Catalytic mechanisms and reaction intermediates along the hydrolytic pathway of a plant β-D-glucan glucohydrolase

Background: Barley beta -D-glucan glucohydrolases represent family 3 glycoside hydrolases that catalyze the hydrolytic removal of nonreducing glucosyl residues from beta -D-glucans and beta -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-beta -D-glucopyranoside are diffused into enzyme crystals, they displace the bound glucose and form covalent glycosyl-enzyme complexes through the O delta1 of D285, which is thereby identified as the catalytic nucleophile. A nonhydrolyzable S-glycosyl analog, 4(I),4(III),4(V)-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 Angstrom) with the O epsilon2 of E491, which is likely to be the catalytic acid/base. The glucopyranosyl residues of the S-cellobloside moiety are not distorted from the low-energy C-4(1) 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 beta -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.