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Dissecting differential binding of fructose and phosphate as leaving group/nucleophile of glucosyl transfer catalyzed by sucrose phosphorylase
Author(s) -
Mueller Mario,
Nidetzky Bernd
Publication year - 2007
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2007.07.004
Subject(s) - chemistry , leuconostoc mesenteroides , fructose , sucrose , enzyme , biochemistry , leaving group , phosphate , nucleophile , glycogen phosphorylase , stereochemistry , catalysis , biology , lactic acid , genetics , bacteria
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.