Mechanistic plasticity of DNA topoisomerase IB: Phosphate electrostatics dictate the need for a catalytic arginine

Ligeng Tian, Christopher D. Claeboe, Sidney M. Hecht, Stewart Shuman

Research output: Contribution to journalArticlepeer-review

30 Scopus citations

Abstract

Four conserved amino acids of type IB topoisomerases (Arg130, Lys167, Arg223, and His265 in vaccinia topoisomerase) catalyze the attack by tyrosine on the scissile phosphodiester to form a DNA-(3′-phosphotyrosyl)-enzyme intermediate. The mechanism entails general acid catalysis (by Lys167 and Arg130) and transition-state stabilization (via contact of His265 with the pro-Sp oxygen). Here we query the function of Arg223, which accelerates transesterification by a factor of 105. The requirement for Arg223 is alleviated by a neutral Sp methylphosphonate (MeP) linkage at the cleavage site. Arg223 is not required for the 30,000-fold activation of the latent endonuclease activity of topoisomerase by the Sp MeP. The rate of autohydrolysis by the DNA-(3′-MeP)-topoisomerase intermediate approaches 10% of the rate of religation to a 5′-OH DNA strand. These findings underscore the importance of transition-state electrostatics in determining the composition of the active site and dictating the balance between strand transferase and hydrolase functions.

Original languageEnglish (US)
Pages (from-to)513-520
Number of pages8
JournalStructure
Volume13
Issue number4
DOIs
StatePublished - Apr 2005
Externally publishedYes

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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