2ga3: Difference between revisions

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[[Image:2ga3.gif|left|200px]]
{{Seed}}
[[Image:2ga3.png|left|200px]]


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{{STRUCTURE_2ga3|  PDB=2ga3  |  SCENE=  }}  
{{STRUCTURE_2ga3|  PDB=2ga3  |  SCENE=  }}  


'''Structure of S102T E. coli Alkaline Phosphatase-phosphate intermediate at 2.20A resolution'''
===Structure of S102T E. coli Alkaline Phosphatase-phosphate intermediate at 2.20A resolution===




==Overview==
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We report here the construction of a mutant version of Escherichia coli alkaline phosphatase (AP) in which the active site Ser was replaced by Thr (S102T), in order to investigate whether the enzyme can utilize Thr as the nucleophile and whether the rates of the critical steps in the mechanism are altered by the substitution. The mutant AP with Thr at position 102 exhibited an approximately 4000-fold decrease in k(cat) along with a small decrease in Km. The decrease in catalytic efficiency of approximately 2000-fold was a much smaller drop than that observed when Ala or Gly were substituted at position 102. The mechanism by which Thr can substitute for Ser in AP was further investigated by determining the X-ray structure of the S102T enzyme in the presence of the Pi (S102T_Pi), and after soaking the crystals with substrate (S102T_sub). In the S102T_Pi structure, the Pi was coordinated differently with its position shifted by 1.3 A compared to the structure of the wild-type enzyme in the presence of Pi. In the S102T_sub structure, a covalent Thr-Pi intermediate was observed, instead of the expected bound substrate. The stereochemistry of the phosphorus in the S102T_sub structure was inverted compared to the stereochemistry in the wild-type structure, as would be expected after the first step of a double in-line displacement mechanism. We conclude that the S102T mutation resulted in a shift in the rate-determining step in the mechanism allowing us to trap the covalent intermediate of the reaction in the crystal.
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{{ABSTRACT_PUBMED_17008720}}


==About this Structure==
==About this Structure==
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[[Category: Side chain conformation]]
[[Category: Side chain conformation]]
[[Category: X-ray crystallography]]
[[Category: X-ray crystallography]]
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun May  4 04:52:25 2008''
 
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Jul 28 15:55:13 2008''

Revision as of 12:55, 28 July 2008

File:2ga3.png

Template:STRUCTURE 2ga3

Structure of S102T E. coli Alkaline Phosphatase-phosphate intermediate at 2.20A resolution

Template:ABSTRACT PUBMED 17008720

About this Structure

2GA3 is a Single protein structure of sequence from Escherichia coli. Full crystallographic information is available from OCA.

Reference

Trapping the tetrahedral intermediate in the alkaline phosphatase reaction by substitution of the active site serine with threonine., Wang J, Kantrowitz ER, Protein Sci. 2006 Oct;15(10):2395-401. PMID:17008720

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