標題: Catalytic mechanism of a family 3 beta-glucosidase and mutagenesis study on residue Asp-247
作者: Li, YK
Chir, J
Chen, FY
應用化學系
Department of Applied Chemistry
關鍵字: Bronsted plot;family 3 glycohydrolase;Flavobacterium meningosepticum;secondary isotope effect;site-directed mutagenesis
公開日期: 1-五月-2001
摘要: A family 3 beta -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 stereochemistry of enzymic hydrolysis was identified as occurring with the retention of an anomeric configuration, indicating a double-displacement reaction. Based on the k(cat) 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 (beta = -0.85) for the leaving-group-dependent portion (pK(a) of leaving phenols > 7) indicates substantial bond cleavage at the transition slate. Secondary deuterium kinetic isotope effects with 2,4-dinitrophenyl beta -D-glucopyanoside, o-nitrophenyl beta -3-D-glucopyranoside and p-cyanophenyl beta -D-glucopyanoside as substrates were 1.17 +/- 0.02, 1.19 +/- 0.02 and 1.04 +/- 0.02 respectively. Theseresults support an S(N)1-like mechanism for the deglucosylation step and an S(N)2-like mechanism for the glucosylation step. Site-directed mutagenesis was also performed to study essential amino acid residues. The activities (k(cat)/K-m) 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.
URI: http://hdl.handle.net/11536/29671
ISSN: 0264-6021
期刊: BIOCHEMICAL JOURNAL
Volume: 355
Issue: 
起始頁: 835
結束頁: 840
顯示於類別:期刊論文


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  1. 000168636200034.pdf