Catalytic Mechanism of DNA Topoisomerase IB

Type IB topoisomerases and tyrosine recombinases are structurally homologous strand transferases that act through DNA-(3′-phosphotyrosyl)-enzyme intermediates. A constellation of conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265 in vaccinia topoisomerase) catalyzes transesterification of tyrosine to the scissile phosphodiester. We used 5′-bridging phosphorothiolate-modified DNAs to implicate Lys-167 as a general acid catalyst. The lower pK a of the 5′-S leaving group versus 5′-O restored activity to the K167A mutant, whereas there was no positive thio effect for mutants R223A and H265A. The lysine is located atop a flexible hairpin loop, and it shifts into the minor groove upon DNA binding. Coupling of conformational changes in a general acid loop to covalent catalysis of phosphoryl transfer is one of several mechanistic features shared by the topoisomerase/recombinase and protein phosphatase superfamilies.