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.
Bell
I M
IM
Department of Chemistry, Scripps Research Institute, La Jolla, California 92037.
Fisher
M L
ML
Wu
Z P
ZP
Hilvert
D
D
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