Thermodynamics and kinetics of the cleavage of DNA catalyzed by bleomycin A5.
Microcalorimetry and UV-vis spectroscopy 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-Menten constant for calf thymus DNA and the turnover number of BLM-A5 were calculated by a novel thermokinetic method for an enzyme-catalyzed reaction to be -577 +/- 19 kJ.mol-1, 20.4 +/- 3.8 microm and 2.28 +/- 0.49 x 10-2 s-1, respectively, at 37.0 degrees C. This DNA cleavage was a largely exothermic reaction. The catalytic efficiency of BLM-A5 is of the same order of magnitude as that of lysozyme but several orders of magnitude lower than those of TaqI restriction endonuclease, NaeI endonuclease and BamHI endonuclease. 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. These results suggest that BLM-A5 is not as efficient as a DNA-cleaving enzyme although the cleavage of DNA by BLM-A5 follows Michaelis-Menten kinetics. Binding of BLM-A5 to calf thymus DNA is driven by a favorable entropy increase with a less favorable enthalpy decrease, in line with a partial intercalation mode involved in BLM-catalyzed breakage of DNA.
Liang
Yi
Y
College of Life Sciences and College of Chemistry and Molecular Science, Wuhan University, China. liangyi@whu.edu.cn
Du
Fen
F
Zhou
Bing-Rui
BR
Zhou
Hui
H
Zou
Guo-Lin
GL
Wang
Cun-Xin
CX
Qu
Song-Sheng
SS
D000818:Animals; D001665:Binding Sites; D001761:Bleomycin / Q000031:analogs & derivatives* / Q000378:metabolism*; D002151:Calorimetry; D002384:Catalysis; D002417:Cattle; D004247:DNA / Q000378:metabolism*; D018736:DNA Adducts / Q000737:chemistry; D004249:DNA Damage; D004317:Doxorubicin / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D010618:Phenanthrolines / Q000737:chemistry; D013053:Spectrophotometry; D013816:Thermodynamics
D018736:DNA Adducts; D005296:Ferrous Compounds; D010618:Phenanthrolines; C031902:doxorubicin-DNA; D001761:Bleomycin; C025703:bleomycetin; D004317:Doxorubicin; D004247:DNA; C427203:calf thymus DNA; C025205:1,10-phenanthroline