Mechanistic plasticity of DNA topoisomerase IB: phosphate electrostatics dictate the need for a catalytic arginine.
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 10(5). 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.
Tian
Ligeng
L
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Claeboe
Christopher D
CD
Hecht
Sidney M
SM
Shuman
Stewart
S
D001120:Arginine / Q000378:metabolism*; D002384:Catalysis; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D055672:Static Electricity
D010710:Phosphates; D001120:Arginine; D004264:DNA Topoisomerases, Type I