title: Aromatic interactions at the catalytic subsite of sucrose phosphorylase: their roles in enzymatic glucosyl transfer probed with Phe52→Ala and Phe52→Asn mutants.
abstract: 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 (≥3.5kcal/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.
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Graz, Austria.
authors.forename: Patricia
authors.initials: P
authors.lastname: Wildberger
authors.forename: Christiane
authors.initials: C
authors.lastname: Luley-Goedl
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D005947:Glucose / Q000031:analogs & derivatives / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; C030364:glucosyl fluoride; D010649:Phenylalanine; D005964:Glucosyltransferases; C025901:sucrose phosphorylase; D005947:Glucose