Interactions between Barley a-Amylases, Substrates, Inhibitors and Regulatory Proteins

Maher Abou Hachem; Sophie Bozonnet; Martin Willemoës; Birgit C. Bønsager; Morten Munch Nielsen; Kenji Fukuda; Birte Kramhøft; Kenji Maeda; Bent Walther Sigurskjold; Per Hägglund; Christine Finnie; Haruhide Mori; Xavier Robert; Malene H. Jensen; Samuel Tranier; Nushin Aghajari; Richard Haser; Birte Svensson

Interactions between Barley a-Amylases, Substrates, Inhibitors and Regulatory Proteins

Maher Abou Hachem, Sophie Bozonnet, Martin Willemoës, Birgit C. Bønsager, Morten Munch Nielsen, Kenji Fukuda, Birte Kramhøft, Kenji Maeda, Bent Walther Sigurskjold, Per Hägglund, Christine Finnie, Haruhide Mori, Xavier Robert, Malene H. Jensen, Samuel Tranier, Nushin Aghajari, Richard Haser, Birte Svensson

Abstract

Barley a-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 K_d values determined for ß-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. ß-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 a-amylases of different origin. In the complex of barley a-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 dimeric amylase inhibitors that both belong to the CM-proteins and inhibit animal a-amylase.

Original language	English
Journal	Journal of Applied Glycoscience
Volume	53
Issue number	2
Pages (from-to)	163-169
ISSN	1344-7882
Publication status	Published - 2006

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Hachem, M. A., Bozonnet, S., Willemoës, M., Bønsager, B. C., Nielsen, M. M., Fukuda, K., Kramhøft, B., Maeda, K., Sigurskjold, B. W., Hägglund, P., Finnie, C., Mori, H., Robert, X., Jensen, M. H., Tranier, S., Aghajari, N., Haser, R., & Svensson, B. (2006). Interactions between Barley a-Amylases, Substrates, Inhibitors and Regulatory Proteins. Journal of Applied Glycoscience, 53(2), 163-169. http://www.jstage.jst.go.jp/article/jag/53/2/163/_pdf

Hachem, MA, Bozonnet, S, Willemoës, M, Bønsager, BC, Nielsen, MM, Fukuda, K, Kramhøft, B, Maeda, K, Sigurskjold, BW, Hägglund, P, Finnie, C, Mori, H, Robert, X, Jensen, MH, Tranier, S, Aghajari, N, Haser, R & Svensson, B 2006, 'Interactions between Barley a-Amylases, Substrates, Inhibitors and Regulatory Proteins', Journal of Applied Glycoscience, vol. 53, no. 2, pp. 163-169. <http://www.jstage.jst.go.jp/article/jag/53/2/163/_pdf>

@article{a2550a006f8111de8bc9000ea68e967b,

title = "Interactions between Barley a-Amylases, Substrates, Inhibitors and Regulatory Proteins",

abstract = "Barley a-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 {\ss}-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. {\ss}-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 a-amylases of different origin. In the complex of barley a-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 dimeric amylase inhibitors that both belong to the CM-proteins and inhibit animal a-amylase.",

author = "Hachem, {Maher Abou} and Sophie Bozonnet and Martin Willemo{\"e}s and B{\o}nsager, {Birgit C.} and Nielsen, {Morten Munch} and Kenji Fukuda and Birte Kramh{\o}ft and Kenji Maeda and Sigurskjold, {Bent Walther} and Per H{\"a}gglund and Christine Finnie and Haruhide Mori and Xavier Robert and Jensen, {Malene H.} and Samuel Tranier and Nushin Aghajari and Richard Haser and Birte Svensson",

note = "Key words: glycoside hydrolase family13, secondary binding sites, surface plasmon resonance, calcium effects, regulatory proteins",

year = "2006",

language = "English",

volume = "53",

pages = "163--169",

journal = "Journal of Applied Glycoscience",

issn = "1344-7882",

publisher = "Japanese Society of Applied Glycoscience",

number = "2",

}

TY - JOUR

T1 - Interactions between Barley a-Amylases, Substrates, Inhibitors and Regulatory Proteins

AU - Hachem, Maher Abou

AU - Bozonnet, Sophie

AU - Willemoës, Martin

AU - Bønsager, Birgit C.

AU - Nielsen, Morten Munch

AU - Fukuda, Kenji

AU - Kramhøft, Birte

AU - Maeda, Kenji

AU - Sigurskjold, Bent Walther

AU - Hägglund, Per

AU - Finnie, Christine

AU - Mori, Haruhide

AU - Robert, Xavier

AU - Jensen, Malene H.

AU - Tranier, Samuel

AU - Aghajari, Nushin

AU - Haser, Richard

AU - Svensson, Birte

N1 - Key words: glycoside hydrolase family13, secondary binding sites, surface plasmon resonance, calcium effects, regulatory proteins

PY - 2006

Y1 - 2006

N2 - Barley a-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 ß-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. ß-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 a-amylases of different origin. In the complex of barley a-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 dimeric amylase inhibitors that both belong to the CM-proteins and inhibit animal a-amylase.

AB - Barley a-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 ß-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. ß-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 a-amylases of different origin. In the complex of barley a-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 dimeric amylase inhibitors that both belong to the CM-proteins and inhibit animal a-amylase.

M3 - Journal article

SN - 1344-7882

VL - 53

SP - 163

EP - 169

JO - Journal of Applied Glycoscience

JF - Journal of Applied Glycoscience

IS - 2

ER -

Interactions between Barley a-Amylases, Substrates, Inhibitors and Regulatory Proteins

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