 |
PDBsum entry 1iew
|
|
|
|
|
 |
Contents |
 |
|
|
|
|
|
|
|
|
|
|
|
|
* Residue conservation analysis
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
Catalytic mechanisms and reaction intermediates along the hydrolytic pathway of a plant beta-D-Glucan glucohydrolase.
|
|
|
Authors
|
|
M.Hrmova,
J.N.Varghese,
R.De gori,
B.J.Smith,
H.Driguez,
G.B.Fincher.
|
|
|
Ref.
|
|
Structure, 2001,
9,
1005-1016.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
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.
|
|
|
|
|
|
Figure 4.
Figure 4. Stereo Representation of Ligands Bound in the
Active Site of b Image glucan GlucohydrolaseMOLSCRIPT [47]
diagrams of the nearest hydrogen bonding interactions (dashed
lines) between:(a) Glucose.(b) Cyclohexitol ring.(c)
2-deoxy-2-fluoro-a- Image -glucosyl moiety.(d) S-cellobioside
moiety.and the contact amino acid residues.Ligands are colored
in cyan. The molecular surfaces of domains 1 and 2 are
represented by transparent cyan and magenta surfaces,
respectively, and are generated using GRASP [48]. Black, red,
blue, yellow, and gray spheres represent carbon, oxygen,
nitrogen, sulfur, and fluorine atoms, respectively. Water
molecules are represented as red spheres. In (c), residues E220,
E287, R291, and E491, along with Wat2 and Wat3, are not
included, to improve the clarity of the data. The entrance to
the active site in (b) and (c) is located perpendicularly to the
page and is located toward the lower left hand corner in (a) and
(d)
|
|
|
|
|
|
The above figure is
reprinted
by permission from Cell Press:
Structure
(2001,
9,
1005-1016)
copyright 2001.
|
|
|
|
|
|
|
