Active site studies on Bacillus amyloliquefaciens alpha-amylase (I)

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.