A new approach is proposed for probing the polarity of the immediate environment of—SH groups in thiolenzymes, based on the alkylation of the—SH group with methyl iodide, a relatively small and non‐polar molecule. Rate and activation parameters (δH*, δS*) for the reaction of the enzyme are compared to those of glutathione, a simple—SH compound alkylated in aqueous medium. The enzyme and model compound are also reacted with iodoacetamide, a polar counterpart of the non‐polar methyl iodide. The above method was applied to thiolsubtilisin, an artificial thiolenzyme. The ratio of the rates of alkylation of thiolsubtilisin and glutathione is about 20 times as high with methyl iodide as with iodoacetamide. δH* and δS* for enzyme alkylation, as compared to those for glutathione, are remarkably lower with methyl iodide whereas they are slightly higher with iodoacetamide. δH* and δS* for alkylation of thiolsubtilisin with methyl iodide are similar to those found with glutathione in 40% dioxane/water mixture. The activation enthalpy and entropy values for the reaction of thiolsubtilisin with d‐2‐bromo‐n‐valeramide are lower than those for glutathione reaction. Consequently, in this respect, d‐2‐bromo‐n‐valeramide is similar to methyl iodide rather than to iodoacetamide. It is concluded that the—SH group of thiolsubtilisin is located in an environment less polar than water. The concentration of methyl iodide in this non‐polar layer is higher than in the bulk solution, which results in an enhanced reaction rate.
|Number of pages||7|
|Journal||European Journal of Biochemistry|
|Publication status||Published - Dec 1976|
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