Site-directed mutagenesis reveals critical importance of the catalytic site in the binding of alpha-amylase by wheat proteinaceous inhibitor.

A bacterial alpha-amylase from Bacillus subtilis was found to be strongly inhibited by wheat alpha-amylase inhibitors 0.53 and 0.19, which had previously been thought specific for animal alpha-amylase. Inhibition and gel filtration studies of site-directed mutants of B. subtilis alpha-amylase with the inhibitors indicated a direct correlation between the alpha-amylase activity and the inhibitory effect of inhibitor binding. A mutant enzyme His 180-->Asn, which was 20 times less active in terms of kcat than the wild type, was less sensitive to inhibition by similar degrees, while the specificity for 0.53 and 0.19 changed significantly as a result of the mutation. Catalytic-site mutants that were completely devoid of catalytic activity virtually lost the ability to bind inhibitors, even though they retained high affinities for substrates. The results show that the integrity of the catalytic site is crucial for inhibitor binding and, despite the previously observed tight binding, reveal a subtle nature of the interaction between alpha-amylase and the wheat inhibitor, which leads to a proposal of a two-step mechanism for the binding interaction.