Dissecting differential binding of fructose and phosphate as leaving group/nucleophile of glucosyl transfer catalyzed by sucrose phosphorylase

Site‐directed mutagenesis was used to examine the specificity of Leuconostoc mesenteroides sucrose phosphorylase for utilization of fructose and phosphate as leaving group/nucleophile of the reaction. The largest catalytic defect in Arg 137 → Ala (≈60‐fold) and Tyr 340 → Ala (≈2500‐fold) concerned phosphate dependant half‐reactions whereas that in Asp 338 → Asn (≈7000‐fold) derived from disruption of steps where fructose departs or attacks. The relative efficiencies for enzyme glucosylation by sucrose compared with α‐ d ‐glucose‐1‐phosphate and enzyme deglucosylation by phosphate compared with fructose were 5.5 and 6.2 for wild‐type, 19 and 2.0 for Arg 137 → Ala, 950 and 0.17 for Tyr 340 → Ala, and 0.05 and 180 for Asp 338 → Asn, respectively. Asp 338 and Tyr 340 have a key role in differential binding of fructose and phosphate, respectively.