Degradation of the starch components amylopectin and amylose by barley α-amylase 1: Role of surface binding site 2

Publication: Research - peer-reviewJournal article – Annual report year: 2012

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Degradation of the starch components amylopectin and amylose by barley α-amylase 1: Role of surface binding site 2. / Nielsen, Jonas Willum; Kramhøft, Birte; Bozonnet, Sophie; Abou Hachem, Maher ; Stipp, Susan Louise Svane; Svensson, Birte; Willemoes, Martin.

In: Archives of Biochemistry and Biophysics, Vol. 528, No. 1, 2012, p. 1-6.

Publication: Research - peer-reviewJournal article – Annual report year: 2012

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Nielsen, Jonas Willum; Kramhøft, Birte; Bozonnet, Sophie; Abou Hachem, Maher ; Stipp, Susan Louise Svane; Svensson, Birte; Willemoes, Martin / Degradation of the starch components amylopectin and amylose by barley α-amylase 1: Role of surface binding site 2.

In: Archives of Biochemistry and Biophysics, Vol. 528, No. 1, 2012, p. 1-6.

Publication: Research - peer-reviewJournal article – Annual report year: 2012

Bibtex

@article{ef42b17cb85f41449ea97708d9ab8e10,
title = "Degradation of the starch components amylopectin and amylose by barley α-amylase 1: Role of surface binding site 2",
keywords = "Starch binding, Secondary carbohydrate binding sites, Bi-exponential progress curves, Carbohydrate degradation, Complex substrate enzyme kinetics",
publisher = "Academic Press",
author = "Nielsen, {Jonas Willum} and Birte Kramhøft and Sophie Bozonnet and {Abou Hachem}, Maher and Stipp, {Susan Louise Svane} and Birte Svensson and Martin Willemoes",
year = "2012",
doi = "10.1016/j.abb.2012.08.005",
volume = "528",
number = "1",
pages = "1--6",
journal = "Archives of Biochemistry and Biophysics",
issn = "0003-9861",

}

RIS

TY - JOUR

T1 - Degradation of the starch components amylopectin and amylose by barley α-amylase 1: Role of surface binding site 2

A1 - Nielsen,Jonas Willum

A1 - Kramhøft,Birte

A1 - Bozonnet,Sophie

A1 - Abou Hachem,Maher

A1 - Stipp,Susan Louise Svane

A1 - Svensson,Birte

A1 - Willemoes,Martin

AU - Nielsen,Jonas Willum

AU - Kramhøft,Birte

AU - Bozonnet,Sophie

AU - Abou Hachem,Maher

AU - Stipp,Susan Louise Svane

AU - Svensson,Birte

AU - Willemoes,Martin

PB - Academic Press

PY - 2012

Y1 - 2012

N2 - Barley α-amylase isozyme 1 (AMY1, EC 3.2.1.1) contains two surface binding sites, SBS1 and SBS2, involved in the degradation of starch granules. The distinct role of SBS1 and SBS2 remains to be fully understood. Mutational analysis of Tyr-380 situated at SBS2 previously revealed that Tyr-380 is required for binding of the amylose helix mimic, β-cyclodextrin. Also, mutant enzymes altered at position 380 displayed reduced binding to starch granules. Similarly, binding of wild type AMY1 to starch granules was suppressed in the presence of β-cyclodextrin. We investigated the role of SBS2 by comparing kinetic properties of the wild type AMY1 and the Y380A mutant enzyme in hydrolysis of amylopectin, amylose and β-limit dextrin, and the inhibition by β-cyclodextrin. Progress curves of the release of reducing ends revealed a bi-exponential hydrolysis of amylopectin and β-limit dextrin, whereas hydrolysis of amylose progressed mono-exponentially. β-Cyclodextrin, however, inhibited only one of the two reaction rates of amylopectin and β-limit dextrin hydrolysis, whereas hydrolysis of amylose was unaffected. The Y380A enzyme showed no detectable inhibition by β-cyclodextrin but displayed similar kinetics to the inhibited wild type AMY1. These results point to SBS2 as an important binding site in amylopectin depolymerization.

AB - Barley α-amylase isozyme 1 (AMY1, EC 3.2.1.1) contains two surface binding sites, SBS1 and SBS2, involved in the degradation of starch granules. The distinct role of SBS1 and SBS2 remains to be fully understood. Mutational analysis of Tyr-380 situated at SBS2 previously revealed that Tyr-380 is required for binding of the amylose helix mimic, β-cyclodextrin. Also, mutant enzymes altered at position 380 displayed reduced binding to starch granules. Similarly, binding of wild type AMY1 to starch granules was suppressed in the presence of β-cyclodextrin. We investigated the role of SBS2 by comparing kinetic properties of the wild type AMY1 and the Y380A mutant enzyme in hydrolysis of amylopectin, amylose and β-limit dextrin, and the inhibition by β-cyclodextrin. Progress curves of the release of reducing ends revealed a bi-exponential hydrolysis of amylopectin and β-limit dextrin, whereas hydrolysis of amylose progressed mono-exponentially. β-Cyclodextrin, however, inhibited only one of the two reaction rates of amylopectin and β-limit dextrin hydrolysis, whereas hydrolysis of amylose was unaffected. The Y380A enzyme showed no detectable inhibition by β-cyclodextrin but displayed similar kinetics to the inhibited wild type AMY1. These results point to SBS2 as an important binding site in amylopectin depolymerization.

KW - Starch binding

KW - Secondary carbohydrate binding sites

KW - Bi-exponential progress curves

KW - Carbohydrate degradation

KW - Complex substrate enzyme kinetics

U2 - 10.1016/j.abb.2012.08.005

DO - 10.1016/j.abb.2012.08.005

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 1

VL - 528

SP - 1

EP - 6

ER -