Activated cyclophosphamide: An enzyme-mechanism-based suicide inactivator of DNA polymerase/3??5? exonuclease

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Abstract
Summary DNA polymerase I fromE. coli can toxify activated cyclophosphamide (CP) by means of the 3′–5′ exonuclease activity associated with the enzyme. Acrolein and an alkylating moiety are released in the process. Preincubation of DNA polymerase I with activated CP for 15–60 min leads to an increasing inhibition of DNA polymerase activity, which can be prevented when preincubation of DNA polymerase I with activated CP is carried out in the presence of 5′ AMP, a competitive inhibitor of the 3′–5′ exonuclease subsite of the enzyme. This demonstrates that toxification of activated CP by the 3′–5′ exonuclease subsite of DNA polymerase is a prerequisite for the inhibition of DNA polymerase activity. The kinetics and the degree of DNA polymerase inhibition suggest that the alkylating moiety rather than acrolein released from activated CP during toxification is responsible for the inhibition of DNA polymerase. DNA polymerase with associated 3′–5′ exonuclease activity has also been isolated from eukaryotic cells, and toxification of activated CP by such an enzyme (DNA polymerase δ from rabbit bone marrow) has been shown previously. Thus we suggest that toxification of activated CP by DNA polymerases/3′-5′ exonucleases present mainly in proliferating cells might lead to the specific alkylation of macromolecules involved in the cell proliferation process, such as the DNA polymerase subsite of these enzymes and probably also the DNA bound to the enzymes. The relatively high cancerotoxic selectivity and cytotoxic specificity of activated CP could be based on this specific enzyme-mediated alkylation.