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| - | [[Image:1jci.gif|left|200px]] | + | {{Seed}} |
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| | {{STRUCTURE_1jci| PDB=1jci | SCENE= }} | | {{STRUCTURE_1jci| PDB=1jci | SCENE= }} |
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| - | '''Stabilization of the Engineered Cation-binding Loop in Cytochrome c Peroxidase (CcP)'''
| + | ===Stabilization of the Engineered Cation-binding Loop in Cytochrome c Peroxidase (CcP)=== |
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| - | ==Overview==
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| - | We have previously shown that the K(+) site found in the proximal heme pocket of ascorbate peroxidase (APX) could be successfully engineered into the closely homologous cytochrome c peroxidase (CcP) [Bonagura et al., (1996) Biochemistry 35, 6107-6115; Bonagura et al. (1999) Biochemistry 38, 5538-5545]. In addition, specificity could be switched to binding Ca(2+) as found in other peroxidases [Bonagura et al. (1999) J. Biol. Chem. 274, 37827-37833]. The introduction of a proximal cation-binding site also promotes conversion of the Trp191 containing cation-binding loop from a "closed" to an "open" conformer. In the present study we have changed a crucial hinge residue of the cation-binding loop, Asn195, to Pro which stabilizes the loop, albeit, only in the presence of bound K(+). The crystal structure of this mutant, N195PK2, has been refined to 1.9 A. As predicted, introduction of this crucial hinge residue stabilizes the cation-binding loop in the presence of the bound K(+). As in earlier work, the characteristic EPR signal of Trp191 cation radical becomes progressively weaker with increasing [K(+)] and the lifetime of the Trp191 radical also has been considerably shortened in this mutant. This mutant CcP exhibits reduced enzyme activity, which could be titrated to lower levels with increasing [K(+)] when horse heart cytochrome c is the substrate. However, with yeast cytochrome c as the substrate, the mutant was as active as wild-type at low ionic strength, but 40-fold lower at high ionic strength. We attribute this difference to a change in the rate-limiting step as a function of ionic strength when yeast cytochrome c is the substrate.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_11851415}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 11851415 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_11851415}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Open/closed conformer]] | | [[Category: Open/closed conformer]] |
| | [[Category: Trp191 cationic radical]] | | [[Category: Trp191 cationic radical]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 21:03:25 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 1 20:01:33 2008'' |
Revision as of 17:01, 1 July 2008
Template:STRUCTURE 1jci
Stabilization of the Engineered Cation-binding Loop in Cytochrome c Peroxidase (CcP)
Template:ABSTRACT PUBMED 11851415
About this Structure
1JCI is a Single protein structure of sequence from Saccharomyces cerevisiae. Full crystallographic information is available from OCA.
Reference
Cation-induced stabilization of the engineered cation-binding loop in cytochrome c peroxidase (CcP)., Bhaskar B, Bonagura CA, Li H, Poulos TL, Biochemistry. 2002 Feb 26;41(8):2684-93. PMID:11851415
Page seeded by OCA on Tue Jul 1 20:01:33 2008
