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Aromatic interactions at the catalytic subsite of sucrose phosphorylase: Their roles in enzymatic glucosyl transfer probed with Phe 52 → Ala and Phe 52 → Asn mutants
Author(s) -
Wildberger Patricia,
Luley-Goedl Christiane,
Nidetzky Bernd
Publication year - 2011
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.2010.12.041
Subject(s) - chemistry , oxocarbenium , leuconostoc mesenteroides , glycogen phosphorylase , covalent bond , stereochemistry , enzyme , catalysis , fructose , biochemistry , organic chemistry , lactic acid , biology , bacteria , nucleophile , genetics
Mutants of Leuconostoc mesenteroides sucrose phosphorylase having active‐site Phe 52 replaced by Ala (F52A) or Asn (F52N) were characterized by free energy profile analysis for catalytic glucosyl transfer from sucrose to phosphate. Despite large destabilization (⩾3.5 kcal/mol) of the transition states for enzyme glucosylation and deglucosylation in both mutants as compared to wild‐type, the relative stability of the glucosyl enzyme intermediate was weakly affected by substitution of Phe 52 . In reverse reaction where fructose becomes glucocylated, “error hydrolysis” was the preponderant path of breakdown of the covalent intermediate of F52A and F52N. It is proposed, therefore, that Phe 52 facilitates reaction of the phosphorylase through (1) positioning of the transferred glucosyl moiety at the catalytic subsite and (2) strong cation‐π stabilization of the oxocarbenium ion‐like transition states flanking the covalent enzyme intermediate.