Bleomycin A5 is analogous in mechanism to a DNA-cleaving enzyme: a microcalorimetric study

Abstract

Microcalorimetry, UV–VIS spectroscopy and HPLC were used to conduct thermodynamic and kinetic investigations of the scission of calf thymus DNA catalyzed by bleomycin A5 (BLM-A5) in the presence of ferrous ion and oxygen. The molar reaction enthalpy for the cleavage, the Michaelis constant for calf thymus DNA and the turnover number of BLM-A5 were calculated by a novel thermokinetic method for enzyme-catalyzed reactions to be −577±19kJmol−1, (2.04±0.38)×10−5moldm−3 and (2.28±0.49)×10−2s−1, respectively, at 37.00°C. This DNA cleavage was a fast and exothermic reaction. The catalytic efficiencies of BLM-A5 and of some DNA-cleaving enzymes, such as TaqI restriction endonuclease and NaeI endonuclease, are of the same order of magnitude. By comparing the molar enthalpy change for the cleavage of calf thymus DNA induced by BLM-A5 with those for the scission of calf thymus DNA mediated by adriamycin and by (1,10-phenanthroline)-copper, it was found that BLM-A5 possessed the highest DNA cleavage efficiency among these DNA-damaging agents. The UV–VIS spectral experiments showed that BLM-A5 itself remained unchanged chemically after the cleavage. The products of this scission, including four kinds of free nucleic acid bases, were separated and detected by HPLC. These results strongly suggest that BLM-A5 is analogous in mechanism to a DNA-cleaving enzyme, although it is much smaller than such type of enzymes. A possible mechanism for the cleavage of DNA catalyzed by BLM was suggested in this paper.