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Investigations of Escherichia coli a...

Hughes, Ivana Nikolic.

Investigations of Escherichia coli alkaline phosphatase and phosphoryl transfer via comparative analysis.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Investigations of Escherichia coli alkaline phosphatase and phosphoryl transfer via comparative analysis.
Author:	Hughes, Ivana Nikolic.
Description:	183 p.
Notes:	Adviser: Daniel Herschlag.
Notes:	Source: Dissertation Abstracts International, Volume: 66-08, Section: B, page: 4357.
Contained By:	Dissertation Abstracts International66-08B.
Subject:	Engineering, Chemical.
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3187296
ISBN:	9780542295140

Investigations of Escherichia coli alkaline phosphatase and phosphoryl transfer via comparative analysis.
Hughes, Ivana Nikolic.

Investigations of Escherichia coli alkaline phosphatase and phosphoryl transfer via comparative analysis. - 183 p.

Adviser: Daniel Herschlag.

Thesis (Ph.D.)--Stanford University, 2005.

In addition to transfer of the phosphoryl group, AP's native activity, AP catalyzes hydrolysis of sulfate monoesters. To understand why AP prefers phosphate monoesters over its other substrates and what contributes to its unsurpassed catalytic proficiency for the native reaction, we have analyzed in detail AP's phosphatase and sulfatase activities. We have probed the energetic contribution to catalysis of phosphate and sulfate monoester hydrolysis of each presumed transition state interaction between AP and substrate. Despite more than a 1011-fold greater catalysis of the phosphate monoester reaction, relative to the sulfate monoester reaction, several catalytic interactions and strategies appear to provide the same or similar rate enhancements: stabilization and positioning of non-bridging oxygens by Arg166; stabilization of the leaving group by one of the active site Zn2+ ions; and activation of the Ser102 nucleophile by the other active site Zn2+ ion. The transition state interaction that appears to be substantially different for the two substrates is that of a non-bridging oxygen atom sequestered between the two active site zinc ions. The role of this electrostatic interaction in catalysis is investigated and discussed in detail.

ISBN: 9780542295140Subjects--Topical Terms:

226989
Engineering, Chemical.

Investigations of Escherichia coli alkaline phosphatase and phosphoryl transfer via comparative analysis.
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100 0 $a Hughes, Ivana Nikolic. $3 244877
245 1 0 $a Investigations of Escherichia coli alkaline phosphatase and phosphoryl transfer via comparative analysis.
300 $a 183 p.
500 $a Adviser: Daniel Herschlag.
500 $a Source: Dissertation Abstracts International, Volume: 66-08, Section: B, page: 4357.
502 $a Thesis (Ph.D.)--Stanford University, 2005.
520 # $a In addition to transfer of the phosphoryl group, AP's native activity, AP catalyzes hydrolysis of sulfate monoesters. To understand why AP prefers phosphate monoesters over its other substrates and what contributes to its unsurpassed catalytic proficiency for the native reaction, we have analyzed in detail AP's phosphatase and sulfatase activities. We have probed the energetic contribution to catalysis of phosphate and sulfate monoester hydrolysis of each presumed transition state interaction between AP and substrate. Despite more than a 1011-fold greater catalysis of the phosphate monoester reaction, relative to the sulfate monoester reaction, several catalytic interactions and strategies appear to provide the same or similar rate enhancements: stabilization and positioning of non-bridging oxygens by Arg166; stabilization of the leaving group by one of the active site Zn2+ ions; and activation of the Ser102 nucleophile by the other active site Zn2+ ion. The transition state interaction that appears to be substantially different for the two substrates is that of a non-bridging oxygen atom sequestered between the two active site zinc ions. The role of this electrostatic interaction in catalysis is investigated and discussed in detail.
520 # $a In the last part of our work, we assessed the ability of vibrational spectroscopy to provide a probe of the local electrostatic environment experienced by the phosphoryl group. We examined differences in the bonding properties of the phosphoryl group, via vibrational spectroscopy, from changes in the phosphoryl group substituent and solvent environment. Our results and analysis suggest that ground-state distortions of substrates bound to enzymes can provide a readout of the electrostatic environment of active sites.
520 # $a Today's man-made catalysts lag far behind their biological counterparts in their ability to accelerate chemical reactions. An understanding of how enzymes achieve tremendous rate enhancements may aid in efforts to engineer better and/or new catalysts. Herein we study the mechanism of E. coli Alkaline Phosphatase (AP), a representative of what appears to be the most potent class of catalysts, two-metal-ion enzymes. Although much prior structural and biochemical data is available for this enzyme, the origin of its extraordinary catalytic proficiency [(kcat/KM/k w, unitless] > 1020 is poorly understood.
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650 # 0 $a Chemistry, Biochemistry. $3 226900
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773 0 # $g 66-08B. $t Dissertation Abstracts International
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790 1 0 $a Herschlag, Daniel, $e advisor
791 $a Ph.D.
792 $a 2005
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