Mutational Analysis of Target Enzyme Recognition of the β-Trefoil Fold Barley α-Amylase/Subtilisin Inhibitor

Abstract

The barley ␣-amylase/subtilisin inhibitor (BASI) inhibits ␣-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 ␣-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.The double-headed barley ␣-amylase/subtilisin inhibitor (BASI) 1 of the Kunitz soybean trypsin inhibitor family acts on proteases of the subtilisin family and the endogenous high pI ␣-amylase (AMY2) but has no effect on the minor isozyme AMY1 that shares 80% sequence identity with AMY2

Dissecting Molecular Interactions Involved in Recognition of Target Disulfides by the Barley Thioredoxin System

“…The latter residue has a functional role in the inhibition of barley α-amylase as shown using the BASI mutants E168Q and E168T. 33 …”

An enzyme family reunion — similarities, differences and eccentricities in actions on α-glucans

“…The BASI-AMY2 complex has been well described using site-directed mutagenesis, crystallography, surface plasmon resonance, and activity inhibition analyses (Vallée et al 1998;Rodenburg et al 2000;Nielsen et al 2003;Bønsager et al 2005) and found to have high stability, sub-nanomolar affinity; specific residues were assigned functional roles both in enzyme and inhibitor for the complex formation. Analysis of the LDI-limit dextrinase complex is just initiated thanks to breakthroughs with successful heterologous production of both LDI and limit dextrinase (M. VesterChristensen et al, manuscript in preparation) and data now emerge showing sub-nanomolar affinity also for this complex (J.M.…”

Cerealn-glycoproteins Enrichment by Lectin Affinity Monolithic Chromatography

“…CM16, CMb (identified in barley and spelt) and CM3 and CM1 (discovered in durum) belong to "CM-proteins". Wheat α-amylase/subtilisin inhibitor (WASI; detected in spelt) inactivates alphaamylase from animal and insect origin and it is also able to inhibit subtilisin (Bønsager et al 2005). CM proteins as well as WASI are connected with IgE wheat allergy (Sotkovsky et al 2011).…”