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.

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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.

The title compounds (VI) and (‐)‐(VIII) are potential inhibitors of the title enzyme.

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