Interplay of catalytic subsite residues in the positioning of α-d-glucose 1-phosphate in sucrose phosphorylase.

Kinetic and molecular docking studies were performed to characterize the binding of α-d-glucose 1-phosphate (αGlc 1-) at the catalytic subsite of a family GH-13 sucrose phosphorylase (from ) in wild-type and mutated form. The best-fit binding mode of αGlc 1- dianion had the phosphate group placed relative to the glucosyl moiety (adopting a relaxed chair conformation) and was stabilized mainly by hydrogen bonds from residues of the enzyme׳s catalytic triad (Asp, Glu and Asp) and from Arg. Additional feature of the αGlc 1- docking 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- did 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-. Molecular docking results also support kinetic data in showing that mutation of Phe, a key residue at the catalytic subsite involved in transition state stabilization, had little effect on the ground-state binding of αGlc 1- by the phosphorylase. However, when combined with a second mutation involving one of the catalytic triad residues, the mutation of Phe by Ala caused complete (F52A_D196A; F52A_E237A) or very large (F52A_D295A) disruption of the proposed productive binding mode of αGlc 1- with consequent effects on the enzyme activity. Effects of positioning of αGlc 1- for efficient glucosyl transfer from phosphate to the catalytic nucleophile of the enzyme (Asp) are suggested. High similarity between the αGlc 1- conformers bound to sucrose phosphorylase (modeled) and the structurally and mechanistically unrelated maltodextrin phosphorylase (experimental) is revealed.