Thermodynamics and kinetics of the cleavage of DNA catalyzed by bleomycin A₅

Abstract

Microcalorimetry and UV-vis spectroscopy were used to conduct thermodynamic and kinetic investigations of the scission of calf thymus DNA catalyzed by bleomycin A 5 (BLM-A 5 ) in the presence of ferrous ion and oxygen. The molar reaction enthalpy for the cleavage, the MichaelisMenten constant for calf thymus DNA and the turnover number of BLM-A 5 were calculated by a novel thermokinetic method for an enzyme-catalyzed reaction to be )577 ± 19 kJAEmol )1 , 20.4 ± 3.8 lM and 2.28 ± 0.49 · 10 )2 s )1 , respectively, at 37.0°C. This DNA cleavage was a largely exothermic reaction. The catalytic efficiency of BLM-A 5 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-A 5 with those for the scission of calf thymus DNA mediated by adriamycin and by (1,10-phenanthroline)-copper, it was found that BLM-A 5 possessed the highest DNA cleavage efficiency among these DNA-damaging agents. These results suggest that BLM-A 5 is not as efficient as a DNA-cleaving enzyme although the cleavage of DNA by BLM-A 5 follows Michaelis-Menten kinetics. Binding of BLM-A 5 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.Keywords: bleomycin; DNA cleavage; kinetics; microcalorimetry; thermodynamics.The bleomycins (BLMs, Fig. 1) are a family of naturally occurring, structurally related, glycopeptide-derived antitumor antibiotics discovered by Umezawa and coworkers from cultures of Streptomyces verticillus in 1966 [1], which have more than 200 members, such as A 2 , A 5 and B 2 [2]. BLMs consist of an unusual linear hexapeptide, a disaccharide and a terminal amine (the R group in Fig. 1). Mixtures of BLMs are presently used for the clinical treatment of a variety of cancers, notably squamous cell carcinomas, testicular tumors and nonHodgkin's lymphoma [2]. The therapeutic effect of BLM is believed to result from its ability to induce single-and double-strand breakage of DNA molecules by oxidation of the deoxyribose moiety in the presence of oxygen and a redox-active metal ion, e.g. Fe and Co [2-6]. On the other hand, RNA is also considered as a therapeutically relevant target for BLM [7,8]. It has been found that BLM-induced autoxidation of ferrous iron follows the Michaelis-Menten kinetics [9,10]. Although a significant number of experimental approaches have been used to elucidate the mechanism of DNA cleavage by BLM in the past two decades [2][3][4][5][6][11][12][13][14][15][16][17][18][19][20], thermodynamic information for the scission, which is necessary for a thorough understanding of the mechanism, is eagerly awaited. The purpose of this investigation is to provide detailed thermodynamic and kinetic data for BLMmediated DNA degradation to furnish insights into the anticancer mechanism of BL...

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