1yep: Difference between revisions

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Structural and biochemical analysis of the link between enzymatic activity and olgomerization in AhpC, a bacterial peroxiredoxin.

OverviewOverview

Peroxiredoxins (Prxs) make up a ubiquitous class (proposed EC 1.11.1.15) of cysteine-dependent peroxidases with roles in oxidant protection and signal transduction. An intriguing biophysical property of typical 2-Cys Prxs is the redox-dependent modulation of their oligomeric state between decamers and dimers at physiological concentrations. The functional consequences of this linkage are unknown, but on the basis of structural considerations, we hypothesized that decamer-building (dimer-dimer) interactions serve to stabilize a loop that forms the peroxidatic active site. Here, we address this important issue by studying mutations of Thr77 at the decamer-building interface of AhpC from Salmonella typhimurium. Ultracentrifugation studies revealed that two of the substitutions (T77I and T77D) successfully disrupted the decamer, while the third (T77V) actually enhanced decamer stability. Crystal structures of the decameric forms of all three mutant proteins provide a rationale for their properties. A new assay allowed the first ever measurement of the true k(cat) and K(m) values of wild-type AhpC with H(2)O(2), placing the catalytic efficiency at 4 x 10(7) M(-)(1) s(-)(1). T77V had slightly higher activity than wild-type enzyme, and both T77I and T77D exhibited ca. 100-fold lower catalytic efficiency, indicating that the decameric structure is quite important for, but not essential to, activity. The interplay between decamer formation and active site loop dynamics is emphasized by a decreased susceptibility of T77I and T77D to peroxide-mediated inactivation, and by an increase in the crystallographic B-factors in the active site loop, rather than at the site of the mutation, in the T77D variant.

About this StructureAbout this Structure

1YEP is a Single protein structure of sequence from Salmonella typhimurium with as ligand. This structure supersedes the now removed PDB entry 1KYG. Full crystallographic information is available from OCA.

ReferenceReference

Analysis of the link between enzymatic activity and oligomeric state in AhpC, a bacterial peroxiredoxin., Parsonage D, Youngblood DS, Sarma GN, Wood ZA, Karplus PA, Poole LB, Biochemistry. 2005 Aug 9;44(31):10583-92. PMID:16060667

Page seeded by OCA on Thu Feb 21 16:04:32 2008

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-[[Image:1yep.gif|left|200px]]<br /><applet load="1yep" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1yep.gif|left|200px]]<br /><applet load="1yep" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1yep, resolution 2.5&Aring;" />
 '''Structural and biochemical analysis of the link between enzymatic activity and olgomerization in AhpC, a bacterial peroxiredoxin.'''<br />
 ==Overview==
-Peroxiredoxins (Prxs) make up a ubiquitous class (proposed EC 1.11.1.15), of cysteine-dependent peroxidases with roles in oxidant protection and, signal transduction. An intriguing biophysical property of typical 2-Cys, Prxs is the redox-dependent modulation of their oligomeric state between, decamers and dimers at physiological concentrations. The functional, consequences of this linkage are unknown, but on the basis of structural, considerations, we hypothesized that decamer-building (dimer-dimer), interactions serve to stabilize a loop that forms the peroxidatic active, site. Here, we address this important issue by studying mutations of Thr77, at the decamer-building interface of AhpC from Salmonella typhimurium., Ultracentrifugation studies revealed that two of the substitutions (T77I, and T77D) successfully disrupted the decamer, while the third (T77V), actually enhanced decamer stability. Crystal structures of the decameric, forms of all three mutant proteins provide a rationale for their, properties. A new assay allowed the first ever measurement of the true, k(cat) and K(m) values of wild-type AhpC with H(2)O(2), placing the, catalytic efficiency at 4 x 10(7) M(-)(1) s(-)(1). T77V had slightly, higher activity than wild-type enzyme, and both T77I and T77D exhibited, ca. 100-fold lower catalytic efficiency, indicating that the decameric, structure is quite important for, but not essential to, activity. The, interplay between decamer formation and active site loop dynamics is, emphasized by a decreased susceptibility of T77I and T77D to, peroxide-mediated inactivation, and by an increase in the crystallographic, B-factors in the active site loop, rather than at the site of the, mutation, in the T77D variant.
+Peroxiredoxins (Prxs) make up a ubiquitous class (proposed EC 1.11.1.15) of cysteine-dependent peroxidases with roles in oxidant protection and signal transduction. An intriguing biophysical property of typical 2-Cys Prxs is the redox-dependent modulation of their oligomeric state between decamers and dimers at physiological concentrations. The functional consequences of this linkage are unknown, but on the basis of structural considerations, we hypothesized that decamer-building (dimer-dimer) interactions serve to stabilize a loop that forms the peroxidatic active site. Here, we address this important issue by studying mutations of Thr77 at the decamer-building interface of AhpC from Salmonella typhimurium. Ultracentrifugation studies revealed that two of the substitutions (T77I and T77D) successfully disrupted the decamer, while the third (T77V) actually enhanced decamer stability. Crystal structures of the decameric forms of all three mutant proteins provide a rationale for their properties. A new assay allowed the first ever measurement of the true k(cat) and K(m) values of wild-type AhpC with H(2)O(2), placing the catalytic efficiency at 4 x 10(7) M(-)(1) s(-)(1). T77V had slightly higher activity than wild-type enzyme, and both T77I and T77D exhibited ca. 100-fold lower catalytic efficiency, indicating that the decameric structure is quite important for, but not essential to, activity. The interplay between decamer formation and active site loop dynamics is emphasized by a decreased susceptibility of T77I and T77D to peroxide-mediated inactivation, and by an increase in the crystallographic B-factors in the active site loop, rather than at the site of the mutation, in the T77D variant.
 ==About this Structure==
-YEP is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Salmonella_typhimurium Salmonella typhimurium] with SO4 as [http://en.wikipedia.org/wiki/ligand ligand]. This structure superseeds the now removed PDB entry 1KYG. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1YEP OCA].
+YEP is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Salmonella_typhimurium Salmonella typhimurium] with <scene name='pdbligand=SO4:'>SO4</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. This structure supersedes the now removed PDB entry 1KYG. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1YEP OCA].
 ==Reference==
@@ Line 13: / Line 13: @@
 [[Category: Salmonella typhimurium]]
 [[Category: Single protein]]
-[[Category: Karplus, P.A.]]
+[[Category: Karplus, P A.]]
 [[Category: Parsonage, D.]]
-[[Category: Poole, L.B.]]
+[[Category: Poole, L B.]]
-[[Category: Sarma, G.N.]]
+[[Category: Sarma, G N.]]
-[[Category: Wood, Z.A.]]
+[[Category: Wood, Z A.]]
-[[Category: Youngblood, D.S.]]
+[[Category: Youngblood, D S.]]
 [[Category: SO4]]
 [[Category: ahpc]]
@@ Line 24: / Line 24: @@
 [[Category: peroxiredoxin]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 06:43:16 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 16:04:32 2008''