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Mechanistic insights into catalysis ...

Kamadurai, Hari Bascar.

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Mechanistic insights into catalysis and allosteric enzyme activation in bacteriophage lambda integrase.

Record Type:	Language materials, printed : Monograph/item
Title/Author:	Mechanistic insights into catalysis and allosteric enzyme activation in bacteriophage lambda integrase./
Author:	Kamadurai, Hari Bascar.
Description:	195 p.
Notes:	Adviser: Mark Foster.
Contained By:	Dissertation Abstracts International68-01B.
Subject:	Biology, Virology. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3247926

Mechanistic insights into catalysis and allosteric enzyme activation in bacteriophage lambda integrase.
Kamadurai, Hari Bascar.

Mechanistic insights into catalysis and allosteric enzyme activation in bacteriophage lambda integrase. - 195 p.

Adviser: Mark Foster.

Thesis (Ph.D.)--The Ohio State University, 2007.

Bacteriophage lambda integrase (lambda-Int) is a tyrosine-dependent DNA recombinase that enables site-specific integration and excision of the bacteriophage viral genome into and out of its bacterial host genome. Recombination is brought about by the action of four lambda-Int molecules via two series of DNA cleavage, strand exchange, and ligation reactions across specific sequences. Of the three domains in lambda-Int, the core-binding domain (Int CB) and the catalytic domain (IntCat) are required for site-specific DNA recognition and cleavage. A conserved tyrosine (Tyr342) in IntCat mediates the DNA cleavage reaction through a nucleophilic attack on the scissile phosphate, which results in its covalent linkage to the cleaved DNA through a stable phosphotyrosine bond. Previous solution and crystallographic studies suggest that in the absence of the substrate DNA, IntCat may exist in an inactive conformation in which the tyrosine nucleophile is positioned away from the other critical residues involved in catalysis and that a structural rearrangement would be required to adopt a cleavage-competent conformation. A mass spectrometry-based study in this dissertation, through examining the changes in the electrospray ionization pattern of Int Cat due to the presence of DNA, provides support to such structural arrangements occurring in IntCat during its interaction with DNA. However, subsequent studies suggest that DNA binding alone might be insufficient to populate the active conformation of IntCat molecules, but show that IntCB could stimulate the activity of Int Cat in a DNA sequence-dependent manner. Structural and biochemical studies suggest a novel mechanism for this stimulatory effect, in which Int CB stimulates the activity of IntCat by deforming the substrate DNA that is bound between the two domains. This dissertation provides experimental evidence for IntCB-mediated structural changes in the DNA that occur concomitantly with IntCB folding. This study also provides critical insights into the binding energetics and sequence-specificity in IntCB-DNA interactions as well as important leads towards understanding the structural basis for the allostery.Subjects--Topical Terms:

1019068
Biology, Virology.

Mechanistic insights into catalysis and allosteric enzyme activation in bacteriophage lambda integrase.
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035 $a (UMI)AAI3247926
035 $a AAI3247926
040 $a UMI $c UMI
100 1 $a Kamadurai, Hari Bascar. $3 1263201
245 1 0 $a Mechanistic insights into catalysis and allosteric enzyme activation in bacteriophage lambda integrase.
300 $a 195 p.
500 $a Adviser: Mark Foster.
500 $a Source: Dissertation Abstracts International, Volume: 68-01, Section: B, page: 0273.
502 $a Thesis (Ph.D.)--The Ohio State University, 2007.
520 $a Bacteriophage lambda integrase (lambda-Int) is a tyrosine-dependent DNA recombinase that enables site-specific integration and excision of the bacteriophage viral genome into and out of its bacterial host genome. Recombination is brought about by the action of four lambda-Int molecules via two series of DNA cleavage, strand exchange, and ligation reactions across specific sequences. Of the three domains in lambda-Int, the core-binding domain (Int CB) and the catalytic domain (IntCat) are required for site-specific DNA recognition and cleavage. A conserved tyrosine (Tyr342) in IntCat mediates the DNA cleavage reaction through a nucleophilic attack on the scissile phosphate, which results in its covalent linkage to the cleaved DNA through a stable phosphotyrosine bond. Previous solution and crystallographic studies suggest that in the absence of the substrate DNA, IntCat may exist in an inactive conformation in which the tyrosine nucleophile is positioned away from the other critical residues involved in catalysis and that a structural rearrangement would be required to adopt a cleavage-competent conformation. A mass spectrometry-based study in this dissertation, through examining the changes in the electrospray ionization pattern of Int Cat due to the presence of DNA, provides support to such structural arrangements occurring in IntCat during its interaction with DNA. However, subsequent studies suggest that DNA binding alone might be insufficient to populate the active conformation of IntCat molecules, but show that IntCB could stimulate the activity of Int Cat in a DNA sequence-dependent manner. Structural and biochemical studies suggest a novel mechanism for this stimulatory effect, in which Int CB stimulates the activity of IntCat by deforming the substrate DNA that is bound between the two domains. This dissertation provides experimental evidence for IntCB-mediated structural changes in the DNA that occur concomitantly with IntCB folding. This study also provides critical insights into the binding energetics and sequence-specificity in IntCB-DNA interactions as well as important leads towards understanding the structural basis for the allostery.
590 $a School code: 0168.
650 4 $a Biology, Virology. $3 1019068
650 4 $a Biophysics, General. $3 1019105
650 4 $a Chemistry, Biochemistry. $3 1017722
690 $a 0487
690 $a 0720
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710 2 $a The Ohio State University. $3 718944
773 0 $t Dissertation Abstracts International $g 68-01B.
790 $a 0168
790 1 0 $a Foster, Mark, $e advisor
791 $a Ph.D.
792 $a 2007
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3247926