Browsing by Author "Chen, FY"

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Catalytic mechanism of a family 3 beta-glucosidase and mutagenesis study on residue Asp-247
(2001-05-01)
Li, YK
;
Chir, J
;
Chen, FY
;
應用化學系
;
Department of Applied Chemistry
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.
Identification of the general acid/base catalyst of a family 3 beta-glucosidase from Flavobacterium meningosepticum
(2002-02-26)
Li, YK
;
Chir, J
;
Tanaka, S
;
Chen, FY
;
應用化學系
beta-Glucosidase from Flavobacterium meningosepticum (Fbgl) (also known as Chryseobacterium meningosepticum) has been classified as a member of the family 3 glycohydrolases. It is a retaining enzyme involving a two-step, double-displacement mechanism. 0247 was shown to function as the nucleophile of the enzymatic reaction [Li, Y.-K., Chir, J., and Chen, F.-Y. (2001) Biochem. J. 355, 835-840]. However, the general acid/base catalyst of this enzyme and of all other family 3 glycohydrolases has not yet been identified. On the basis of amino acid sequence alignment of 15 family 3 enzymes, 11 residues (071, 8129, E132, E136, D137, K168, H169, E177, D247, D458, and E473) are highly conserved. All of these residues are studied by site-directed mutagenesis and kinetic investigation. Analyzing the catalytic power of all mutants reveals E473 residue is the best candidate of the acid/base catalyst. Detailed studies supporting this suggestion are summarized as follows. (1) The k(cat) and K-m values for the hydrolysis of 2,4-dinitrophenyl beta-D-glucopyranoside (2,4-DNPG) by E473G are reduced 3300- and 900-fold, respectively, compared with those of the wild type (WT). (2) The k(cat) values of E473G-catalyzed hydrolysis are virtually invariant with pH over the range of 5.0-9.0. (3) The activity of E473G with 2,4-DNPG is enhanced by the addition of azide, and beta-glucosyl azide is formed. (4) The k(cat) of the reaction of 2-carboxyphenyl beta-glucoside catalyzed by E473G is comparable to that for hydrolysis by wild-type Fbgl and is 100- and 320-fold better than the k(cat) values for the E473G-catalyzed hydrolysis of 4-carboxyphenyl beta-glucoside and the corresponding methyl ester, respectively. (5) The accumulated glucosyl-enzyme intermediate was directly observed by mass analysis in the reaction of 2,4-DNPG with E473G. All of these results confirm that E473 is the general acid/base catalyst of Fbgl.