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Type: Journal article
Title: Catalytic mechanisms and reaction intermediates along the hydrolytic pathway of a plant beta-D-glucan glucohydrolase
Author: Hrmova, M.
Varghese, J.
De Gori, R.
Smith, B.
Driguez, H.
Fincher, G.
Citation: Structure, 2001; 9(11):1005-1016
Publisher: Cell Press
Issue Date: 2001
ISSN: 0969-2126
Statement of
Maria Hrmova, Joseph N. Varghese, Ross De Gori, Brian J. Smith, Hugues Driguez and Geoffrey B. Fincher
Abstract: Background: Barley β-D-glucan glucohydrolases represent family 3 glycoside hydrolases that catalyze the hydrolytic removal of nonreducing glucosyl residues from β-D-glucans and β-D-glucooligosaccharides. After hydrolysis is completed, glucose remains bound in the active site. Results: When conduritol B epoxide and 2′, 4′-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucopyranoside are diffused into enzyme crystals, they displace the bound glucose and form covalent glycosyl-enzyme complexes through the Oδ1 of D285, which is thereby identified as the catalytic nucleophile. A nonhydrolyzable S-glycosyl analog, 4I, 4III, 4V-S-trithiocellohexaose, also diffuses into the active site, and a S-cellobioside moiety positions itself at the −1 and +1 subsites. The glycosidic S atom of the S-cellobioside moiety forms a short contact (2.75 Å) with the Oε2 of E491, which is likely to be the catalytic acid/base. The glucopyranosyl residues of the S-cellobioside moiety are not distorted from the low-energy 4C1 conformation, but the glucopyranosyl ring at the +1 subsite is rotated and translated about the linkage. Conclusions: X-ray crystallography is used to define the three key intermediates during catalysis by β-D-glucan glucohydrolase. Before a new hydrolytic event begins, the bound product (glucose) from the previous catalytic reaction is displaced by the incoming substrate, and a new enzyme-substrate complex is formed. The second stage of the hydrolytic pathway involves glycosidic bond cleavage, which proceeds through a double-displacement reaction mechanism. The crystallographic analysis of the S-cellobioside-enzyme complex with quantum mechanical modeling suggests that the complex might mimic the oxonium intermediate rather than the enzyme-substrate complex. Author Keywords: catalytic acid/base; catalytic nucleophile; enzyme kinetics; family 3 glycoside hydrolases; mechanism-based inhibitors; S-glycosyl substrate analog.
Keywords: catalytic acid/base
catalytic nucleophile
enzyme kinetics
family 3 glycoside hydrolases
mechanism-based inhibitors
S-glycosyl substrate analog
DOI: 10.1016/S0969-2126(01)00673-6
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