The role of Asp-295 in the catalytic mechanism of Leuconostoc mesenteroides sucrose phosphorylase probed with site-directed mutagenesis

Replacements of Asp-295 by Asn (D295N) and Glu (D295E) decreased the catalytic center activity of Leuconostoc mesenteroides sucrose phosphorylase to about 0.01% of the wild-type level (kcat = 200 s−1). Glucosylation and deglucosylation steps of D295N were affected uniformly, ∼104.3-fold, and independently of leaving group ability and nucleophilic reactivity of the substrate, respectively. pH dependences of the catalytic steps were similar for D295N and wild-type. The 105-fold preference of the wild-type for glucosyl transfer compared with mannosyl transfer from phosphate to fructose was lost in D295N and D295E. Selective disruption of catalysis to glucosyl but not mannosyl transfer in the two mutants suggests that the side chain of Asp-295, through a strong hydrogen bond with the equatorial sugar 2-hydroxyl, stabilizes the transition states flanking the β-glucosyl enzyme intermediate by ⩾23 kJ/mol.