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1f8w
From Proteopedia
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|PDB= 1f8w |SIZE=350|CAPTION= <scene name='initialview01'>1f8w</scene>, resolution 2.45Å | |PDB= 1f8w |SIZE=350|CAPTION= <scene name='initialview01'>1f8w</scene>, resolution 2.45Å | ||
|SITE= | |SITE= | ||
| - | |LIGAND= <scene name='pdbligand=FAD:FLAVIN-ADENINE DINUCLEOTIDE'>FAD</scene> | + | |LIGAND= <scene name='pdbligand=CSX:S-OXY+CYSTEINE'>CSX</scene>, <scene name='pdbligand=FAD:FLAVIN-ADENINE+DINUCLEOTIDE'>FAD</scene> |
| - | |ACTIVITY= [http://en.wikipedia.org/wiki/NADH_peroxidase NADH peroxidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.11.1.1 1.11.1.1] | + | |ACTIVITY= <span class='plainlinks'>[http://en.wikipedia.org/wiki/NADH_peroxidase NADH peroxidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.11.1.1 1.11.1.1] </span> |
|GENE= | |GENE= | ||
| + | |DOMAIN= | ||
| + | |RELATEDENTRY=[[1joa|1JOA]], [[1nhp|1NHP]], [[1nhq|1NHQ]], [[1nhr|1NHR]], [[1nhs|1NHS]], [[1npx|1NPX]] | ||
| + | |RESOURCES=<span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=1f8w FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1f8w OCA], [http://www.ebi.ac.uk/pdbsum/1f8w PDBsum], [http://www.rcsb.org/pdb/explore.do?structureId=1f8w RCSB]</span> | ||
}} | }} | ||
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[[Category: Hol, W G.J.]] | [[Category: Hol, W G.J.]] | ||
[[Category: Yeh, J I.]] | [[Category: Yeh, J I.]] | ||
| - | [[Category: FAD]] | ||
[[Category: fad]] | [[Category: fad]] | ||
[[Category: interface]] | [[Category: interface]] | ||
[[Category: nad-binding domain]] | [[Category: nad-binding domain]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun Mar 30 20:18:16 2008'' |
Revision as of 17:18, 30 March 2008
| |||||||
| , resolution 2.45Å | |||||||
|---|---|---|---|---|---|---|---|
| Ligands: | , | ||||||
| Activity: | NADH peroxidase, with EC number 1.11.1.1 | ||||||
| Related: | 1JOA, 1NHP, 1NHQ, 1NHR, 1NHS, 1NPX
| ||||||
| Resources: | FirstGlance, OCA, PDBsum, RCSB | ||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||
CRYSTAL STRUCTURE OF NADH PEROXIDASE MUTANT: R303M
Overview
The crystal structure of the flavoprotein NADH peroxidase shows that the Arg303 side chain forms a hydrogen bond with the active-site His10 imidazole and is therefore likely to influence the catalytic mechanism. Dithionite titration of an R303M mutant [E(FAD, Cys42-sulfenic acid)] yields a two-electron reduced intermediate (EH(2)) with enhanced flavin fluorescence and almost no charge-transfer absorbance at pH 7.0; the pK(a) for the nascent Cys42-SH is increased by over 3.5 units in comparison with the wild-type EH(2) pK(a) of </=4.5. NADH titration of the mutant peroxidase yields the same EH(2) intermediate, but in contrast to the behavior of wild-type enzyme, this species can be reduced directly to an EH(4).NAD(+) complex. Kinetic analyses demonstrate that the R303M mutant is severely compromised, although active, with k(cat) = 3 s(-)(1) at pH 7.0, 5 degrees C; enzyme-monitored turnover results indicate that the steady-state consists predominantly of an E-FADH(2).NAD(+) species. When the oxidized mutant is reacted anaerobically with 0.9 equiv of NADH/FAD, a clearly biphasic pattern is observed at 450 nm; relatively rapid flavin reduction is followed by reoxidation at 2.6-2.7 s(-)(1) ( approximately k(cat)). Thus replacement of Arg303 with Met leads to an altered peroxidase form in which the rate-limiting step in turnover is the intramolecular transfer of electrons from FADH(2) --> Cys42-SOH. The crystal structure of the R303M peroxidase has been refined at 2.45 A resolution. In addition to eliminating the Arg303 interactions with His10 and Glu14, the mutant exhibits a significant change in the conformation of the Cys42-SOH side chain relative to FAD and His10 in particular. These and other results provide a detailed understanding of Arg303 and its role in the structure and mechanism of this unique flavoprotein peroxidase.
About this Structure
1F8W is a Single protein structure of sequence from Enterococcus faecalis. Full crystallographic information is available from OCA.
Reference
Analysis of the kinetic and redox properties of the NADH peroxidase R303M mutant: correlation with the crystal structure., Crane EJ 3rd, Yeh JI, Luba J, Claiborne A, Biochemistry. 2000 Aug 29;39(34):10353-64. PMID:10956025
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