Abstract
Abstract The effects of hydrogen peroxide on a bacterial serine protease, subtilisin Carlsberg (EC 3.4.4.16), were investigated. Of the 5 methionine residues in this molecule, only 1, Residue 222, was oxidized to methionine sulfoxide. No other changes in amino acid composition were found. The oxidized enzyme was enzymatically active, but the efficiency was less than that of the native subtilisin. The extent of decrease in kcat was between 57 and 92%, depending on the substrate used. Km(app) increased for most substrates used, but it decreased for the N-benzyloxycarbonylglycine p-nitrophenyl ester. The change in individual reaction rate parameters was investigated using N-cinnamoylimidazole. The dissociation constant of the ES complex, K8, was greater for oxidized subtilisin than for native subtilisin, while the rate constants for the acylation step, k2, and the deacylation step, k3, were decreased.
Highlights
The oxidized enzyme was enzymatically active, but the efficiency was less than that of the native subtilisin
We examined the consequences of oxidizing subtilisin with HzOz
Oxidation of Subtilisin by HzOz-The oxidized enzyme was prepared in the following way
Summary
The oxidized enzyme was enzymatically active, but the efficiency was less than that of the native subtilisin. The extent of decrease in &at was between 57 and 92%, depending on the substrate used. KMcapp) increased for most substrates used, but it decreased for the N-benzyloxycarbonylglycine p-nitrophenyl ester. The change in individual reaction rate parameters was investigated using N-cinnamoylimidazole. The dissociation constant of the ES complex, KB, was greater for oxidized subtilisin than for native subtilisin, while the rate constants for the acylation step, kz, and the deacylation step, k3, were decreased
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