Document Detail

Reduced activity of bamhi variants c54i, c64w, and c54d/c64r is consistent with the substrate-assisted catalysis model.
MedLine Citation:
PMID:  11549269     Owner:  NLM     Status:  MEDLINE    
Three specific mutants, C54I, C54W, and a double-mutant C54D:C64R of restriction endonuclease BamHI, were generated and studied to investigate the role, if any, of the 54th and 64th cysteine residues in the catalysis of BamHI. The mutation was achieved using the megaprimer approach for PCR. The mutant genes were cloned and characterized by sequencing. The mutant and the wild-type proteins were expressed and purified and their kinetic parameters were determined using short synthetic oligonucleotides as substrates. All mutants had higher K(m) values than that of the wild-type enzyme suggesting a decrease in the affinity of the enzyme for its substrate. The mutant protein C54W showed significant changes in the CD spectra vis-a-vis wild-type enzyme and had the lowest K(m)/K(cat) value among the mutants indicative of changes in the secondary structure of the protein. The melting curves of the mutant proteins overlapped that of the wild-type enzyme. Analysis of the K(cat) values in the context of cocrystal structure suggests that the effect of Cys54 mutation is probably through the perturbation of the local structure whereas reduced activity of the double mutant is consistent with the substrate-assisted catalysis mechanism.
A S Acharya; K B Roy
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biochemical and biophysical research communications     Volume:  287     ISSN:  0006-291X     ISO Abbreviation:  Biochem. Biophys. Res. Commun.     Publication Date:  2001 Sep 
Date Detail:
Created Date:  2001-09-10     Completed Date:  2001-10-04     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0372516     Medline TA:  Biochem Biophys Res Commun     Country:  United States    
Other Details:
Languages:  eng     Pagination:  153-9     Citation Subset:  IM    
Copyright Information:
Copyright 2001 Academic Press.
Centre for Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India.
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MeSH Terms
Amino Acid Substitution
Cloning, Molecular
Deoxyribonuclease BamHI / genetics,  metabolism*
Escherichia coli
Mutagenesis, Site-Directed
Protein Conformation
Recombinant Proteins / metabolism
SOS Response (Genetics) / physiology
Substrate Specificity
Reg. No./Substance:
0/Recombinant Proteins; EC 3.1.21.-/Deoxyribonuclease BamHI

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