1l0f: Difference between revisions

Revision as of 14:39, 21 February 2008

X-ray Crystal Structure of AmpC N152H Mutant beta-Lactamase

OverviewOverview

The structures of enzymes reflect two tendencies that appear opposed. On one hand, they fold into compact, stable structures; on the other hand, they bind a ligand and catalyze a reaction. To be stable, enzymes fold to maximize favorable interactions, forming a tightly packed hydrophobic core, exposing hydrophilic groups, and optimizing intramolecular hydrogen-bonding. To be functional, enzymes carve out an active site for ligand binding, exposing hydrophobic surface area, clustering like charges, and providing unfulfilled hydrogen bond donors and acceptors. Using AmpC beta-lactamase, an enzyme that is well-characterized structurally and mechanistically, the relationship between enzyme stability and function was investigated by substituting key active-site residues and measuring the changes in stability and activity. Substitutions of catalytic residues Ser64, Lys67, Tyr150, Asn152, and Lys315 decrease the activity of the enzyme by 10(3)-10(5)-fold compared to wild-type. Concomitantly, many of these substitutions increase the stability of the enzyme significantly, by up to 4.7kcal/mol. To determine the structural origins of stabilization, the crystal structures of four mutant enzymes were determined to between 1.90A and 1.50A resolution. These structures revealed several mechanisms by which stability was increased, including mimicry of the substrate by the substituted residue (S64D), relief of steric strain (S64G), relief of electrostatic strain (K67Q), and improved polar complementarity (N152H). These results suggest that the preorganization of functionality characteristic of active sites has come at a considerable cost to enzyme stability. In proteins of unknown function, the presence of such destabilized regions may indicate the presence of a binding site.

About this StructureAbout this Structure

1L0F is a Single protein structure of sequence from Escherichia coli with as ligand. Active as Beta-lactamase, with EC number 3.5.2.6 Full crystallographic information is available from OCA.

ReferenceReference

Structural bases of stability-function tradeoffs in enzymes., Beadle BM, Shoichet BK, J Mol Biol. 2002 Aug 9;321(2):285-96. PMID:12144785

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-[[Image:1l0f.jpg|left|200px]]<br /><applet load="1l0f" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1l0f.jpg|left|200px]]<br /><applet load="1l0f" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1l0f, resolution 1.66&Aring;" />
 '''X-ray Crystal Structure of AmpC N152H Mutant beta-Lactamase'''<br />
 ==Overview==
-The structures of enzymes reflect two tendencies that appear opposed. On, one hand, they fold into compact, stable structures; on the other hand, they bind a ligand and catalyze a reaction. To be stable, enzymes fold to, maximize favorable interactions, forming a tightly packed hydrophobic, core, exposing hydrophilic groups, and optimizing intramolecular, hydrogen-bonding. To be functional, enzymes carve out an active site for, ligand binding, exposing hydrophobic surface area, clustering like, charges, and providing unfulfilled hydrogen bond donors and acceptors., Using AmpC beta-lactamase, an enzyme that is well-characterized, structurally and mechanistically, the relationship between enzyme, stability and function was investigated by substituting key active-site, residues and measuring the changes in stability and activity., Substitutions of catalytic residues Ser64, Lys67, Tyr150, Asn152, and, Lys315 decrease the activity of the enzyme by 10(3)-10(5)-fold compared to, wild-type. Concomitantly, many of these substitutions increase the, stability of the enzyme significantly, by up to 4.7kcal/mol. To determine, the structural origins of stabilization, the crystal structures of four, mutant enzymes were determined to between 1.90A and 1.50A resolution., These structures revealed several mechanisms by which stability was, increased, including mimicry of the substrate by the substituted residue, (S64D), relief of steric strain (S64G), relief of electrostatic strain, (K67Q), and improved polar complementarity (N152H). These results suggest, that the preorganization of functionality characteristic of active sites, has come at a considerable cost to enzyme stability. In proteins of, unknown function, the presence of such destabilized regions may indicate, the presence of a binding site.
+The structures of enzymes reflect two tendencies that appear opposed. On one hand, they fold into compact, stable structures; on the other hand, they bind a ligand and catalyze a reaction. To be stable, enzymes fold to maximize favorable interactions, forming a tightly packed hydrophobic core, exposing hydrophilic groups, and optimizing intramolecular hydrogen-bonding. To be functional, enzymes carve out an active site for ligand binding, exposing hydrophobic surface area, clustering like charges, and providing unfulfilled hydrogen bond donors and acceptors. Using AmpC beta-lactamase, an enzyme that is well-characterized structurally and mechanistically, the relationship between enzyme stability and function was investigated by substituting key active-site residues and measuring the changes in stability and activity. Substitutions of catalytic residues Ser64, Lys67, Tyr150, Asn152, and Lys315 decrease the activity of the enzyme by 10(3)-10(5)-fold compared to wild-type. Concomitantly, many of these substitutions increase the stability of the enzyme significantly, by up to 4.7kcal/mol. To determine the structural origins of stabilization, the crystal structures of four mutant enzymes were determined to between 1.90A and 1.50A resolution. These structures revealed several mechanisms by which stability was increased, including mimicry of the substrate by the substituted residue (S64D), relief of steric strain (S64G), relief of electrostatic strain (K67Q), and improved polar complementarity (N152H). These results suggest that the preorganization of functionality characteristic of active sites has come at a considerable cost to enzyme stability. In proteins of unknown function, the presence of such destabilized regions may indicate the presence of a binding site.
 ==About this Structure==
-L0F is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with PO4 as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Beta-lactamase Beta-lactamase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.2.6 3.5.2.6] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1L0F OCA].
+L0F is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=PO4:'>PO4</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Beta-lactamase Beta-lactamase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.2.6 3.5.2.6] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1L0F OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Escherichia coli]]
 [[Category: Single protein]]
-[[Category: Beadle, B.M.]]
+[[Category: Beadle, B M.]]
-[[Category: Shoichet, B.K.]]
+[[Category: Shoichet, B K.]]
 [[Category: PO4]]
 [[Category: amide hydrolase]]
@@ Line 21: / Line 21: @@
 [[Category: mutant enzyme]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 20:07:57 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:39:55 2008''