1cmt: Difference between revisions

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THE ROLE OF ASPARTATE-235 IN THE BINDING OF CATIONS TO AN ARTIFICIAL CAVITY AT THE RADICAL SITE OF CYTOCHROME C PEROXIDASE

OverviewOverview

The activated state of cytochrome c peroxidase, compound ES, contains a cation radical on the Trp-191 side chain. We recently reported that replacing this tryptophan with glycine creates a buried cavity at the active site that contains ordered solvent and that will specifically bind substituted imidazoles in their protonated cationic forms (Fitzgerald MM, Churchill MJ, McRee DE, Goodin DB, 1994, Biochemistry 33:3807-3818). Proposals that a nearby carboxylate, Asp-235, and competing monovalent cations should modulate the affinity of the W191G cavity for ligand binding are addressed in this study. Competitive binding titrations of the imidazolium ion to W191G as a function of [K+] show that potassium competes weakly with the binding of imidazoles. The dissociation constant observed for potassium binding (18 mM) is more than 3,000-fold higher than that for 1,2-dimethylimidazole (5.5 microM) in the absence of competing cations. Significantly, the W191G-D235N double mutant shows no evidence for binding imidazoles in their cationic or neutral forms, even though the structure of the cavity remains largely unperturbed by replacement of the carboxylate. Refined crystallographic B-values of solvent positions indicate that the weakly bound potassium in W191G is significantly depopulated in the double mutant. These results demonstrate that the buried negative charge of Asp-235 is an essential feature of the cation binding determinant and indicate that this carboxylate plays a critical role in stabilizing the formation of the Trp-191 radical cation.

About this StructureAbout this Structure

1CMT is a Single protein structure of sequence from Saccharomyces cerevisiae with as ligand. Active as Cytochrome-c peroxidase, with EC number 1.11.1.5 Full crystallographic information is available from OCA.

ReferenceReference

The role of aspartate-235 in the binding of cations to an artificial cavity at the radical site of cytochrome c peroxidase., Fitzgerald MM, Trester ML, Jensen GM, McRee DE, Goodin DB, Protein Sci. 1995 Sep;4(9):1844-50. PMID:8528082

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-[[Image:1cmt.gif|left|200px]]<br /><applet load="1cmt" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1cmt.gif|left|200px]]<br /><applet load="1cmt" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1cmt, resolution 2.1&Aring;" />
 '''THE ROLE OF ASPARTATE-235 IN THE BINDING OF CATIONS TO AN ARTIFICIAL CAVITY AT THE RADICAL SITE OF CYTOCHROME C PEROXIDASE'''<br />
 ==Overview==
-The activated state of cytochrome c peroxidase, compound ES, contains a, cation radical on the Trp-191 side chain. We recently reported that, replacing this tryptophan with glycine creates a buried cavity at the, active site that contains ordered solvent and that will specifically bind, substituted imidazoles in their protonated cationic forms (Fitzgerald MM, Churchill MJ, McRee DE, Goodin DB, 1994, Biochemistry 33:3807-3818)., Proposals that a nearby carboxylate, Asp-235, and competing monovalent, cations should modulate the affinity of the W191G cavity for ligand, binding are addressed in this study. Competitive binding titrations of the, imidazolium ion to W191G as a function of [K+] show that potassium, competes weakly with the binding of imidazoles. The dissociation constant, observed for potassium binding (18 mM) is more than 3,000-fold higher than, that for 1,2-dimethylimidazole (5.5 microM) in the absence of competing, cations. Significantly, the W191G-D235N double mutant shows no evidence, for binding imidazoles in their cationic or neutral forms, even though the, structure of the cavity remains largely unperturbed by replacement of the, carboxylate. Refined crystallographic B-values of solvent positions, indicate that the weakly bound potassium in W191G is significantly, depopulated in the double mutant. These results demonstrate that the, buried negative charge of Asp-235 is an essential feature of the cation, binding determinant and indicate that this carboxylate plays a critical, role in stabilizing the formation of the Trp-191 radical cation.
+The activated state of cytochrome c peroxidase, compound ES, contains a cation radical on the Trp-191 side chain. We recently reported that replacing this tryptophan with glycine creates a buried cavity at the active site that contains ordered solvent and that will specifically bind substituted imidazoles in their protonated cationic forms (Fitzgerald MM, Churchill MJ, McRee DE, Goodin DB, 1994, Biochemistry 33:3807-3818). Proposals that a nearby carboxylate, Asp-235, and competing monovalent cations should modulate the affinity of the W191G cavity for ligand binding are addressed in this study. Competitive binding titrations of the imidazolium ion to W191G as a function of [K+] show that potassium competes weakly with the binding of imidazoles. The dissociation constant observed for potassium binding (18 mM) is more than 3,000-fold higher than that for 1,2-dimethylimidazole (5.5 microM) in the absence of competing cations. Significantly, the W191G-D235N double mutant shows no evidence for binding imidazoles in their cationic or neutral forms, even though the structure of the cavity remains largely unperturbed by replacement of the carboxylate. Refined crystallographic B-values of solvent positions indicate that the weakly bound potassium in W191G is significantly depopulated in the double mutant. These results demonstrate that the buried negative charge of Asp-235 is an essential feature of the cation binding determinant and indicate that this carboxylate plays a critical role in stabilizing the formation of the Trp-191 radical cation.
 ==About this Structure==
-CMT is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae Saccharomyces cerevisiae] with HEM as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Cytochrome-c_peroxidase Cytochrome-c peroxidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.11.1.5 1.11.1.5] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1CMT OCA].
+CMT is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae Saccharomyces cerevisiae] with <scene name='pdbligand=HEM:'>HEM</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Cytochrome-c_peroxidase Cytochrome-c peroxidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.11.1.5 1.11.1.5] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1CMT OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Saccharomyces cerevisiae]]
 [[Category: Single protein]]
-[[Category: Fitzgerald, M.M.]]
+[[Category: Fitzgerald, M M.]]
-[[Category: Goodin, D.B.]]
+[[Category: Goodin, D B.]]
-[[Category: Jensen, G.M.]]
+[[Category: Jensen, G M.]]
-[[Category: Mcree, D.E.]]
+[[Category: Mcree, D E.]]
-[[Category: Trester, M.L.]]
+[[Category: Trester, M L.]]
 [[Category: HEM]]
 [[Category: oxidoreductase (h2o2(a))]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 12:35:23 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:07:39 2008''