TY - JOUR
T1 - Mechanistic plasticity of DNA topoisomerase IB
T2 - Phosphate electrostatics dictate the need for a catalytic arginine
AU - Tian, Ligeng
AU - Claeboe, Christopher D.
AU - Hecht, Sidney M.
AU - Shuman, Stewart
N1 - Funding Information:
Supported by NIH Grants GM46330 (S.S.) and CA78415 (S.M.H.).
PY - 2005/4
Y1 - 2005/4
N2 - 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.
AB - 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.
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U2 - 10.1016/j.str.2005.02.001
DO - 10.1016/j.str.2005.02.001
M3 - Article
C2 - 15837190
AN - SCOPUS:17044399694
SN - 0969-2126
VL - 13
SP - 513
EP - 520
JO - Structure
JF - Structure
IS - 4
ER -