Document Detail


Modification of residue 42 of the active site loop with a lysine-mimetic side chain rescues isochorismate-pyruvate lyase activity in Pseudomonas aeruginosa PchB.
MedLine Citation:
PMID:  22970849     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
PchB is an isochorismate-pyruvate lyase from Pseudomonas aeruginosa. A positively charged lysine residue is located in a flexible loop that behaves as a lid to the active site, and the lysine residue is required for efficient production of salicylate. A variant of PchB that lacks the lysine at residue 42 has a reduced catalytic free energy of activation of up to 4.4 kcal/mol. Construction of a lysine isosteric residue bearing a positive charge at the appropriate position leads to the recovery of 2.5-2.7 kcal/mol (about 60%) of the 4.4 kcal/mol by chemical rescue. Exogenous addition of ethylamine to the K42A variant leads to a neglible recovery of activity (0.180 kcal/mol, roughly 7% rescue), whereas addition of propylamine caused an additional modest loss in catalytic power (0.056 kcal/mol, or 2% loss). This is consistent with the view that (a) the lysine-42 residue is required in a specific conformation to stabilize the transition state and (b) the correct conformation is achieved for a lysine-mimetic side chain at site 42 in the course of loop closure, as expected for transition-state stabilization by the side chain ammonio function. That the positive charge is the main effector of transition state stabilization is shown by the construction of a lysine-isosteric residue capable of exerting steric effects and hydrogen bonding but not electrostatic effects, leading to a modest increase of catalytic power (0.267-0.505 kcal/mol of catalytic free energy, or roughly 6-11% rescue).
Authors:
José Olucha; Kathleen M Meneely; Audrey L Lamb
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2012-09-12
Journal Detail:
Title:  Biochemistry     Volume:  51     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2012 Sep 
Date Detail:
Created Date:  2013-02-07     Completed Date:  2013-04-01     Revised Date:  2013-10-17    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  7525-32     Citation Subset:  IM    
Affiliation:
Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA.
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MeSH Terms
Descriptor/Qualifier:
Base Sequence
Carbon-Oxygen Lyases / chemistry,  metabolism*
Catalysis
Catalytic Domain
Circular Dichroism
DNA Primers
Kinetics
Lysine / chemistry*
Models, Molecular
Molecular Mimicry*
Pseudomonas aeruginosa / enzymology*
Thermodynamics
Grant Support
ID/Acronym/Agency:
K02 AI093675/AI/NIAID NIH HHS; K02 AI093675/AI/NIAID NIH HHS; P20 RR016475/RR/NCRR NIH HHS; P20 RR016475/RR/NCRR NIH HHS; R01 AI077725/AI/NIAID NIH HHS; R01 AI77725/AI/NIAID NIH HHS; T32 GM008545/GM/NIGMS NIH HHS; T32 GM08545/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/DNA Primers; 56-87-1/Lysine; EC 4.2.-/Carbon-Oxygen Lyases
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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