Document Detail

Chemical mutagenesis of vaccinia DNA topoisomerase lysine 167 provides insights to the catalysis of DNA transesterification.
MedLine Citation:
PMID:  23317114     Owner:  NLM     Status:  MEDLINE    
Vaccinia DNA topoisomerase IB (TopIB) relaxes supercoils by forming and resealing a covalent DNA-(3'-phosphotyrosyl(274))-enzyme intermediate. Conserved active site side chains promote the attack of Tyr274 on the scissile phosphodiester via transition state stabilization and general acid catalysis. Two essential side chains, Lys167 and Arg130, act in concert to protonate and expel the 5'-O leaving group. Here we gained new insights to catalysis through chemical mutagenesis of Lys167. Changing Lys167 to cysteine crippled the DNA cleavage and religation transesterification steps (k(cl) = 4.3 × 10(-4) s(-1); k(rel) = 9 × 10(-4) s(-1)). The transesterification activities of the K167C enzyme were revived by in vitro alkylation with 2-bromoethylamine (k(cl) = 0.031 s(-1); k(rel) ≥ 0.4 s(-1)) and 3-bromopropylamine (k(cl) = 0.013 s(-1); k(rel) = 0.22 s(-1)), which convert the cysteines to γ-thialysine and γ-thiahomolysine, respectively. These chemically installed lysine analogues were more effective than a genetically programmed arginine 167 substitution characterized previously. The modest differences in the transesterification rates of the 2-bromoethylamine- and 3-bromopropylamine-treated enzymes highlight that TopIB is tolerant of a longer homolysine side chain for assembly of the active site and formation of the transition state.
Lyudmila Yakovleva; Stewart Shuman
Related Documents :
16000444 - Suspension microarray with dendrimer signal amplification allows direct and high-throug...
12089254 - Detection and genotyping of human group a rotaviruses by oligonucleotide microarray hyb...
7790734 - Large stationary microstrip arrays for superficial microwave hyperthermia at 433 mhz: s...
12626724 - Nucleic acid capture assay, a new method for direct quantitation of nucleic acids.
2258054 - Simplified colorimetric analysis of polymerase chain reactions: detection of hiv sequen...
7557074 - Flow cytometric analysis of the dna content in colorectal adenomas with focal cancers.
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2013-01-23
Journal Detail:
Title:  Biochemistry     Volume:  52     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2013 Feb 
Date Detail:
Created Date:  2013-02-05     Completed Date:  2013-03-29     Revised Date:  2014-02-06    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  984-91     Citation Subset:  IM    
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Base Sequence
Cysteine / analogs & derivatives,  genetics,  metabolism
DNA Topoisomerases, Type I / chemistry*,  genetics,  metabolism*
DNA, Viral / chemistry,  metabolism*
Lysine / analogs & derivatives,  genetics*,  metabolism
Models, Molecular
Point Mutation
Vaccinia / virology*
Vaccinia virus / chemistry,  enzymology*,  genetics,  metabolism
Grant Support
Reg. No./Substance:
0/DNA, Viral; EC Topoisomerases, Type I; K3Z4F929H6/Lysine; K848JZ4886/Cysteine

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

Previous Document:  Asymptomatic primary tuberculous pleurisy with intense 18-fluorodeoxyglucose uptake mimicking malign...
Next Document:  N200 and P300 as orthogonal and integrable indicators of distinct awareness and recognition processe...