1cyf: Difference between revisions
New page: left|200px<br /><applet load="1cyf" size="450" color="white" frame="true" align="right" spinBox="true" caption="1cyf, resolution 2.35Å" /> '''IDENTIFYING THE PHYS... |
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[[Image:1cyf.jpg|left|200px]]<br /><applet load="1cyf" size=" | [[Image:1cyf.jpg|left|200px]]<br /><applet load="1cyf" size="350" color="white" frame="true" align="right" spinBox="true" | ||
caption="1cyf, resolution 2.35Å" /> | caption="1cyf, resolution 2.35Å" /> | ||
'''IDENTIFYING THE PHYSIOLOGICAL ELECTRON TRANSFER SITE OF CYTOCHROME C PEROXIDASE BY STRUCTURE-BASED ENGINEERING'''<br /> | '''IDENTIFYING THE PHYSIOLOGICAL ELECTRON TRANSFER SITE OF CYTOCHROME C PEROXIDASE BY STRUCTURE-BASED ENGINEERING'''<br /> | ||
==Overview== | ==Overview== | ||
A technique was developed to evaluate whether electron transfer (ET) | A technique was developed to evaluate whether electron transfer (ET) complexes formed in solution by the cloned cytochrome c peroxidase [CcP(MI)] and cytochromes c from yeast (yCc) and horse (hCc) are structurally similar to those seen in the respective crystal structures. Site-directed mutagenesis was used to convert the sole Cys of the parent enzyme (Cys 128) to Ala, and a Cys residue was introduced at position 193 of CcP(MI), the point of closest contact between CcP(MI) and yCc in the crystal structure. Cys 193 was then modified with a bulky sulfhydryl reagent, 3-(N-maleimidylpropionyl)-biocytin (MPB), to prevent yCc from binding at the site seen in the crystal. The MPB modification has no effect on overall enzyme structure but causes 20-100-fold decreases in transient and steady-state ET reaction rates with yCc. The MPB modification causes only 2-3-fold decreases in ET reaction rates with hCc, however. This differential effect is predicted by modeling studies based on the crystal structures and indicates that solution phase ET complexes closely resemble the crystalline complexes. The low rate of catalysis of the MPB-enzyme was constant for yCc in buffers of 20-160 mM ionic strength. This indicates that the low affinity complex formed between CcP(MI) and yCc at low ionic strength is not reactive in ET. | ||
==About this Structure== | ==About this Structure== | ||
1CYF 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:// | 1CYF 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=1CYF OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: Saccharomyces cerevisiae]] | [[Category: Saccharomyces cerevisiae]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Han, G | [[Category: Han, G W.]] | ||
[[Category: Kraut, J.]] | [[Category: Kraut, J.]] | ||
[[Category: Miller, M | [[Category: Miller, M A.]] | ||
[[Category: HEM]] | [[Category: HEM]] | ||
[[Category: oxidoreductase (h2o2(a))]] | [[Category: oxidoreductase (h2o2(a))]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:10:58 2008'' | ||
Revision as of 13:11, 21 February 2008
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IDENTIFYING THE PHYSIOLOGICAL ELECTRON TRANSFER SITE OF CYTOCHROME C PEROXIDASE BY STRUCTURE-BASED ENGINEERING
OverviewOverview
A technique was developed to evaluate whether electron transfer (ET) complexes formed in solution by the cloned cytochrome c peroxidase [CcP(MI)] and cytochromes c from yeast (yCc) and horse (hCc) are structurally similar to those seen in the respective crystal structures. Site-directed mutagenesis was used to convert the sole Cys of the parent enzyme (Cys 128) to Ala, and a Cys residue was introduced at position 193 of CcP(MI), the point of closest contact between CcP(MI) and yCc in the crystal structure. Cys 193 was then modified with a bulky sulfhydryl reagent, 3-(N-maleimidylpropionyl)-biocytin (MPB), to prevent yCc from binding at the site seen in the crystal. The MPB modification has no effect on overall enzyme structure but causes 20-100-fold decreases in transient and steady-state ET reaction rates with yCc. The MPB modification causes only 2-3-fold decreases in ET reaction rates with hCc, however. This differential effect is predicted by modeling studies based on the crystal structures and indicates that solution phase ET complexes closely resemble the crystalline complexes. The low rate of catalysis of the MPB-enzyme was constant for yCc in buffers of 20-160 mM ionic strength. This indicates that the low affinity complex formed between CcP(MI) and yCc at low ionic strength is not reactive in ET.
About this StructureAbout this Structure
1CYF 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
Identifying the physiological electron transfer site of cytochrome c peroxidase by structure-based engineering., Miller MA, Geren L, Han GW, Saunders A, Beasley J, Pielak GJ, Durham B, Millett F, Kraut J, Biochemistry. 1996 Jan 23;35(3):667-73. PMID:8547245
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