Catalytic phosphoryl interactions of topoisomerase IB.
The reversible nucleophilic substitution reaction catalyzed by the vaccinia virus type IB topoisomerase has been investigated by measuring the equilibrium and rate effects of stereospecific sulfur substitution at the two nonbridging oxygen atoms of the attacked phosphodiester group. An energetic analysis of the combined effects of sulfur substitution and site-directed mutagenesis of active site residues of the enzyme has identified enzyme interactions with each oxygen in the ground state and transition state. We use these findings in combination with previous structural and 5'-bridging sulfur substitution results to deduce the web of enzymatic interactions with the nonbridging oxygens as well as the 5'-hydroxyl leaving group. A key finding is the central role of Arg130, which forms electrostatic interactions with both nonbridging oxygens and the 5'-leaving group.
Nagarajan
Rajesh
R
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205-2185, USA.
Kwon
Keehwan
K
Nawrot
Barbara
B
Stec
Wojciech J
WJ
Stivers
James T
JT
D019943:Amino Acid Substitution; D002384:Catalysis; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D004264:DNA Topoisomerases, Type I