Journal article
Kinetic analysis of inhibition of glucoamylase and active site mutants via chemoselective oxime immobilization of acarbose on SPR chip surfaces
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 104M−1s−1 and 103s−1, respectively, and that the conformational change to a tight enzyme–inhibitor complex affects the dissociation rate constant by a factor of 102s−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.
Language: | English |
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Year: | 2013 |
Pages: | 21-28 |
ISSN: | 1873426x and 00086215 |
Types: | Journal article |
DOI: | 10.1016/j.carres.2013.04.012 |
ORCIDs: | Abou Hachem, Maher , Svensson, Birte , 0000-0003-3525-5452 and 0000-0002-0158-2802 |