Aromatic interactions at the catalytic subsite of sucrose phosphorylase: Their roles in enzymatic glucosyl transfer probed with Phe⁵² → Ala and Phe⁵² → Asn mutants

Abstract

a b s t r a c tMutants 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 (P3.5 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-p stabilization of the oxocarbenium ion-like transition states flanking the covalent enzyme intermediate.

Probing enzyme–substrate interactions at the catalytic subsite ofLeuconostoc mesenteroidessucrose phosphorylase with site-directed mutagenesis: the roles of Asp49and Arg395

“…Compared to the wild-type enzyme whose specifi c activity was 98 U/mg under the conditions used (Wildberger et al 2011), the R395L mutant was 650-fold less active. The specifi c activity of D49A was even lower than that of R395L, refl ecting a 50,000-fold activity loss in comparison to the specifi c activity of wild-type Lm SPase.…”

“…The results are summarised in Table I along with the relevant kinetic data for the wild-type enzyme, which were taken from one of our recent papers (Wildberger et al 2011). Interpretation is done on the basis of the Ping-pong bi-bi kinetic mechanism of wild-type Lm SPase (Goedl & Nidetzky 2009).…”

“…Point mutations Asp 49 ® Ala (D49A) and Arg 395 ® Leu (R395L) were introduced using a reported twostage PCR protocol (Wang & Malcolm 1999) in which a pASK-IBA7 ϩ (IBA GmbH) expression vector encoding wild-type Lm SPase was used as template (Wildberger et al 2011). The following pairs of oligonucleotide primers were used to amplify the entire plasmid with Pfu DNA polymerase for D49A and Phusion High-Fidelity DNA polymerase for R395L, respectively, whereby the mismatched codons are indicated in bold.…”

“…Sequenced plasmid vectors harbouring the mutagenised gene were transformed into Escherichia coli BL21-Gold (DE3) cells, and recipient strains were grown at 37 ° C in Lysogeny broth (LB)-medium containing 0.15 mg/L ampicillin. Induction of gene expression and recombinant protein production was carried out as reported elsewhere (Wildberger et al 2011).…”

“…for K i was equal to or smaller than 18.7%. a Sucrose was varied at 14 levels between 0.01 and 120 mM for the wild-type enzyme (Wildberger et al 2011). For D49A, sucrose was varied at 8 levels between 50 and 800 mM.…”