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| - | [[Image:1xo1.jpg|left|200px]] | + | {{Seed}} |
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| | {{STRUCTURE_1xo1| PDB=1xo1 | SCENE= }} | | {{STRUCTURE_1xo1| PDB=1xo1 | SCENE= }} |
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| - | '''T5 5'-EXONUCLEASE MUTANT K83A'''
| + | ===T5 5'-EXONUCLEASE MUTANT K83A=== |
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| - | ==Overview==
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| - | Efficient cellular DNA replication requires the activity of a 5'-3' exonuclease. These enzymes are able to hydrolyze DNA.DNA and RNA.DNA substrates exonucleolytically, and they are structure-specific endonucleases. The 5'-3' exonucleases are conserved in organisms as diverse as bacteriophage and mammals. Crystal structures of three representative enzymes identify two divalent-metal-binding sites typically separated by 8-10 A. Site-directed mutagenesis was used to investigate the roles of three lysine residues (K83, K196, and K215) situated near two metal-binding sites in bacteriophage T5 5'-3' exonuclease. Neither K196 nor K215 was essential for either the exo- or the endonuclease activity, but mutation of these residues increased the dissociation constant for the substrate from 5 nM to 200 nM (K196A) and 50 nM (K215A). Biochemical analysis demonstrated that K83 is absolutely required for exonucleolytic activity on single-stranded DNA but is not required for endonucleolytic cleavage of flap structures. Structural analysis of this mutant by x-ray crystallography showed no significant perturbations around the metal-binding sites in the active site. The wild-type protein has different pH optima for endonuclease and exonuclease activities. Taken together, these results suggest that different mechanisms for endo- and exonucleolytic hydrolysis are used by this multifunctional enzyme.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_9874768}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 9874768 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_9874768}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Hydrolase]] | | [[Category: Hydrolase]] |
| | [[Category: Nuclease]] | | [[Category: Nuclease]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 15:16:57 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Jul 28 19:44:55 2008'' |
Revision as of 16:44, 28 July 2008
Template:STRUCTURE 1xo1
T5 5'-EXONUCLEASE MUTANT K83A
Template:ABSTRACT PUBMED 9874768
About this Structure
1XO1 is a Single protein structure of sequence from Enterobacteria phage t5. Full crystallographic information is available from OCA.
Reference
Mutagenesis of conserved lysine residues in bacteriophage T5 5'-3' exonuclease suggests separate mechanisms of endo-and exonucleolytic cleavage., Garforth SJ, Ceska TA, Suck D, Sayers JR, Proc Natl Acad Sci U S A. 1999 Jan 5;96(1):38-43. PMID:9874768
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