1jd9: Difference between revisions
New page: left|200px<br /><applet load="1jd9" size="450" color="white" frame="true" align="right" spinBox="true" caption="1jd9, resolution 2.5Å" /> '''CRYSTAL STRUCTURE ANA... |
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[[Image:1jd9.jpg|left|200px]]<br /><applet load="1jd9" size=" | [[Image:1jd9.jpg|left|200px]]<br /><applet load="1jd9" size="350" color="white" frame="true" align="right" spinBox="true" | ||
caption="1jd9, resolution 2.5Å" /> | caption="1jd9, resolution 2.5Å" /> | ||
'''CRYSTAL STRUCTURE ANALYSIS OF THE MUTANT K300Q OF PSEUDOALTEROMONAS HALOPLANCTIS ALPHA-AMYLASE'''<br /> | '''CRYSTAL STRUCTURE ANALYSIS OF THE MUTANT K300Q OF PSEUDOALTEROMONAS HALOPLANCTIS ALPHA-AMYLASE'''<br /> | ||
==Overview== | ==Overview== | ||
To further investigate the mechanism and function of allosteric activation | To further investigate the mechanism and function of allosteric activation by chloride in some alpha-amylases, the structure of the bacterial alpha-amylase from the psychrophilic micro-organism Pseudoalteromonas haloplanktis in complex with nitrate has been solved at 2.1 A degrees, as well as the structure of the mutants Lys300Gln (2.5 A degrees ) and Lys300Arg (2.25 A degrees ). Nitrate binds strongly to alpha-amylase but is a weak activator. Mutation of the critical chloride ligand Lys300 into Gln results in a chloride-independent enzyme, whereas the mutation into Arg mimics the binding site as is found in animal alpha-amylases with, however, a lower affinity for chloride. These structures reveal that the triangular conformation of the chloride ligands and the nearly equatorial coordination allow the perfect accommodation of planar trigonal monovalent anions such as NO3-, explaining their unusual strong binding. It is also shown that a localized negative charge such as that of Cl-, rather than a delocalized charge as in the case of nitrate, is essential for maximal activation. The chloride-free mutant Lys300Gln indicates that chloride is not mandatory for the catalytic mechanism but strongly increases the reactivity at the active site. Disappearance of the putative catalytic water molecule in this weakly active mutant supports the view that chloride helps to polarize the hydrolytic water molecule and enhances the rate of the second step in the catalytic reaction. | ||
==About this Structure== | ==About this Structure== | ||
1JD9 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Pseudoalteromonas_haloplanktis Pseudoalteromonas haloplanktis] with CA as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Alpha-amylase Alpha-amylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.1 3.2.1.1] Full crystallographic information is available from [http:// | 1JD9 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Pseudoalteromonas_haloplanktis Pseudoalteromonas haloplanktis] with <scene name='pdbligand=CA:'>CA</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Alpha-amylase Alpha-amylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.1 3.2.1.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1JD9 OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: glycosyl hydrolase]] | [[Category: glycosyl hydrolase]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:21:22 2008'' | ||
Revision as of 14:21, 21 February 2008
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CRYSTAL STRUCTURE ANALYSIS OF THE MUTANT K300Q OF PSEUDOALTEROMONAS HALOPLANCTIS ALPHA-AMYLASE
OverviewOverview
To further investigate the mechanism and function of allosteric activation by chloride in some alpha-amylases, the structure of the bacterial alpha-amylase from the psychrophilic micro-organism Pseudoalteromonas haloplanktis in complex with nitrate has been solved at 2.1 A degrees, as well as the structure of the mutants Lys300Gln (2.5 A degrees ) and Lys300Arg (2.25 A degrees ). Nitrate binds strongly to alpha-amylase but is a weak activator. Mutation of the critical chloride ligand Lys300 into Gln results in a chloride-independent enzyme, whereas the mutation into Arg mimics the binding site as is found in animal alpha-amylases with, however, a lower affinity for chloride. These structures reveal that the triangular conformation of the chloride ligands and the nearly equatorial coordination allow the perfect accommodation of planar trigonal monovalent anions such as NO3-, explaining their unusual strong binding. It is also shown that a localized negative charge such as that of Cl-, rather than a delocalized charge as in the case of nitrate, is essential for maximal activation. The chloride-free mutant Lys300Gln indicates that chloride is not mandatory for the catalytic mechanism but strongly increases the reactivity at the active site. Disappearance of the putative catalytic water molecule in this weakly active mutant supports the view that chloride helps to polarize the hydrolytic water molecule and enhances the rate of the second step in the catalytic reaction.
About this StructureAbout this Structure
1JD9 is a Single protein structure of sequence from Pseudoalteromonas haloplanktis with as ligand. Active as Alpha-amylase, with EC number 3.2.1.1 Full crystallographic information is available from OCA.
ReferenceReference
Structural basis of alpha-amylase activation by chloride., Aghajari N, Feller G, Gerday C, Haser R, Protein Sci. 2002 Jun;11(6):1435-41. PMID:12021442
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