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PDBsum entry 1v3h
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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The roles of glu186 and glu380 in the catalytic reaction of soybean beta-Amylase.
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Authors
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Y.N.Kang,
M.Adachi,
S.Utsumi,
B.Mikami.
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Ref.
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J Mol Biol, 2004,
339,
1129-1140.
[DOI no: ]
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PubMed id
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Abstract
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It has previously been suggested that the glutamic acid residues Glu186 and
Glu380 of soybean beta-amylase play critical roles as a general acid and a
general base catalyst, respectively. In order to confirm the roles of Glu186 and
Glu380, each residue was mutated to a glutamine residue and the crystal
structures of the substrate (E186Q/maltopentaose) and product (E380Q/maltose)
complexes were determined at resolutions of 1.6 Angstrom and 1.9 Angstrom,
respectively. Both mutant enzymes exhibited 16,000- and 37,000-fold decreased
activity relative to that of the wild-type enzyme. The crystal structure of the
E186Q/maltopentaose complex revealed an unambiguous five-glucose unit at
subsites -2 to +3. Two maltose molecules bind on subsites -2 to -1 and +2 to +3
in the E380Q/maltose complex, whereas they bind in tandem to -2 to -1 and +1 to
+2 in the wild-type/maltose complex. The conformation of the glucose residue at
subsite -1 was identified as a stable (4)C(1) alpha-anomer in the E380Q/maltose
complex, whereas a distorted ring conformation was observed in the
wild-type/maltose complex. The side-chain movement of Gln380 to the position of
a putative attacking water molecule seen in the wild-type enzyme caused the
inactivation of the E380Q mutant and an altered binding pattern of maltose
molecules. These results confirm the critical roles played by Glu186 in the
donation of a proton to the glycosidic oxygen of the substrate, and by Glu380 in
the activation of an attacking water molecule. The observed difference between
the backbones of E186Q/maltopentaose and E380Q/maltose in terms of Thr342
suggests that the side-chain of Thr342 may stabilize the deprotonated form of
Glu186 after the cleavage of the glycosidic bond.
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Figure 2.
Figure 2. (a) Stereo view of the E186Q/maltopentaose (shown
in magenta) superimposed on the wild-type/maltose (shown in
yellow) structure at the active site. The protein residues of
each complex were superimposed using the RIGID program of
TURBO-FRODO. Comparison of the two structures revealed almost
the same conformation at the active site, except for in the
main-chain region around the Thr342 residue. (b) Comparison of
the hydrogen bonding pattern between the structures of
E186Q/maltopentaose (magenta) and wild-type/maltose (yellow)
around subsites -1 and +1 in stereo. The dotted lines indicate
the hydrogen bond interactions. A putative attacking water
molecule (H[2]O 712) was observed at the position corresponding
to the O1 atom of Glc( -1) in the wild-type/maltose structure.
The intramolecular hydrogen bonds between Glu186, Arg188, and
Tyr192 were not greatly altered, but there was no interaction
between Gln186 and Thr342 due to the main-chain conformational
change in the 340-343 residues. The NE2 atom of Gln186 formed a
hydrogen bond with the O4 atom of Glc(+1) at a distance of 2.9
Å.
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Figure 4.
Figure 4. A schematic representation of the hydrogen
bonding networks of (a) the wild-type enzyme in the attack by a
catalytic water molecule on the C1 atom of Glc( -1) from above,
and (b) the wild-type enzyme after the inversion at the O1 atom.
Note that the Thr342 residue assumes different conformations in
(a) and (b). (c) The hydrogen bonding network rearranged in the
E380Q/maltose structure. The catalytic water molecule was
eliminated and a new water molecule was introduced between
Gln380 and Asn340. No interaction between Gln380 and Lys295 was
observed.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2004,
339,
1129-1140)
copyright 2004.
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