Nonessential active site residues modulate selenosubtilisin's kinetic mechanism.
Selenosubtilisin, a semisynthetic enzyme produced by chemical modification of subtilisin's catalytic serine, mimics the antioxidant enzyme glutathione peroxidase, catalyzing the reduction of hydroperoxides by 3-carboxy-4-nitrobenzenethiol. In analogy with the unmodified protease, selenosubtilisins derived from distantly related subtilisin templates exhibit significantly different kinetic properties. Selenosubtilisin BPN' not only is less active than the previously studied Carlsberg selenoenzyme but exhibits sequential rather than ping-pong kinetics, indicating the formation of a ternary complex between enzyme, thiol, and peroxide prior to product release. Experiments with subtilisin E and the BPN' Y217L variant show that the observed differences in kinetic mechanism and chemical efficiency can be attributed largely to amino acid substitutions in the enzyme's S1 and S1' binding sites, respectively. These contributions appear to be roughly additive, and a BPN' triple mutant (E156S/G169A/Y217L) has properties that closely approximate those of selenosubtilisin Carlsberg. The kinetic mechanism of selenosubtilisin can thus be controlled by limited mutagenesis of several active site residues not directly involved in the redox chemistry.
Peterson
E B
EB
Department of Chemistry, Scripps Research Institute, La Jolla, California 92037, USA.
Hilvert
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D
D001665:Binding Sites; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010545:Peroxides / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000737:chemistry* / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D020122:tert-Butylhydroperoxide
D010545:Peroxides; D013438:Sulfhydryl Compounds; D012694:Serine; D020122:tert-Butylhydroperoxide; D013381:Subtilisins; C080590:selenosubtilisin; C093688:3-carboxy-4-nitrobenzenethiol