HOMEPRODUCTSSERVICESCOMPANYCONTACTFAQResearchDictionaryPharmaMobileSign Up FREE or Login

Chemical mutagenesis of vaccinia DNA topoisomerase lysine 167 provides insights to the catalysis of DNA transesterification.

Abstract
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.
AuthorsLyudmila Yakovleva, Stewart Shuman
JournalBiochemistry (Biochemistry) Vol. 52 Issue 5 Pg. 984-91 (Feb 5 2013) ISSN: 1520-4995 [Electronic] United States
PMID23317114 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)
Chemical References
  • DNA, Viral
  • DNA Topoisomerases, Type I
  • Lysine
  • Cysteine
Topics
  • Base Sequence
  • Cysteine (analogs & derivatives, genetics, metabolism)
  • DNA Topoisomerases, Type I (chemistry, genetics, metabolism)
  • DNA, Viral (chemistry, metabolism)
  • Esterification
  • Lysine (analogs & derivatives, genetics, metabolism)
  • Models, Molecular
  • Mutagenesis
  • Point Mutation
  • Vaccinia (virology)
  • Vaccinia virus (chemistry, enzymology, genetics, metabolism)

Join CureHunter, for free Research Interface BASIC access!

Take advantage of free CureHunter research engine access to explore the best drug and treatment options for any disease. Find out why thousands of doctors, pharma researchers and patient activists around the world use CureHunter every day.
Realize the full power of the drug-disease research network!


Choose Username:
Email:
Password:
Verify Password: