Kinetics and Energetics of the Binding between Barley α-Amylase/Subtilisin Inhibitor and Barley α-Amylase 2 Analyzed by Surface Plasmon Resonance and Isothermal Titration Calorimetry

Nielsen, Peter Kresten; Bønsager, Birgit Christine; Berland, Carolyn R.; Sigurskjold, Bent W.; Svensson, Birte

doi:10.1021/bi020508+

Cited by 46 publications

(37 citation statements)

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“…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.…”

Section: New Roles and Diversity Of α-Glucan-active Enzymes In Biologmentioning

confidence: 99%

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

et al. 2008

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α-Glucans in general, including starch, glycogen and their derived oligosaccharides are processed by a host of more or less closely related enzymes that represent wide diversity in structure, mechanism, specificity and biological role. Sophisticated three-dimensional structures continue to emerge hand-in-hand with the gaining of novel insight in modes of action. We are witnessing the "test of time" blending with remaining questions and new relationships for these enzymes. Information from both within and outside of ALAMY 3 Symposium will provide examples on what the family contains and outline some future directions. In 2007 a quantum leap crowned the structural biology by the glucansucrase crystal structure. This initiates the disclosure of the mystery on the organisation of the multidomain structure and the "robotics mechanism" of this group of enzymes. The central issue on architecture and domain interplay in multidomain enzymes is also relevant in connection with the recent focus on carbohydrate-binding domains as well as on surface binding sites and their long underrated potential. Other questions include, how different or similar are glycoside hydrolase families 13 and 31 and is the lid finally lifted off the disguise of the starch lyase, also belonging to family 31? Is family 57 holding back secret specificities? Will the different families be sporting new "eccentric" functions, are there new families out there, and why are crystal structures of "simple" enzymes still missing? Indeed new understanding and discovery of biological roles continuously emphasize value of the collections of enzyme models, sequences, and evolutionary trees which will also be enabling advancement in design for useful and novel applications.

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Section: New Roles and Diversity Of α-Glucan-active Enzymes In Biologmentioning

confidence: 99%

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

et al. 2008

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“…The K m of savinase for azoalbumin was 14 mg ϫ ml Ϫ1 as determined from the initial hydrolysis rates at seven substrate concentrations (0.5-12.5 mg ϫ ml Ϫ1 ). Surface Plasmon Resonance-Binding kinetics of BASI and either AMY2 or savinase was determined by using a BIAcore 3000 (Biosensor, Amersham Biosciences) (4,8). The sensor chips were charged by 300 -1000 response units of either biotinylated AMY2 (streptavidin-sensor chips), BASI, or savinase bound by using the amine coupling procedure (CM5 sensor chips) (8).…”

Section: Methodsmentioning

confidence: 99%

“…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 (1)(2)(3)(4)(5)(6)(7)(8). Under favorable conditions, BASI inhibits the AMY2-catalyzed hydrolysis of starch with a K i ϳ0.1 nM (2,4).…”

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confidence: 99%

“…An exception is AMY2-BASI, which has been the subject of extensive studies with respect to binding mechanisms and structure/function relationships (2,5,6,9). Recently data from mutants of AMY2 residues binding BASI (7), SPR, and isothermal titration calorimetry of AMY2-BASI wild-type protein interactions have demonstrated the effects of ionic strength, pH, and Ca 2ϩ on kinetics and thermodynamics of the complex formation (5,8), but many important questions are still unanswered. The newly established heterologous expression system in Escherichia coli (4) prompted dissection of the contribution of individual regions and side chains to the overall inhibitory activity of BASI.…”

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

Bønsager

Nielsen

Hachem

et al. 2005

Journal of Biological Chemistry

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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

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“…The complex formation of AMY2 and BASI was described using surface plasmon resonance analysis which also facilitated studies of the effect of calcium on the stability of the AMY2 BASI complex. 24,25) While increasing [Ca 2+ ] from the low µM range to 20 mM evidently stabilised the complex by an order of magnitude, the dependence of the stability on pH suggested that a group in BASI of pKa around 6 participates in the binding to AMY2. The three dimensional structure of the AMY2 BASI pointed to Glu168 as an obvious candidate, since this residue through hydrogen bonds with the molecules in the water shell around Ca503 was indirectly in contact with the enzyme.…”

Section: Reactions Involving α-Amylases α-Amylase Inhibitors and Thimentioning

confidence: 99%

Interactions between Barley .ALPHA.-Amylases, Substrates, Inhibitors and Regulatory Proteins

et al. 2006

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α Amylases belong to the glycoside hydrolase family 13 (GH13) that together with glycoside hydrolase families 70 and 77 constitute clan H of glycoside hydrolases (GH H).1) GH H contains approximately 30 different enzyme specificities acting in hydrolase and transglucosidase reactions on α 1,4 and or α 1,6 glucan and α glucoside substrates.2)The three dimensional structures of α amylases mostly contain three domains; domain A, a (β α)8 barrel; domain B a small protruding loop situated between β strand and α helix 3 in the (β α)8 barrel; and domain C, a C terminal anti parallel β sheet composed of 5 10 strands. The substrate binding site is made from residues of domains A and B and the three acid residues involved in catalysis are situated at the C terminal ends of β strands 4, 5 and 7.2) These three catalytic residues are the only invariant residues in GH H.3) A few new complexes between amylolytic enzymes and substrates or substrate analogues have been recently published. 4,5) These structures provide novel insight in particular with respect to Abstract: Barley α-amylase binds sugars at two sites on the enzyme surface in addition to the active site. Crystallography and site-directed mutagenesis highlight the importance of aromatic residues at these surface sites as demonstrated by Kd values determined for β-cyclodextrin by surface plasmon resonance and for starch granules by adsorption analysis. Activity towards amylopectin and amylose follows two different kinetic models, degradation of amylopectin being composed of a fast and a slow component, perhaps reflecting attack on A and B chains, respectively, whereas amylose hydrolysis follows a simple Michaelian kinetics. β-cyclodextrin binding at surface sites inhibits only the fast reaction in amylopectin degradation. Site-directed mutagenesis and activity analysis, furthermore show that one of the surface binding sites as well as individual subsites in the active site cleft have distinct roles in the multiple attack on amylose. Although the two isozymes AMY1 and AMY2 share ligands for three structural calcium ions, they differ importantly in the effect of calcium on activity and stability, AMY1 having the higher affinity and the lower stability. The role of the individual calcium ions is studied by mutagenesis, crystallography and microcalorimetry. Further improvement of recombinant AMY2 production allows future direct mutational analysis in this isozyme. Specific proteinaceous inhibitors act on α-amylases of different origin. In the complex of barley α-amylase subtilisin inhibitor (BASI) with AMY2, a fully hydrated calcium ion at the protein interface mediates contact between inhibitor residues and the enzyme catalytic groups in a manner that depends on calcium and which can be suppressed by site-directed mutagenesis of Glu168 in BASI. Finally certain inhibitors and enzymes are targets of the disulphide reductase thioredoxin h that attacks a specific disulphide bond in BASI and, remarkably, reduces two different disulphide bonds in the barley monomeric and dimeri...

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Kinetics and Energetics of the Binding between Barley α-Amylase/Subtilisin Inhibitor and Barley α-Amylase 2 Analyzed by Surface Plasmon Resonance and Isothermal Titration Calorimetry

Cited by 46 publications

References 55 publications

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

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

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

Interactions between Barley .ALPHA.-Amylases, Substrates, Inhibitors and Regulatory Proteins

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