Chemical modification of the α-amylase ofBacillus caldovelox with diethyl pyrocarbonate: Evidence for an essential histidine at the active site

Abstract

The a-amylase of Bacillus caldovelox is inactivated by diethyl pyrocarbonate at pH 6.6 and 20 ~ by a monomolecular reaction with a second-order rate constant of 41.7 M -l. rain-i. The rate of inactivation increases with decreasing pH, suggesting participation of an amino acid residue with a pKa of 6.6. The increase in absorbance at 240 nm, unchanged absorbance at 280 nm and reactivation in the presence of hydroxylamine suggest the participation of a histidine residue. Statistical analyses of inactivation suggest that only one histidine residue is essential for activity. Substrate afforded complete protection against inactivation, indicating the involvement of the histidine residue at the active site of the enzyme.

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