Document Detail

Probing the regulatory site of Escherichia coli aspartate transcarbamoylase by site-specific mutagenesis.
MedLine Citation:
PMID:  1731936     Owner:  NLM     Status:  MEDLINE    
The effector binding site of Escherichia coli aspartate transcarbamoylase, composed of the triphosphate and ribose-base subsites, is located on the regulatory (r) chains of the enzyme. In order to probe the function of amino acid side chains at this nucleotide triphosphate site, site-specific mutagenesis was used to create three mutant versions of the enzyme. On the basis of the three-dimensional structure of the enzyme with CTP bound, three residues were selected. Specifically, Arg-96r was replaced with Gln, and His-20r and Tyr-89r were both replaced with Ala. Analyses of these mutant enzymes indicate that none of these substitutions significantly alter the catalytic properties of the enzyme. However, the mutations at His-20r and Tyr-89r produced altered response to the regulatory nucleotides. For the His-20r----Ala enzyme, the affinities of the enzyme for ATP and CTP are reduced 40-fold and 10-fold, respectively, when compared with the wild-type enzyme. Furthermore, CTP is able to inhibit the His-20r----Ala enzyme 40% more than the wild-type enzyme. In the case of the Tyr-89r----Ala enzyme. ATP can increase the mutant enzyme's activity 181% compared to 157% for the wild-type enzyme, while simultaneously the affinity of this enzyme for ATP decreases about 70%. These results suggest that Tyr-89r does have an indirect role in the discrimination between ATP and CTP. The His-20r----Ala enzyme shows no UTP synergistic inhibition in the presence of CTP.(ABSTRACT TRUNCATED AT 250 WORDS)
Y Zhang; E R Kantrowitz
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Biochemistry     Volume:  31     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  1992 Jan 
Date Detail:
Created Date:  1992-02-25     Completed Date:  1992-02-25     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  792-8     Citation Subset:  IM    
Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02167.
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MeSH Terms
Adenosine Triphosphate / metabolism
Amino Acid Sequence
Aspartate Carbamoyltransferase / genetics,  metabolism*
Binding Sites
Cytidine Triphosphate / metabolism
Escherichia coli / enzymology*,  genetics
Models, Molecular
Mutagenesis, Site-Directed*
Protein Conformation
Recombinant Proteins / metabolism
Uridine Triphosphate / metabolism
Grant Support
Reg. No./Substance:
0/Recombinant Proteins; 56-65-5/Adenosine Triphosphate; 63-39-8/Uridine Triphosphate; 65-47-4/Cytidine Triphosphate; EC Carbamoyltransferase

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

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