Kinetic studies on the peroxidase activity of selenosubtilisin.
Selenosubtilisin, a semisynthetic selenoenzyme produced by chemical modification of the serine protease subtilisin, acts as a mimic of glutathione peroxidase, catalyzing the reduction of tert-butyl hydroperoxide by 3-carboxy-4-nitrobenzenethiol. To clarify the mechanism of action of this catalyst, detailed kinetic studies have been carried out. Thiol-mediated reduction converts the seleninic acid form of selenosubtilisin (ESeO2H) into a selenenyl sulfide (ESeSAr). Investigations into the reduction of ESeO2H by the aromatic thiol revealed saturation kinetics and were consistent with a significant lowering of the pKa of the seleninic acid in the enzyme active site. While the reduction of ESeO2H was slow compared with a simple model system, the reduced selenoenzyme (ESeSAr) exhibited a much greater peroxidase activity than model compounds. The enzymic selenocysteine residue was shown to be crucial for this activity, and ping-pong kinetics were observed. A catalytic cycle involving interconversion of the ESeSAr, ESeH, and ESeOH forms of the enzyme has been proposed that is consistent with all the available data. The pH-rate profile for the peroxidase activity indicates the involvement of the active site histidine (His64) in the rate-determining step, which these investigations suggest is attack of ArS- on ESeSAr. The results presented here correlated well with crystallographic and spectroscopic data and provide more detailed information about crucial interactions within the active site of selenosubtilisin.
Department of Chemistry, Scripps Research Institute, La Jolla, California 92037.
I M
IM
Bell
M L
ML
Fisher
Z P
ZP
Wu
D
D
Hilvert
D001665:Binding Sites; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D005979:Glutathione Peroxidase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000378:metabolism*; D010545:Peroxides / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014961:X-Ray Diffraction; D020122:tert-Butylhydroperoxide
D010545:Peroxides; D013438:Sulfhydryl Compounds; D020122:tert-Butylhydroperoxide; D009195:Peroxidase; D005979:Glutathione Peroxidase; D013381:Subtilisins; C080590:selenosubtilisin; D007460:Iodoacetamide