Pacaud M, Richaud C
J Biol Chem. 1975 Oct 10;250(19):7771-9.
We have previously demonstrated the existence of two types of endopeptidase in Escherichia coli. A purification procedure is described for one of these, designated protease II. It has been purified about 13,500-fold with a recovery of 24%. The isolated enzyme appears homogeneous by electrophoresis and gel filtration. Its molecular weight is estimated by three different methods to be about 58,000. Its optimal pH is around 8. Protease II activity is unaffected by chelating agents and sulfhydryl reagents. Amidase and proteolytic activities are stimulated by calcium ion, which decreases the enzyme stability. Like pancreatic trypsin, this endopeptidase catalyses the hydrolysis of alpha-amino-substituted lysine and arginine esters. It appears distinct from the previously isolated protease I, which is a chymotrypsin-like enzyme. The apparent Michaelis constant for hydrolysis of N-benzoyl-L-arginine ethyl ester is 4.7 X 10(-4) M. The esterase activity is inhibited by diisopryopylphosphorofluoridate (Ki(app) equals 2.7 X 10(-3) M) and tosyl lysine chloromethyl ketone (Ki(app) equals 1.8 X 10(-5) M), indicating that serine and histidine residues may be present in the active site. However, protease II is insensitive to phenylmethanesulfonyl fluoride and several natural trypsin inhibitors. Its amidase and esterase activities are competitively inhibited by free arginine and aromatic amidines. The proteolytic activity measured on axocasein is very low. In contrast to trypsin, protease II is without effect on native beta-galactosidase. It easily degrades aspartokinase I and III. Nevertheless both enzymes are resistant to proteolysis in the presence of their respective allosteric effectors. These results provide further evidence that such differences in protease susceptibility can be related to the conformational state of the substrate. The possible implication of structural changes in the mechanism of preferential proteolysis in vivo, is discussed.
我们之前已证明大肠杆菌中存在两种类型的内肽酶。本文描述了其中一种名为蛋白酶II的纯化方法。它已被纯化约13500倍，回收率为24%。通过电泳和凝胶过滤，分离出的酶看起来是纯一的。用三种不同方法估计其分子量约为58000。其最适pH约为8。蛋白酶II的活性不受螯合剂和巯基试剂的影响。酰胺酶和蛋白水解活性受钙离子刺激，钙离子会降低酶的稳定性。与胰蛋白酶一样，这种内肽酶催化α-氨基取代的赖氨酸和精氨酸酯的水解。它似乎与之前分离出的蛋白酶I不同，蛋白酶I是一种类似胰凝乳蛋白酶的酶。水解N-苯甲酰-L-精氨酸乙酯的表观米氏常数为4.7×10⁻⁴M。酯酶活性受二异丙基氟磷酸酯（Ki(app)等于2.7×10⁻³M）和甲苯磺酰赖氨酸氯甲基酮（Ki(app)等于1.8×10⁻⁵M）抑制，表明活性位点可能存在丝氨酸和组氨酸残基。然而，蛋白酶II对苯甲基磺酰氟和几种天然胰蛋白酶抑制剂不敏感。其酰胺酶和酯酶活性受游离精氨酸和芳香脒竞争性抑制。在轴酪蛋白上测得的蛋白水解活性非常低。与胰蛋白酶不同，蛋白酶II对天然β-半乳糖苷酶没有作用。它很容易降解天冬氨酸激酶I和III。然而，在各自的别构效应剂存在下，这两种酶都对蛋白水解有抗性。这些结果进一步证明，蛋白酶敏感性的这种差异可能与底物的构象状态有关。讨论了体内优先蛋白水解机制中结构变化的可能影响。
