1v3h: Difference between revisions

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The roles of Glu186 and Glu380 in the catalytic reaction of soybean beta-amylase

OverviewOverview

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.

About this StructureAbout this Structure

1V3H is a Single protein structure of sequence from Glycine max with as ligand. Active as Beta-amylase, with EC number 3.2.1.2 Full crystallographic information is available from OCA.

ReferenceReference

The roles of Glu186 and Glu380 in the catalytic reaction of soybean beta-amylase., Kang YN, Adachi M, Utsumi S, Mikami B, J Mol Biol. 2004 Jun 18;339(5):1129-40. PMID:15178253

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 caption="1v3h, resolution 1.60&Aring;" />
 '''The roles of Glu186 and Glu380 in the catalytic reaction of soybean beta-amylase'''<br />
 ==Overview==
-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.
+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.
 ==About this Structure==
-V3H is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Glycine_max Glycine max] with SO4 as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Beta-amylase Beta-amylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.2 3.2.1.2] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1V3H OCA].
+V3H is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Glycine_max Glycine max] with <scene name='pdbligand=SO4:'>SO4</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Beta-amylase Beta-amylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.2 3.2.1.2] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1V3H OCA].
 ==Reference==
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 [[Category: Single protein]]
 [[Category: Adachi, M.]]
-[[Category: Kang, Y.N.]]
+[[Category: Kang, Y N.]]
 [[Category: Mikami, B.]]
 [[Category: Utsumi, S.]]
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 [[Category: (beta/alpha)8 barrel]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 04:24:57 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 15:31:08 2008''