Document Detail


Mutational analysis of metallo-beta-lactamase CcrA from Bacteroides fragilis.
MedLine Citation:
PMID:  10985778     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
In an effort to evaluate the roles of Lys184, Asn193, and Asp103 in the binding and catalysis of metallo-beta-lactamase CcrA from Bacteroides fragilis, site-directed mutants of CcrA were generated and characterized using metal analyses, CD spectroscopy, and kinetic studies. Three Lys184 mutants were generated where the lysine was replaced with alanine, leucine, and glutamate, and the analysis of these mutants indicates that Lys184 is not greatly involved in binding of cephalosporins to CcrA; however, this residue does have a significant role in binding of penicillin G. Three Asn193 mutants were generated where the asparagine was replaced with alanine, leucine, and aspartate, and these mutants exhibited <4-fold decrease in k(cat), suggesting that Asn193 does not play a large role in catalysis. However, stopped-flow visible kinetic studies showed that the Asn193 mutants exhibit a slower substrate decay rate and no change in the product formation rate as compared with wild-type CcrA. These results support the proposed role of Asn193 in interacting with and activating substrate during catalysis. Two Asp103 mutants were generated where the aspartate was replaced with serine and cysteine. The D103C and D103S mutants bind the same amount of Zn(II) as wild-type CcrA and exhibited a 10(2)-fold and 10(5)-fold decrease in activity, respectively. Results from solvent isotope, proton inventory, and rapid-scanning visible studies suggest that Asp103 plays a role in generating the enzyme intermediate but does not donate a proton to the enzyme intermediate during the rate-limiting step of the catalytic mechanism.
Authors:
M P Yanchak; R A Taylor; M W Crowder
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Biochemistry     Volume:  39     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  2000 Sep 
Date Detail:
Created Date:  2000-10-19     Completed Date:  2000-10-19     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  11330-9     Citation Subset:  IM    
Affiliation:
Department of Chemistry and Biochemistry, 112 Hughes Hall, Miami University, Oxford, Ohio 45056, USA.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Substitution / genetics
Asparagine / genetics
Aspartic Acid / genetics
Bacterial Proteins*
Bacteroides fragilis / enzymology*,  genetics*
Cysteine / genetics
DNA Mutational Analysis
DNA, Bacterial / analysis*,  biosynthesis,  isolation & purification
Deuterium
Gene Expression Regulation, Bacterial
Hydrogen-Ion Concentration
Kinetics
Metalloproteins / biosynthesis,  genetics*,  isolation & purification
Mutagenesis, Site-Directed
Protons
Recombinant Proteins / biosynthesis,  isolation & purification
Serine / genetics
Solvents
Zinc / analysis
beta-Lactamases / biosynthesis,  genetics*,  isolation & purification
Grant Support
ID/Acronym/Agency:
R29 AI40052/AI/NIAID NIH HHS
Chemical
Reg. No./Substance:
0/Bacterial Proteins; 0/DNA, Bacterial; 0/Metalloproteins; 0/Protons; 0/Recombinant Proteins; 0/Solvents; 52-90-4/Cysteine; 56-45-1/Serine; 56-84-8/Aspartic Acid; 7006-34-0/Asparagine; 7440-66-6/Zinc; 7782-39-0/Deuterium; EC 3.5.2.6/beta-Lactamases; EC 3.5.2.6/carbapenemase

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


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