Topoisomerases are ubiquitous enzymes that modify the topological state of DNA inside the cell and are essential for several cellular processes. Topoisomerase V is the sole member of the type IC topoisomerase subtype. The topoisomerase domain has a unique fold among topoisomerases, and the putative active site residues show a distinct arrangement. The present study was aimed at identifying the roles of the putative active site residues in the DNA cleavage/religation process. Residues Arg-131, Arg-144, His-200, Glu-215, Lys-218, and Tyr-226 were mutated individually to a series of conservative and non-conservative amino acids, and the DNA relaxation activity at different pH values, times, and enzyme concentrations was compared with wild-type activity. The results suggest that Arg-144 is essential for protein stability because any substitution at this position was deleterious and that Arg-131 and His-200 are involved in transition state stabilization. Glu-215 reduces the DNA binding ability of topoisomerase V, especially in shorter fragments with fewer helix-hairpin-helix DNA binding motifs. Finally, Lys-218 appears to play a direct role in catalysis but not in charge stabilization of the protein-DNA intermediate complex. The results suggest that although catalytically important residues are oriented in different fashions in the active sites of type IB and type IC topoisomerases, similar amino acids play equivalent roles in both of these subtypes of enzymes, showing convergent evolution of the catalytic mechanism.