Mikhailova A G, Rumsh L D, Dalgalarrondo M, Chobert J-M, Haertle T
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia.
Biochemistry (Mosc). 2003 Aug;68(8):926-33. doi: 10.1023/a:1025711401572.
Temperature dependences of kinetic constants (k(cat) and K(m)) were studied for enzymatic hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-arginine-p-nitroanilide and N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-lysine-p-nitroanilide by bovine cationic and rat anionic (wild-type and mutant) trypsins. The findings were compared with the corresponding literature data for hydrolysis of N-benzoyl-DL-arginine-p-nitroanilide by bovine cationic trypsin and natural trypsins of cold-adapted fishes. The anionic and cationic trypsins were found to differ in organization of the S(1)-substrate-binding pocket. The difference in the binding of lysine and arginine residues to this site (S(1)) was also displayed by opposite temperature dependences of hydrolysis constants for the corresponding substrates by the anionic and cationic trypsins. The data suggest that the effect of any factor on the binding of substrates (the K(m) value) to the anionic and cationic trypsins and on the catalytic activity k(cat) should be compared only with the corresponding data for the natural enzyme of the same type. Mutants of rat anionic trypsin at residues K188 or Y228 were prepared by site-directed mutagenesis as approximate models of natural psychrophilic trypsins. Substitution of the charged lysine residue in position 188 by hydrophobic phenylalanine residue shifted the pH optimum of the resulting mutant trypsin K188F from 8.0 to 9.0-10.0, similarly to the case of some natural psychrophilic trypsins, and also 1.5-fold increased its catalytic activity at low temperatures as compared to the wild-type enzyme.
研究了牛阳离子胰蛋白酶和大鼠阴离子（野生型和突变型）胰蛋白酶对N-琥珀酰-L-丙氨酰-L-丙氨酰-L-脯氨酰-L-精氨酸对硝基苯胺和N-琥珀酰-L-丙氨酰-L-丙氨酰-L-脯氨酰-L-赖氨酸对硝基苯胺进行酶促水解时动力学常数（k(cat)和K(m)）的温度依赖性。将这些发现与牛阳离子胰蛋白酶和冷适应鱼类天然胰蛋白酶对N-苯甲酰-DL-精氨酸对硝基苯胺水解的相应文献数据进行了比较。发现阴离子和阳离子胰蛋白酶在S(1)底物结合口袋的结构上存在差异。阴离子和阳离子胰蛋白酶对相应底物水解常数的相反温度依赖性也显示了赖氨酸和精氨酸残基与该位点（S(1)）结合的差异。数据表明，任何因素对底物与阴离子和阳离子胰蛋白酶结合（K(m)值）以及对催化活性k(cat)的影响，都应仅与同一类型天然酶的相应数据进行比较。通过定点诱变制备了大鼠阴离子胰蛋白酶在K188或Y228残基处的突变体，作为天然嗜冷胰蛋白酶的近似模型。将188位带电荷的赖氨酸残基替换为疏水性苯丙氨酸残基，使所得突变体胰蛋白酶K188F的最适pH从8.0变为9.0 - 10.0，类似于一些天然嗜冷胰蛋白酶的情况，并且与野生型酶相比，其在低温下的催化活性提高了1.5倍。
