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

• Anchoring of acarbose in situ in the flow cells of SPR instrument by oxime formation.
• Acquisition of binding kinetics for starch-processing enzyme glucoamylase and mutants.
• E180 implicated in conformational change causes 100-fold increase in kd upon mutation.

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 104 M−1 s−1 and 103 s−1, respectively, and that the conformational change to a tight enzyme–inhibitor complex affects the dissociation rate constant by a factor of 102 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.

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