1q6e: Difference between revisions
New page: left|200px<br /><applet load="1q6e" size="450" color="white" frame="true" align="right" spinBox="true" caption="1q6e, resolution 1.95Å" /> '''Crystal Structure of... |
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[[Image:1q6e.gif|left|200px]]<br /><applet load="1q6e" size=" | [[Image:1q6e.gif|left|200px]]<br /><applet load="1q6e" size="350" color="white" frame="true" align="right" spinBox="true" | ||
caption="1q6e, resolution 1.95Å" /> | caption="1q6e, resolution 1.95Å" /> | ||
'''Crystal Structure of Soybean Beta-Amylase Mutant (E178Y) with Increased pH Optimum at pH 5.4'''<br /> | '''Crystal Structure of Soybean Beta-Amylase Mutant (E178Y) with Increased pH Optimum at pH 5.4'''<br /> | ||
==Overview== | ==Overview== | ||
Comparison of the architecture around the active site of soybean | Comparison of the architecture around the active site of soybean beta-amylase and Bacillus cereus beta-amylase showed that the hydrogen bond networks (Glu380-(Lys295-Met51) and Glu380-Asn340-Glu178) in soybean beta-amylase around the base catalytic residue, Glu380, seem to contribute to the lower pH optimum of soybean beta-amylase. To convert the pH optimum of soybean beta-amylase (pH 5.4) to that of the bacterial type enzyme (pH 6.7), three mutants of soybean beta-amylase, M51T, E178Y, and N340T, were constructed such that the hydrogen bond networks were removed by site-directed mutagenesis. The kinetic analysis showed that the pH optimum of all mutants shifted dramatically to a neutral pH (range, from 5.4 to 6.0-6.6). The Km values of the mutants were almost the same as that of soybean beta-amylase except in the case of M51T, while the Vmax values of all mutants were low compared with that of soybean beta-amylase. The crystal structure analysis of the wild type-maltose and mutant-maltose complexes showed that the direct hydrogen bond between Glu380 and Asn340 was completely disrupted in the mutants M51T, E178Y, and N340T. In the case of M51T, the hydrogen bond between Glu380 and Lys295 was also disrupted. These results indicated that the reduced pKa value of Glu380 is stabilized by the hydrogen bond network and is responsible for the lower pH optimum of soybean beta-amylase compared with that of the bacterial beta-amylase. | ||
==About this Structure== | ==About this Structure== | ||
1Q6E 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:// | 1Q6E 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=1Q6E OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: Adachi, M.]] | [[Category: Adachi, M.]] | ||
[[Category: Hirata, A.]] | [[Category: Hirata, A.]] | ||
[[Category: Kang, Y | [[Category: Kang, Y N.]] | ||
[[Category: Mikami, B.]] | [[Category: Mikami, B.]] | ||
[[Category: Sekine, A.]] | [[Category: Sekine, A.]] | ||
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[[Category: maltose complex]] | [[Category: maltose complex]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 14:36:20 2008'' | ||
Revision as of 15:36, 21 February 2008
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Crystal Structure of Soybean Beta-Amylase Mutant (E178Y) with Increased pH Optimum at pH 5.4
OverviewOverview
Comparison of the architecture around the active site of soybean beta-amylase and Bacillus cereus beta-amylase showed that the hydrogen bond networks (Glu380-(Lys295-Met51) and Glu380-Asn340-Glu178) in soybean beta-amylase around the base catalytic residue, Glu380, seem to contribute to the lower pH optimum of soybean beta-amylase. To convert the pH optimum of soybean beta-amylase (pH 5.4) to that of the bacterial type enzyme (pH 6.7), three mutants of soybean beta-amylase, M51T, E178Y, and N340T, were constructed such that the hydrogen bond networks were removed by site-directed mutagenesis. The kinetic analysis showed that the pH optimum of all mutants shifted dramatically to a neutral pH (range, from 5.4 to 6.0-6.6). The Km values of the mutants were almost the same as that of soybean beta-amylase except in the case of M51T, while the Vmax values of all mutants were low compared with that of soybean beta-amylase. The crystal structure analysis of the wild type-maltose and mutant-maltose complexes showed that the direct hydrogen bond between Glu380 and Asn340 was completely disrupted in the mutants M51T, E178Y, and N340T. In the case of M51T, the hydrogen bond between Glu380 and Lys295 was also disrupted. These results indicated that the reduced pKa value of Glu380 is stabilized by the hydrogen bond network and is responsible for the lower pH optimum of soybean beta-amylase compared with that of the bacterial beta-amylase.
About this StructureAbout this Structure
1Q6E 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
Structural and enzymatic analysis of soybean beta-amylase mutants with increased pH optimum., Hirata A, Adachi M, Sekine A, Kang YN, Utsumi S, Mikami B, J Biol Chem. 2004 Feb 20;279(8):7287-95. Epub 2003 Nov 24. PMID:14638688
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