title: Catalytic mechanisms and reaction intermediates along the hydrolytic pathway of a plant beta-D-glucan glucohydrolase.
abstract: 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.
authors.affiliation: Department of Plant Science, University of Adelaide, Waite Campus, 5064, Glen Osmond, SA, Australia. hrmova@adelaide.edu.au
authors.forename: M
authors.initials: M
authors.lastname: Hrmova
authors.forename: J N
authors.initials: JN
authors.lastname: Varghese
authors.forename: R
authors.initials: R
authors.lastname: De Gori
authors.forename: B J
authors.initials: BJ
authors.lastname: Smith
authors.forename: H
authors.initials: H
authors.lastname: Driguez
authors.forename: G B
authors.initials: GB
authors.lastname: Fincher
mesh_terms: 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
chemical_list: D003511:Cyclohexanols; D019788:Fluorodeoxyglucose F18; D007294:Inositol; D005959:Glucosidases; C446280:beta-D-glucan glucohydrolase; D005947:Glucose; C011423:conduritol epoxide