TY - JOUR

T1 - Kinetic analysis of inhibition of glucoamylase and active site mutants via chemoselective oxime immobilization of acarbose on SPR chip surfaces

AU - Sauer, Jørgen

AU - Abou Hachem, Maher

AU - Svensson, Birte

AU - Jensen, Knud Jørgen

AU - Thygesen, Mikkel Boas

N1 - Copyright © 2013 Elsevier Ltd. All rights reserved.

PY - 2013

Y1 - 2013

N2 - We here report a quantitative study on the binding kinetics of inhibition of the enzyme glucoamylase and how individual active site amino acid mutations influence kinetics. To address this challenge, we have developed a fast and efficient method for anchoring native acarbose to gold chip surfaces for surface plasmon resonance studies employing wild type glucoamylase and active site mutants, Y175F, E180Q, and R54L, as analytes. The key method was the chemoselective and protecting group-free oxime functionalization of the pseudo-tetrasaccharide-based inhibitor acarbose. By using this technique we have shown that at pH 7.0 the association and dissociation rate constants for the acarbose-glucoamylase interaction are 10(4)M(-1)s(-1) and 10(3)s(-1), respectively, and that the conformational change to a tight enzyme-inhibitor complex affects the dissociation rate constant by a factor of 10(2)s(-1). Additionally, the acarbose-presenting SPR surfaces could be used as a glucoamylase sensor that allowed rapid, label-free affinity screening of small carbohydrate-based inhibitors in solution, which is otherwise difficult with immobilized enzymes or other proteins.

AB - We here report a quantitative study on the binding kinetics of inhibition of the enzyme glucoamylase and how individual active site amino acid mutations influence kinetics. To address this challenge, we have developed a fast and efficient method for anchoring native acarbose to gold chip surfaces for surface plasmon resonance studies employing wild type glucoamylase and active site mutants, Y175F, E180Q, and R54L, as analytes. The key method was the chemoselective and protecting group-free oxime functionalization of the pseudo-tetrasaccharide-based inhibitor acarbose. By using this technique we have shown that at pH 7.0 the association and dissociation rate constants for the acarbose-glucoamylase interaction are 10(4)M(-1)s(-1) and 10(3)s(-1), respectively, and that the conformational change to a tight enzyme-inhibitor complex affects the dissociation rate constant by a factor of 10(2)s(-1). Additionally, the acarbose-presenting SPR surfaces could be used as a glucoamylase sensor that allowed rapid, label-free affinity screening of small carbohydrate-based inhibitors in solution, which is otherwise difficult with immobilized enzymes or other proteins.

KW - Acarbose

KW - Catalytic Domain

KW - Dose-Response Relationship, Drug

KW - Enzyme Inhibitors

KW - Glucan 1,4-alpha-Glucosidase

KW - Gold

KW - Kinetics

KW - Models, Molecular

KW - Molecular Structure

KW - Mutation

KW - Oximes

KW - Structure-Activity Relationship

KW - Surface Plasmon Resonance

KW - Surface Properties

U2 - 10.1016/j.carres.2013.04.012

DO - 10.1016/j.carres.2013.04.012

M3 - Journal article

C2 - 23680647

VL - 375

SP - 21

EP - 28

JO - Carbohydrate Research

JF - Carbohydrate Research

SN - 0008-6215

ER -