1stq: Difference between revisions

Revision as of 16:05, 21 February 2008

Cyrstal Structure of Cytochrome c Peroxidase Mutant: CcPK2M3

OverviewOverview

Previously a K(+)-binding site, analogous to that found in ascorbate peroxidase (APX), was engineered into cytochrome c peroxidase (CcP) to test the hypothesis that the bound K(+) influences the stability of the Trp191 cation radical formed during the CcP catalytic cycle (Bonagura et al., (1996) Biochemistry 35, 6107 and Bonagura et al., (1999) Biochemistry 38, 5528). Characterization of this mutant, designated CcPK2, showed that the stability of the Trp191 cation radical is dependent on the occupancy of the engineered K(+) site and that the Trp191 radical was much less stable in this mutant than in wild-type CcP. The mutations Met230Leu, Met231Gln, and Met172Ser have now been constructed on the CcPK2 mutant template to test if the Met residues also contribute to the stabilization of the Trp191 cation radical. Crystal structures show that the mutations affect only the local structure near the sites of mutation. Removal of these electronegative residues located less than 8 A from the Trp radical results in a further destabilization of the Trp radical. The characteristic EPR signal associated with the Trp radical is significantly narrowed and is characteristic of a tyrosine radical signal. Double-mixing stopped-flow experiments, where the delay time between the formation of CcP compound I and its mixing with horse heart ferrocytochrome c is varied, show that the stability of the Trp radical decreases as the Met residues are removed from the proximal cavity. When taken together, these results demonstrate a strong correlation between the experimentally determined stability of the Trp191 radical, the enzyme activity, and the calculated electrostatic stabilization of the Trp191 radical.

About this StructureAbout this Structure

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

ReferenceReference

Electrostatic control of the tryptophan radical in cytochrome c peroxidase., Barrows TP, Bhaskar B, Poulos TL, Biochemistry. 2004 Jul 13;43(27):8826-34. PMID:15236591

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OCA

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-[[Image:1stq.jpg|left|200px]]<br /><applet load="1stq" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1stq.jpg|left|200px]]<br /><applet load="1stq" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1stq, resolution 1.82&Aring;" />
 '''Cyrstal Structure of Cytochrome c Peroxidase Mutant: CcPK2M3'''<br />
 ==Overview==
-Previously a K(+)-binding site, analogous to that found in ascorbate, peroxidase (APX), was engineered into cytochrome c peroxidase (CcP) to, test the hypothesis that the bound K(+) influences the stability of the, Trp191 cation radical formed during the CcP catalytic cycle (Bonagura et, al., (1996) Biochemistry 35, 6107 and Bonagura et al., (1999) Biochemistry, 38, 5528). Characterization of this mutant, designated CcPK2, showed that, the stability of the Trp191 cation radical is dependent on the occupancy, of the engineered K(+) site and that the Trp191 radical was much less, stable in this mutant than in wild-type CcP. The mutations Met230Leu, Met231Gln, and Met172Ser have now been constructed on the CcPK2 mutant, template to test if the Met residues also contribute to the stabilization, of the Trp191 cation radical. Crystal structures show that the mutations, affect only the local structure near the sites of mutation. Removal of, these electronegative residues located less than 8 A from the Trp radical, results in a further destabilization of the Trp radical. The, characteristic EPR signal associated with the Trp radical is significantly, narrowed and is characteristic of a tyrosine radical signal. Double-mixing, stopped-flow experiments, where the delay time between the formation of, CcP compound I and its mixing with horse heart ferrocytochrome c is, varied, show that the stability of the Trp radical decreases as the Met, residues are removed from the proximal cavity. When taken together, these, results demonstrate a strong correlation between the experimentally, determined stability of the Trp191 radical, the enzyme activity, and the, calculated electrostatic stabilization of the Trp191 radical.
+Previously a K(+)-binding site, analogous to that found in ascorbate peroxidase (APX), was engineered into cytochrome c peroxidase (CcP) to test the hypothesis that the bound K(+) influences the stability of the Trp191 cation radical formed during the CcP catalytic cycle (Bonagura et al., (1996) Biochemistry 35, 6107 and Bonagura et al., (1999) Biochemistry 38, 5528). Characterization of this mutant, designated CcPK2, showed that the stability of the Trp191 cation radical is dependent on the occupancy of the engineered K(+) site and that the Trp191 radical was much less stable in this mutant than in wild-type CcP. The mutations Met230Leu, Met231Gln, and Met172Ser have now been constructed on the CcPK2 mutant template to test if the Met residues also contribute to the stabilization of the Trp191 cation radical. Crystal structures show that the mutations affect only the local structure near the sites of mutation. Removal of these electronegative residues located less than 8 A from the Trp radical results in a further destabilization of the Trp radical. The characteristic EPR signal associated with the Trp radical is significantly narrowed and is characteristic of a tyrosine radical signal. Double-mixing stopped-flow experiments, where the delay time between the formation of CcP compound I and its mixing with horse heart ferrocytochrome c is varied, show that the stability of the Trp radical decreases as the Met residues are removed from the proximal cavity. When taken together, these results demonstrate a strong correlation between the experimentally determined stability of the Trp191 radical, the enzyme activity, and the calculated electrostatic stabilization of the Trp191 radical.
 ==About this Structure==
-STQ 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 K and HEM as [http://en.wikipedia.org/wiki/ligands ligands]. 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=1STQ OCA].
+STQ 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=K:'>K</scene> and <scene name='pdbligand=HEM:'>HEM</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. 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=1STQ OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Saccharomyces cerevisiae]]
 [[Category: Single protein]]
-[[Category: Barrows, T.P.]]
+[[Category: Barrows, T P.]]
 [[Category: Bhaskar, B.]]
-[[Category: Poulos, T.L.]]
+[[Category: Poulos, T L.]]
 [[Category: HEM]]
 [[Category: K]]
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 [[Category: heme peroxidase]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 02:39:34 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 15:05:04 2008''