We apologize for Proteopedia being slow to respond. For the past two years, a new implementation of Proteopedia has been being built. Soon, it will replace this 18-year old system. All existing content will be moved to the new system at a date that will be announced here.
From Proteopedia
(Difference between revisions)
proteopedia linkproteopedia link
|
|
| Line 1: |
Line 1: |
| - | [[Image:1f8w.jpg|left|200px]] | + | {{Seed}} |
| | + | [[Image:1f8w.png|left|200px]] |
| | | | |
| | <!-- | | <!-- |
| Line 9: |
Line 10: |
| | {{STRUCTURE_1f8w| PDB=1f8w | SCENE= }} | | {{STRUCTURE_1f8w| PDB=1f8w | SCENE= }} |
| | | | |
| - | '''CRYSTAL STRUCTURE OF NADH PEROXIDASE MUTANT: R303M'''
| + | ===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. | + | The line below this paragraph, {{ABSTRACT_PUBMED_10956025}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 10956025 is the PubMed ID number. |
| | + | --> |
| | + | {{ABSTRACT_PUBMED_10956025}} |
| | | | |
| | ==About this Structure== | | ==About this Structure== |
| Line 29: |
Line 33: |
| | [[Category: Interface]] | | [[Category: Interface]] |
| | [[Category: Nad-binding domain]] | | [[Category: Nad-binding domain]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 16:02:41 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 1 02:53:10 2008'' |
Revision as of 23:53, 30 June 2008
Template:STRUCTURE 1f8w
CRYSTAL STRUCTURE OF NADH PEROXIDASE MUTANT: R303M
Template:ABSTRACT PUBMED 10956025
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
Page seeded by OCA on Tue Jul 1 02:53:10 2008
