Document Detail


Identification of regions of rabbit muscle pyruvate kinase important for allosteric regulation by phenylalanine, detected by H/D exchange mass spectrometry.
MedLine Citation:
PMID:  23418858     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Mass spectrometry has been used to determine the number of exchangeable backbone amide protons and the associated rate constants that are altered when rabbit muscle pyruvate kinase (rM1-PYK) binds either the allosteric inhibitor (phenylalanine) or a nonallosteric analogue of the inhibitor. Alanine is used as the nonallosteric analogue because it binds competitively with phenylalanine but elicits a negligible allosteric inhibition, i.e., a negligible reduction in the affinity of rM1-PYK for the substrate, phosphoenolpyruvate. This experimental design is expected to distinguish changes in the protein caused by effector binding (i.e., those changes common upon the addition of alanine vs phenylalanine) from changes associated with allosteric regulation (i.e., those elicited by the addition of phenylalanine binding, but not alanine binding). High-quality peptic fragments covering 98% of the protein were identified. Changes in both the number of exchangeable protons per peptide and in the rate constant associated with exchange highlight regions of the protein with allosteric roles. The set of allosterically relevant peptides identified by this technique includes residues previously identified by mutagenesis to have roles in allosteric regulation by phenylalanine.
Authors:
Charulata B Prasannan; Maria T Villar; Antonio Artigues; Aron W Fenton
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2013-03-06
Journal Detail:
Title:  Biochemistry     Volume:  52     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2013 Mar 
Date Detail:
Created Date:  2013-03-19     Completed Date:  2013-05-09     Revised Date:  2014-03-26    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1998-2006     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Allosteric Regulation
Allosteric Site
Animals
Mass Spectrometry
Models, Molecular
Muscles / chemistry,  enzymology*,  metabolism
Peptides / chemistry,  metabolism
Phenylalanine / metabolism*
Protein Binding
Protein Conformation
Pyruvate Kinase / chemistry*,  metabolism*
Rabbits
Grant Support
ID/Acronym/Agency:
DK78076/DK/NIDDK NIH HHS; R01 DK078076/DK/NIDDK NIH HHS
Chemical
Reg. No./Substance:
0/Peptides; 47E5O17Y3R/Phenylalanine; EC 2.7.1.40/Pyruvate Kinase
Comments/Corrections

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


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