Catalytic mechanisms and reaction intermediates along the hydrolytic pathway of a plant beta-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 Odelta1 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 A) with the Oepsilon2 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 4C(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.
Hrmova
M
M
Department of Plant Science, University of Adelaide, Waite Campus, 5064, Glen Osmond, SA, Australia. hrmova@adelaide.edu.au
Varghese
J N
JN
De Gori
R
R
Smith
B J
BJ
Driguez
H
H
Fincher
G B
GB
D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003511:Cyclohexanols / Q000737:chemistry / Q000378:metabolism; D019788:Fluorodeoxyglucose F18 / Q000737:chemistry / Q000378:metabolism; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D005959:Glucosidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology*; D006868:Hydrolysis; D007294:Inositol / Q000031:analogs & derivatives* / Q000494:pharmacology; D008956:Models, Chemical; D008958:Models, Molecular; D010449:Peptide Mapping; D011789:Quantum Theory
D003511:Cyclohexanols; D019788:Fluorodeoxyglucose F18; D007294:Inositol; D005959:Glucosidases; C446280:beta-D-glucan glucohydrolase; D005947:Glucose; C011423:conduritol epoxide