title: Interplay of catalytic subsite residues in the positioning of α-d-glucose 1-phosphate in sucrose phosphorylase.
abstract: Kinetic and molecular docking studies were performed to characterize the binding of α-d-glucose 1-phosphate (αGlc 1-P) at the catalytic subsite of a family GH-13 sucrose phosphorylase (fromL. mesenteroides) in wild-type and mutated form. The best-fit binding mode of αGlc 1-Pdianion had the phosphate group placedantirelative to the glucosyl moiety (adopting a relaxed4C1chair conformation) and was stabilized mainly by hydrogen bonds from residues of the enzyme׳s catalytic triad (Asp196, Glu237and Asp295) and from Arg137. Additional feature of the αGlc 1-Pdocking pose was an intramolecular hydrogen bond (2.7 Å) between the glucosyl C2-hydroxyl and the phosphate oxygen. An inactive phosphonate analog of αGlc 1-Pdid not show binding to sucrose phosphorylase in different experimental assays (saturation transfer difference NMR, steady-state reversible inhibition), consistent with evidence from molecular docking study that also suggested a completely different and strongly disfavored binding mode of the analog as compared to αGlc 1-P. Molecular docking results also support kinetic data in showing that mutation of Phe52, a key residue at the catalytic subsite involved in transition state stabilization, had little effect on the ground-state binding of αGlc 1-Pby the phosphorylase. However, when combined with a second mutation involving one of the catalytic triad residues, the mutation of Phe52by Ala caused complete (F52A_D196A; F52A_E237A) or very large (F52A_D295A) disruption of the proposed productive binding mode of αGlc 1-Pwith consequent effects on the enzyme activity. Effects of positioning of αGlc 1-Pfor efficient glucosyl transfer from phosphate to the catalytic nucleophile of the enzyme (Asp196) are suggested. High similarity between the αGlc 1-Pconformers bound to sucrose phosphorylase (modeled) and the structurally and mechanistically unrelated maltodextrin phosphorylase (experimental) is revealed.
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, A-8010 Graz, Austria.
authors.forename: Patricia
authors.initials: P
authors.lastname: Wildberger
authors.affiliation: College of Pharmacy, Dalhousie University, PO Box 15,000, 5968 College Street, Halifax, Nova Scotia, Canada B3H 4R2.
authors.forename: Gaia A
authors.initials: GA
authors.lastname: Aish
authors.affiliation: College of Pharmacy, Dalhousie University, PO Box 15,000, 5968 College Street, Halifax, Nova Scotia, Canada B3H 4R2.
authors.forename: David L
authors.initials: DL
authors.lastname: Jakeman
authors.affiliation: Institute of Organic Chemistry, University of Vienna, Währingerstraße 38, A-1090 Vienna, Austria.
authors.forename: Lothar
authors.initials: L
authors.lastname: Brecker
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, A-8010 Graz, Austria ; Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010 Graz, Austria.
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
keywords: Binding recognition; Catalytic subsite; Glycoside hydrolase family GH-13; LmSPase, SPase from Leuconostoc mesenteroides; Molecular docking; SPase, sucrose phosphorylase; Sucrose phosphorylase; α-d-Glucose 1-phosphate; αGlc 1-P, α-d-glucose 1-phosphate