locust cuticle and also possesses activity vs elastin. Pr1 shows a broad primary specificity toward amino acids with hydrophobic side groups in synthetic ester and amide substrates. The kinetic properties of Pr1 demonstrate a preference for extended peptide chains with the active site recognising at least five substrate residues. The S5 and S4 subsites show a preference for negatively charged succinyl and hydrophobic acetyl groups, respectively. The S3 and S2 subsites both discriminated in favor of alanine and against proline. Pr2 rapidly hydrolyzed casein and synthetic substrates containing arginine or lysine. It possessed little or no activity vs cuticle, elastin, or synthetic substrates for chymotrypsin and elastase. Specific active site inhibitors confirmed the similarities between Pr2 and trypsin.
从昆虫病原真菌绿僵菌的培养滤液中分离出了两种糜弹性蛋白酶和三种类胰蛋白酶。通过硫酸铵沉淀、等电聚焦和亲和层析，将一种糜弹性蛋白酶（Pr1）（pI 10.3，Mr 25,000）和胰蛋白酶（Pr2）（pI 4.42，Mr 28,500）纯化至同质。抑制研究表明，这两种酶在活性位点均含有必需的丝氨酸和组氨酸残基。Pr1对蝗虫表皮的活性高于Pr2或哺乳动物的酶，并且对弹性蛋白也有活性。Pr1对合成酯和酰胺底物中带有疏水侧基的氨基酸表现出广泛的一级特异性。Pr1的动力学特性表明其倾向于长肽链，其活性位点可识别至少五个底物残基。S5和S4亚位点分别对带负电荷的琥珀酰基和疏水的乙酰基表现出偏好。S3和S2亚位点都倾向于丙氨酸而不利于脯氨酸。Pr2能快速水解酪蛋白以及含有精氨酸或赖氨酸的合成底物。它对表皮、弹性蛋白或用于胰凝乳蛋白酶和弹性蛋白酶的合成底物几乎没有活性。特异性活性位点抑制剂证实了Pr2与胰蛋白酶之间的相似性。
