Aromatic interactions at the catalytic subsite of sucrose phosphorylase: Their roles in enzymatic glucosyl transfer probed with Phe → Ala and Phe → Asn mutants

Mutants of Leuconostoc mesenteroides sucrose phosphorylase having active-site Phe⁵² 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 Phe⁵². 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 Phe⁵² 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.