Search

Liquefying alpha-amylase from Bacillus amyloliquefaciens was inactivated by treatment with tetranitromethane and N-acetylimidazole. The loss of activity occurred with modification of five tyrosine residues. Preincubation of the enzyme with either the substrate or the competitive inhibitor at saturating levels provided complete protection against inactivation. However, the presence of substrate/inhibitor in the reaction mixture protected only two of the five modifiable tyrosine residues, suggesting the involvement of only two tyrosine residues at the active center. This was confirmed when hydroxylamine treatment of the acetylated enzyme fully restored the enzymatic activity. Both nitration and acetylation increased the apparent Km of the enzyme for soluble starch, which indicated that the tyrosine residues are involved in substrate binding. Reduction of nitrotyrosine residues to aminotyrosine residues failed to restore the enzymatic activity. So, the loss of activity on modification of tyrosine residues was ascribed to conformational perturbances and not simply to the changes in the ionic character of tyrosine residues.

Liquefying alpha-amylase from Bacillus amyloliquefaciens was inactivated on treatment with N-bromosuccinamide. Preincubation of the enzyme with either of the substrate, or competitive inhibitor provided significant protection against inactivation. The relationship between activity loss and the number of tryptophan residues modified, as well as presence of substrate/inhibitor in the reaction mixture, demonstrated that only one of three modifiable tryptophan residues is at or near the active center. The apparent Km of the modified enzyme for soluble starch increased manifold, thus implicating the sensitive tryptophan residue in the substrate binding region of the enzyme.

Modification of liquefying alpha-amylase by diethylpyrocarbonate or its photo-oxidation in the presence of rose bengal caused rapid loss of enzyme activity. The photo-oxidation followed pseudo-first-order kinetics giving maximal value at pH 8.0. The photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm which was directly proportional to the extent of inactivation. Diethylpyrocarbonate at low concentration at pH 6.0 and 30 degrees C completely inactivated alpha-amylase. Inactivation followed pseudo-first-order kinetics. The reaction order with respect to inactivation by diethylpyrocarbonate-modified enzyme showed increased absorbance at 240 nm which was reversed completely upon treatment with NH2OH at 30 degrees C for 16 hr. Calculating the histidine residues being modified from the increase in absorbance at 240 nm showed that three residues were ethoxyformylated on treatment with diethylpyrocarbonate, of which only one was found at the active site. Substrate and competitive inhibitor protects the enzyme against both, photo-oxidation, and modification by diethylpyrocarbonate, confirming that histidine plays an essential role at the alpha-amylase active site.