Essential histidine residues in dextransucrase: Chemical modification by diethyl pyrocarbonate and dye photooxidation

Abstract

Treatment of Leuconostoc mesenteroides B-512F dextransucrase with diethyl pyrocarbonate (DEP) at pH 6.0 and 25° or photo-oxidation in the presence of Rose Bengal or Methylene Blue at pH 6.0 and 25°, caused a rapid decrease of enzyme activity. Both types of inactivation followed pseudo-first-order kinetics. Enzyme partially inactivated by DEP could be completely reactivated by treatment with 100m m hydroxylamine at pH 7 and 4°. The presence of dextran partially protected the enzyme from inactivation. At pH 7 or below, DEP is relatively specific for the modification of histidine. DEP-modified enzyme showed an increased absorbance at 240 nm, indicating the presence of (ethoxyformyl)ated histidine residues. DEP modification of the sulfhydryl group of cysteine and of the phenolic group of tyrosine was ruled out by showing that native and DEP-modified enzyme had the same number of sulfhydryl and phenolic groups. DEP modification of the ε-amino group of lysine was ruled out by reaction at pH 6 and reactivation with hydroxylamine, which has no effect on DEP-modified ε-amino groups. The photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm, also indicating that histidine had been oxidized, and no decrease in the absorbance at 280 nm, indicating that tyrosine and tryptophan were not oxidized. A statistical, kinetic analysis of the data on inactivation by DEP showed that two histidine residues are essential for the enzyme activity. Previously, it was proposed that two nucleophiles at the active site attack bound sucrose, to give two covalent d-glucosyl-enzyme intermediates. We now propose that in addition, two imidazolium groups of histidine at the active site donate protons to the leaving, d-fructosyl moieties. The resulting imidazole groups then facilitate the formation of the α-(1→6)-glycosidic linkage by abstracting protons from the C-6-OH groups, and become reprotonated for the next series of reactions.