Mutational Analysis of Target Enzyme Recognition of the β-Trefoil Fold Barley α-Amylase/Subtilisin Inhibitor
| Autor: | Birgit Christine Bønsager, Peter Kresten Nielsen, Birte Svensson, Mette Prætorius-Ibba, Maher Abou Hachem, Kenji Fukuda |
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| Rok vydání: | 2005 |
| Předmět: |
Models
Molecular Protein Folding Time Factors Glycoside Hydrolases Protein Conformation Stereochemistry medicine.medical_treatment DNA Mutational Analysis Molecular Sequence Data Mutant Biochemistry Protein Structure Secondary Catalytic Domain medicine Glycoside hydrolase Amino Acid Sequence Amylase Surface plasmon resonance Molecular Biology chemistry.chemical_classification Binding Sites Protease Sequence Homology Amino Acid biology Chemistry Subtilisin Wild type Hordeum Stereoisomerism Cell Biology Surface Plasmon Resonance Protein Structure Tertiary Kinetics Enzyme Mutation biology.protein Thermodynamics Calcium Electrophoresis Polyacrylamide Gel Isoelectric Focusing alpha-Amylases Software Peptide Hydrolases Protein Binding |
| Zdroj: | Journal of Biological Chemistry. 280:14855-14864 |
| ISSN: | 0021-9258 |
| Popis: | The barley alpha-amylase/subtilisin inhibitor (BASI) inhibits alpha-amylase 2 (AMY2) with subnanomolar affinity. The contribution of selected side chains of BASI to this high affinity is discerned in this study, and binding to other targets is investigated. Seven BASI residues along the AMY2-BASI interface and four residues in the putative protease-binding loop on the opposite side of the inhibitor were mutated. A total of 15 variants were compared with the wild type by monitoring the alpha-amylase and protease inhibitory activities using Blue Starch and azoalbumin, respectively, and the kinetics of binding to target enzymes by surface plasmon resonance. Generally, the mutations had little effect on k(on), whereas the k(off) values were increased up to 67-fold. The effects on the inhibitory activity, however, were far more pronounced, and the K(i) values of some mutants on the AMY2-binding side increased 2-3 orders of magnitude, whereas mutations on the other side of the inhibitor had virtually no effect. The mutants K140L, D150N, and E168T lost inhibitory activity, revealing the pivotal role of charge interactions for BASI activity on AMY2. A fully hydrated Ca(2+) at the AMY2-BASI interface mediates contacts to the catalytic residues of AMY2. Mutations involving residues contacting the solvent ligands of this Ca(2+) had weaker affinity for AMY2 and reduced sensitivity to the Ca(2+) modulation of the affinity. These results suggest that the Ca(2+) and its solvation sphere are integral components of the AMY2-BASI complex, thus illuminating a novel mode of inhibition and a novel role for calcium in relation to glycoside hydrolases. |
| Databáze: | OpenAIRE |
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