title: Kinetic analysis of inhibition of glucoamylase and active site mutants via chemoselective oxime immobilization of acarbose on SPR chip surfaces.
abstract: 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.
authors.affiliation: Centre for Carbohydrate Recognition and Signalling, Department of Chemistry, Faculty of Science, University of Copenhagen, Frederiksberg C, Denmark.
authors.forename: Jørgen
authors.initials: J
authors.lastname: Sauer
authors.forename: Maher
authors.initials: M
authors.lastname: Abou Hachem
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
authors.forename: Knud J
authors.initials: KJ
authors.lastname: Jensen
authors.forename: Mikkel B
authors.initials: MB
authors.lastname: Thygesen
mesh_terms: D020909:Acarbose / Q000737:chemistry* / Q000494:pharmacology*; D020134:Catalytic Domain / Q000187:drug effects / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D005087:Glucan 1,4-alpha-Glucosidase / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D006046:Gold / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D010091:Oximes / Q000737:chemistry*; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D013499:Surface Properties
chemical_list: D004791:Enzyme Inhibitors; D010091:Oximes; D006046:Gold; D005087:Glucan 1,4-alpha-Glucosidase; D020909:Acarbose