Abstract
Diethylpyrocarbonate modification of endoglucanase D from Clostridium thermocellum, cloned in Escherichia coli, resulted in a rapid but partial (maximally 70-80%) loss of activity. The second-order rate constant of inactivation proved to be exceptionally high (3210 M-1.min-1). A 3-fold reduction of the kcat and a 2-fold increase of the Km for 2'-chloro-4'-nitrophenyl beta-cellobioside were observed. Spectrophotometric analysis indicate the presence of one rapidly (k = 0.45 min-1) and two slower (k = 0.23 min-1) reacting histidyl residues. In the presence of 50 mM methyl beta-cellotrioside, the rate of inactivation was reduced 16-fold, and the kinetics of modification were compatible with the protection of 1 histidyl residue. Since peptide analysis was inconclusive, identification of the critical residue was attempted by site-directed mutagenesis. Each of the 12 histidyl residues present in the endoglucanase D sequence was mutated into either Ala or Ser. Seven of the mutant enzymes had specific activities lower than 50% of the wild-type. Only in the case of the Ser-516 mutant, however, was the residual activity not affected by diethyl pyrocarbonate. These findings suggest an important functional or structural role for His-516 in the wild-type enzyme.
Highlights
Treatment of modified endoglucanase D with 50 mM hydroxylamine resulted in complete reactivation of the enzyme within 20 min (Fig. 2B).Since this reagent is known to reverse N-ethoxycarbonylation of modified Tyr and Hirsesidues only (Burnstein et al, 1974), reactivation excludes the possible involvement of Cys or Lys residues (Melchior and Fahrney, 1970; Miles, 1977)
All the evidence presented above point to thepresence of a His residue in the catalytic center of endoglucanase D from C. thermocellum
His-specific modification of endoglucanase D with (EtOCO),O resulted in 70-80% loss of activity
Summary
Tel.: 32-91- cellobioside; PC buffer, 50 mM sodium phosphate/citric acid buffer, 645272; Fax: 32-91-645342. Isolation and Analysis of Modified Peptides-60-pl samples of endoglucanase D (30-40 p~ in PC buffer) were treated for 10 min with 2 mM (EtOCO),O (23 'C), in the presence or absence of a ligand, and inactivation was followed as described above. Immunoblotting Assay of Endoglucanase D-Enzyme concentrationsin crude extracts of strains expressing mutant genes were estimated from Western blots. Modification of Mutant Proteins with (EtOCO)zO-Protein concentration in the crude extracts (measured according to Sedmak and Grossberg, 1977, using bovine serum albumin as a standard) was adjusted with PC buffer to 1.85 mg/ml, and (EtOCO),O was added to a final concentratioonf 5.5 mM.After incubating at room temperature for 0-8 min, 100-plsamples were withdrawn and quenched by diluting into 400 pl of PC buffer containing 2.5 mM N-acetylimidazole.
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