Grøn H, Bech L M, Branner S, Breddam K
Carlsberg Laboratory, Department of Chemistry, Copenhagen, Denmark.
Eur J Biochem. 1990 Dec 27;194(3):897-901. doi: 10.1111/j.1432-1033.1990.tb19484.x.
The subtilisins are known to be susceptible to chemical oxidation due to the conversion of Met222 into the corresponding sulfoxide. A number of derivatives with resistance towards oxidation have previously been prepared by replacement of this group with the other 19 amino acid residues. Unfortunately, the activities of these enzymes were of the order of 1-10% of that obtained with the wild-type enzyme. In contrast, the oxidation-labile cysteine mutant exhibited much higher activity, suggesting that this is associated with the presence of a sulphur atom in the amino acid at position 222. It is shown here that it is possible to maintain a sulphur atom in the amino acid at position 222 without the enzyme becoming labile towards oxidation. A subtilisin from Bacillus lentus, subtilisin 309, in which Met222 was replaced with a cysteinyl residue by site-directed mutagenesis was modified with thioalkylating reagents. Treatment of such enzyme derivatives with H2O2 revealed that their stabilities towards oxidation had increased significantly compared to both wild-type and unmodified [Cys222]subtilisin. One of the chemically modified enzyme derivatives, [Me-S-Cys222]subtilisin, exhibited a kcat/Km value of 56% of that obtained with the wild-type enzyme when assayed against the substrate Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2 (Suc, succinyl) and it exhibited 89% activity when tested in an assay with dimethyl casein as a substrate. The corresponding values obtained for unmodified [Cys222]subtilisin were lower, i.e. 39% for the dimethyl casein activity and 46% for the kcat/Km for the hydrolysis of Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2. This demonstrates the feasibility of replacing the oxidation-labile methionyl residue group in a subtilisin enzyme with a group stable towards oxidation without substantially reducing the activity.
枯草杆菌蛋白酶已知因Met222转化为相应的亚砜而易于受到化学氧化。先前已通过用其他19种氨基酸残基取代该基团制备了许多抗氧化的衍生物。不幸的是，这些酶的活性约为野生型酶活性的1-10%。相比之下，对氧化不稳定的半胱氨酸突变体表现出更高的活性，这表明这与222位氨基酸中硫原子的存在有关。本文表明，在222位氨基酸中保持硫原子，而酶不会变得对氧化不稳定是可能的。通过定点诱变将来自迟缓芽孢杆菌的枯草杆菌蛋白酶309中的Met222替换为半胱氨酸残基，并用硫烷基化试剂对其进行修饰。用过氧化氢处理这种酶衍生物表明，与野生型和未修饰的[Cys222]枯草杆菌蛋白酶相比，它们对氧化的稳定性显著提高。其中一种化学修饰的酶衍生物，[Me-S-Cys222]枯草杆菌蛋白酶，在以底物Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2（Suc，琥珀酰基）进行测定时，其kcat/Km值为野生型酶的56%，在用二甲基酪蛋白作为底物的测定中测试时，其活性为89%。未修饰的[Cys222]枯草杆菌蛋白酶的相应值较低，即以二甲基酪蛋白为底物时的活性为39%，水解Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2时的kcat/Km为46%。这证明了用对氧化稳定的基团取代枯草杆菌蛋白酶中对氧化不稳定的甲硫氨酰残基基团而不显著降低活性的可行性。
