The structural properties of trypsinogen and trypsin Alkylation and oxidation of methionines

Abstract

The side chains of methionines 92 and 166 are buried in the hydrophobic interior of several of the molecular forms (I, I′, III and III′ (refs. 1 and 2)) of bovine trypsinogen and trypsin; they cannot react with H 2O 2, iodoacetamide and iodoacetate. Reversible denaturation by heat or by urea unmasks both thioether functions which are then readily alkylated or oxidized. The kinetics of these modifications are discussed. For example the rate constant for the alkylation of trypsin in 6 M urea (pH 2.2) and 40° is similar to that found by Stark and Stein for the alkylation of N-acetyl methionine under the same conditions. Dialkylated trypsin is devoid of catalytic activity; it has the physical properties of a denatured form. Disulfoxide trypsin is an active enzyme with peptidase, amidase and esterase activities. However, oxidation decreases the thermal stability of the enzyme, lowers its affinity somewhat for benzamidine, and decreases the rates of acylation and deacylation of the strategic serine in the active center. Disulfoxide trypsinogen remains autoactivable in the presence of Ca 2+; when the cation is omitted inert proteins are formed as for unsubstituted trypsinogen. The specific binding site for Ca 2+ is preserved in disulfoxide trypsinogen.