Photoemission probes of catalysis of benzo[a]pyrene epoxide reactions in complexes with linear, double-stranded and closed-circular, single-stranded DNA

Article abstract of DOI:10.1021/ja00070a028

Fluorescence intensity measurements of overall, pseudo-first-order rate constants for two epoxide-containing metabolites of benzo[a]pyrene (BP) were carried out in Tris, EDTA buffer (pH 7.3) without DNA, and in buffer with double-stranded calf thymus DNA (DS ctDNA) and with closed-circular, single-stranded viral M13mp19 DNA (SS M13 DNA). Highly purified SS M13 DNA was employed in order to avoid polymeric contamination which is present in DNA samples obtained using a standard preparation method relying on phenol extraction and which influences results from measurements of DNA-ligand interactions. The BP metabolites examined were highly carcinogenic (±)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[e]pyrene (BPDE) and less genotoxic benzo[a]pyrene 4,5-oxide (BPO). Without DNA, BPDE hydrolyzes to 7,8,9,10-tetrahydroxytetrahydro-BP, while BPO hydrolyzes to trans-4,5-dihydroxy-4,5-dihydro-BP (BP45D) and rearranges to 4-hydroxy-BP. With DNA, BPDE and BPO hydrolysis and rearrangement are catalyzed, and DNA modification occurs. In DS ctDNA, previous kinetic and binding measurements indicate that catalysis occurs primarily at intercalation sites. In SS M13 DNA (0.20 mM), BPDE has overall, pseudo-first-order rate constants (k) of (12 ± 1) × 10^-3 and (2.8 ± 0.5) × 10^-3 s^-1, at Na⁺ concentrations of 2.0 and 100 mM, respectively. At these Na⁺ concentrations, values of k measured in SS M13 DNA are 3-16 times larger than values measured without DNA, but smaller than values measured in DS ctDNA. For BPO, the ordering of k values without DNA, with SS M13 DNA, and with DS ctDNA is the same as for BPDE. At 2.0 mM Na⁺, the nonreactive diols, trans-7,8-dihydroxy-7,8-dihydro-BP (BP78D) and BP45D, which are model compounds for BPDE and BPO, respectively, have SS M13 DNA association constants [(7.2 ± 0.5) × 10³ and (2.7 ± 0.5) × 10³ M^-1] that are 2.3 times smaller than DS ctDNA association constants. In contrast, at 100 mM Na⁺, association constants for SS M13 DNA are 2.9-3.1 times larger than for DS ctDNA. Fluorescence lifetime measurements indicate that, in SS M13 DNA, reversible binding involves intercalation into local duplex regions. Estimated catalytic rate constants (k_cat) for BPDE hydrolysis in SS M13 DNA, obtained from BP78D association constants and from k values measured with and without DNA, are (22.8 ± 2.5) × 10^-3 and (3.5 ± 0.7) × 10^-3 s^-1, at 2.0 and 100 mM Na⁺, respectively. For this Na⁺ concentration range, the ratio of k_cat values for DS ctDNA versus SS M13 DNA is almost constant (1.7 ± 0.6) even though the absolute k_cat values vary by more than a factor of 5. The similar magnitudes of k_cat values for SS M13 DNA and DS ctDNA provide evidence that catalytic sites in SS M13 DNA are similar to intercalated catalytic sites in DS ctDNA.