A highly active and oxidation-resistant subtilisin-like enzyme produced by a combination of site-directed mutagenesis and chemical modification.
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.
Grøn
H
H
Carlsberg Laboratory, Department of Chemistry, Copenhagen, Denmark.
Bech
L M
LM
Branner
S
S
Breddam
K
K
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D007700:Kinetics; D008715:Methionine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D008715:Methionine; D013381:Subtilisins; D003545:Cysteine