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.
Spin-echo NMR spectroscopy was used to record the cleavage of a gamma-glutamyl--amino-acid by (5-L-glutamyl)-L-amino-acid 5-glutamyltransferase (cyclizing) (gamma-glutamylcyclotransferase) in human erythrocyte hemolysates. The Michaelis-Menten steady-state kinetic parameters were obtained by fitting the integrated Michaelis-Menten equation to the reaction time curves. The product, L-5-oxoproline, was shown to be an inhibitor of the reaction. The active site of the enzyme was probed by studies of the inhibition by D- and L-beta-aminoglutaryl-L-alanine which are the beta-amino-acid isomers of D- and L-gamma-glutamyl-L-alanine (the latter being a natural substrate of the enzyme); the D-isomer was the more potent inhibitor (Ki = 0.30 +/- 0.02 mmol/l water). When the alanyl alpha-carboxyl of the inhibitor was reduced to a hydroxyl (i.e. to give D-beta-aminoglutaryl-L-alaninol) the potency of inhibition was reduced. The previously reported kinetic isotope effect of solvent 2H2O on the enzyme-catalyzed reaction has been further studied using a proton inventory. We propose that the solvent kinetic isotope effect is due to an intramolecular proton transfer between the glutamyl amino group and the peptide bond nitrogen.
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