1au9: Difference between revisions

Revision as of 10:41, 2 May 2008

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. 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 Page seeded by OCA on Fri May 2 10:41:52 2008

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OCA

@@ Line 1: / Line 1: @@
 [[Image:1au9.gif|left|200px]]
- {{Structure
+<!--
-|PDB= 1au9 |SIZE=350|CAPTION= <scene name='initialview01'>1au9</scene>, resolution 1.80&Aring;
+The line below this paragraph, containing "STRUCTURE_1au9", creates the "Structure Box" on the page.
-|SITE= <scene name='pdbsite=169:Mutation+From+GLY+To+ALA+At+Residue+169'>169</scene>, <scene name='pdbsite=206:Mutation+From+GLN+To+CYS+At+Residue+206.+An+Unknown+Thio+...'>206</scene>, <scene name='pdbsite=217:Mutation+From+TYR+To+LYS+At+Residue+217'>217</scene>, <scene name='pdbsite=218:Mutation+From+ASN+To+SER+At+Residue+218'>218</scene>, <scene name='pdbsite=C22:Mutation+From+THR+To+CYS+At+Residue+22.+CYS+22+Forms+A+D+...'>C22</scene>, <scene name='pdbsite=C87:Mutation+From+SER+To+CYS+At+Residue+87.+CYS+87+Forms+A+D+...'>C87</scene>, <scene name='pdbsite=CA1:High+Affinity+Ca+Binding+Site'>CA1</scene>, <scene name='pdbsite=CA2:Low+Affinity+Ca+Binding+Site'>CA2</scene> and <scene name='pdbsite=F50:Mutation+From+MET+To+PHE+At+Residue+50'>F50</scene>
+You may change the PDB parameter (which sets the PDB file loaded into the applet)
-|LIGAND= <scene name='pdbligand=CA:CALCIUM+ION'>CA</scene>, <scene name='pdbligand=IPA:ISOPROPYL+ALCOHOL'>IPA</scene>, <scene name='pdbligand=UNX:UNKNOWN+ATOM+OR+ION'>UNX</scene>
+or the SCENE parameter (which sets the initial scene displayed when the page is loaded),
-|ACTIVITY= <span class='plainlinks'>[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] </span>
+or leave the SCENE parameter empty for the default display.
-|GENE=
+-->
-|DOMAIN=
+{{STRUCTURE_1au9|  PDB=1au9  |  SCENE=  }}
-|RELATEDENTRY=
-|RESOURCES=<span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=1au9 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1au9 OCA], [http://www.ebi.ac.uk/pdbsum/1au9 PDBsum], [http://www.rcsb.org/pdb/explore.do?structureId=1au9 RCSB]</span>
-}}
 '''SUBTILISIN BPN' MUTANT 8324 IN CITRATE'''
@@ Line 29: / Line 26: @@
 [[Category: Whitlow, M.]]
 [[Category: Wood, J F.]]
-[[Category: hydrolase]]
+[[Category: Hydrolase]]
-[[Category: serine protease]]
+[[Category: Serine protease]]
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May  2 10:41:52 2008''
-''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun Mar 30 18:47:41 2008''