Abstract
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.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.