Identification of the general acid/base catalyst of a family 3 beta-glucosidase from Flavobacterium meningosepticum.
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. D247 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 (D71, R129, 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.
Li
Yaw-Kuen
YK
Department of Applied Chemistry, National Chiao Tung University, Hsin-Chu, Taiwan 30050. ykl@cc.nctu.edu.tw
Chir
Jiunly
J
Tanaka
Shigeo
S
Chen
Fong-Yi
FY
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D005417:Flavobacterium / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D001617:beta-Glucosidase