Catalytic mechanism of a family 3 β-glucosidase and mutagenesis study on residue Asp-247

Yaw-Kuen Li*, Jiunly Chir, Fong Yi Chen

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

36 Scopus citations

Abstract

A family 3 β-glucosidase (EC 3.2.1.21) from Flavobacterium meningosepticum has been cloned and overexpressed. The mechanistic action of the enzyme was probed by NMR spectroscopy and kinetic investigations, including substrate reactivity, secondary kinetic isotope effects and inhibition studies. The stereo-chemistry of enzymic hydrolysis was identified as occurring with the retention of an anomeric configuration, indicating a double-displacement reaction. Based on the kcat values with a series of aryl glucosides, a Bronsted plot with a concave-downward shape was constructed. This biphasic behaviour is consistent with a two-step mechanism involving the formation and breakdown of a glucosyl - enzyme intermediate. The large Bronsted constant (β = -0.85) for the leaving-group-dependent portion (pKa of leaving phenols > 7) indicates substantial bond cleavage at the transition state. Secondary deuterium kinetic isotope effects with 2,4-dinitrophenyl β-D-glucopyanoside, o-nitrophenyl β-D-glucopyanoside and g-cyanophenyl β-D-glucopyanoside as substrates were 1.17±0.02, 1.19±0.02 and 1.04±0.02 respectively. These results support an SN1-like mechanism for the deglucosylation step and an SN2-like mechanism for the glucosylation step. Site-directed mutagenesis was also performed to study essential amino acid residues. The activities (kcat/Km) of the D247G and D247N mutants were 30000- and 200000-fold lower respectively than that of the wild-type enzyme, whereas the D247E mutant retained 20 % of wild-type activity. These results indicate that Asp-247 is an essential amino acid. It is likely that this residue functions as a nucleophile in the reaction. This conclusion is supported by the kinetics of the irreversible inactivation of the wild-type enzyme by conduritol-B-epoxide, compared with the much slower inhibition of the D247E mutant and the lack of irreversible inhibition of the D247G mutant.

Original languageEnglish
Pages (from-to)835-840
Number of pages6
JournalBiochemical Journal
Volume355
Issue number3
DOIs
StatePublished - 24 Apr 2001

Keywords

  • Bronsted plot
  • Family 3 glycohydrolase
  • Flavobacterium meningosepticum
  • Secondary isotope effect
  • Site-directed mutagenesis

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