Efficient Inorganic Deoxyribonucleases. Greater than 50-Million-Fold Rate Enhancement in Enzyme-Like DNA Cleavage

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Abstract

Glycosylated natural products such as bleomycin, neocarzinostatin, and calicheamicin γ1 are efficient antitumor agents that cleave ds DNA by pathways that involve redox chemistry. In this paper we demonstrate the use of metalloderivatives of natural aminoglycosides as efficient DNA cleavage agents in the absence of external reducing agents. Kinetic characterization of DNA cleavage by copper neamine under Michaelis−Menten-"type" reaction conditions revealed a maximal reaction velocity Vmax' = 0.031 min-1, equivalent to a greater than 50-million-fold rate enhancement in DNA cleavage, when uncorrected for catalyst concentrations. Under true Michaelis conditions, a maximal reaction velocity Vmax = 0.0595 min-1 was obtained (with kcat = 5.95 × 10-4 min-1), corresponding to a million-fold rate enhancement using micromolar concentrations of Cu2+−neamine. The specificity constants for DNA cleavage by copper neamine (kcat/KM = 4.8 × 105 h-1 M-1) are 2 orders of magnitude greater than those reported elsewhere for synthetic compounds, at this time. Cleavage mediated by Cu2+−(kanamycin A) was found to be even more efficient. DNA cleavage was not inhibited by SOD, NaN3, DMSO, or EtOH, nor by handling under anaerobic conditions. The results of gel electrophoretic experiments provide clear evidence for a hydrolytic cleavage pathway with generation of 5'-phosphate and 3'-hydroxyl termini.

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