1au9: Difference between revisions
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==Overview== | ==Overview== | ||
Six individual amino acid substitutions at separate positions in the, tertiary structure of subtilisin BPN' (EC 3.4.21.14) were found to, increase the stability of this enzyme, as judged by differential scanning, calorimetry and decreased rates of thermal inactivation. These stabilizing, changes, N218S, G169A, Y217K, M50F, Q206C, and N76D, were discovered, through the use of five different investigative approaches: (1) random, mutagenesis; (2) design of buried hydrophobic side groups; (3) design of, electrostatic interactions at Ca2+ binding sites; (4) sequence homology, consensus; and (5) serendipity. Individually, the six amino acid, substitutions increase the delta G of unfolding between 0.3 and 1.3, kcal/mol at 58.5 degrees C. The combination of these six individual, stabilizing ... | Six individual amino acid substitutions at separate positions in the, tertiary structure of subtilisin BPN' (EC 3.4.21.14) were found to, increase the stability of this enzyme, as judged by differential scanning, calorimetry and decreased rates of thermal inactivation. These stabilizing, changes, N218S, G169A, Y217K, M50F, Q206C, and N76D, were discovered, through the use of five different investigative approaches: (1) random, mutagenesis; (2) design of buried hydrophobic side groups; (3) design of, electrostatic interactions at Ca2+ binding sites; (4) sequence homology, consensus; and (5) serendipity. Individually, the six amino acid, substitutions increase the delta G of unfolding between 0.3 and 1.3, kcal/mol at 58.5 degrees C. The combination of these six individual, stabilizing mutations together into one subtilisin BPN' molecule was found, to result in approximately independent and additive increases in the delta, G of unfolding to give a net increase of 3.8 kcal/mol (58.5 degrees C)., Thermodynamic stability was also shown to be related to resistance to, irreversible inactivation, which included elevated temperatures (65, degrees C) or extreme alkalinity (pH 12.0). Under these denaturing, conditions, the rate of inactivation of the combination variant is, approximately 300 times slower than that of the wild-type subtilisin BPN'., A comparison of the 1.8-A-resolution crystal structures of mutant and, wild-type enzymes revealed only independent and localized structural, changes around the site of the amino acid side group, substitutions.(ABSTRACT TRUNCATED AT 250 WORDS) | ||
==About this Structure== | ==About this Structure== | ||
1AU9 is a | 1AU9 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bacillus_amyloliquefaciens Bacillus amyloliquefaciens] with CA, UNX and IPA as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Subtilisin Subtilisin], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.4.21.62 3.4.21.62] Structure known Active Sites: 169, 206, 217, 218, C22, C87, CA1, CA2 and F50. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1AU9 OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: serine protease]] | [[Category: serine protease]] | ||
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Revision as of 15:53, 5 November 2007
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SUBTILISIN BPN' MUTANT 8324 IN CITRATE
OverviewOverview
Six individual amino acid substitutions at separate positions in the, tertiary structure of subtilisin BPN' (EC 3.4.21.14) were found to, increase the stability of this enzyme, as judged by differential scanning, calorimetry and decreased rates of thermal inactivation. These stabilizing, changes, N218S, G169A, Y217K, M50F, Q206C, and N76D, were discovered, through the use of five different investigative approaches: (1) random, mutagenesis; (2) design of buried hydrophobic side groups; (3) design of, electrostatic interactions at Ca2+ binding sites; (4) sequence homology, consensus; and (5) serendipity. Individually, the six amino acid, substitutions increase the delta G of unfolding between 0.3 and 1.3, kcal/mol at 58.5 degrees C. The combination of these six individual, stabilizing mutations together into one subtilisin BPN' molecule was found, to result in approximately independent and additive increases in the delta, G of unfolding to give a net increase of 3.8 kcal/mol (58.5 degrees C)., Thermodynamic stability was also shown to be related to resistance to, irreversible inactivation, which included elevated temperatures (65, degrees C) or extreme alkalinity (pH 12.0). Under these denaturing, conditions, the rate of inactivation of the combination variant is, approximately 300 times slower than that of the wild-type subtilisin BPN'., A comparison of the 1.8-A-resolution crystal structures of mutant and, wild-type enzymes revealed only independent and localized structural, changes around the site of the amino acid side group, substitutions.(ABSTRACT TRUNCATED AT 250 WORDS)
About this StructureAbout this Structure
1AU9 is a Single protein structure of sequence from Bacillus amyloliquefaciens with CA, UNX and IPA as ligands. Active as Subtilisin, with EC number 3.4.21.62 Structure known Active Sites: 169, 206, 217, 218, C22, C87, CA1, CA2 and F50. Full crystallographic information is available from OCA.
ReferenceReference
Large increases in general stability for subtilisin BPN' through incremental changes in the free energy of unfolding., Pantoliano MW, Whitlow M, Wood JF, Dodd SW, Hardman KD, Rollence ML, Bryan PN, Biochemistry. 1989 Sep 5;28(18):7205-13. PMID:2684274
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