Kinetic parameters of Citrobacter freundii methionine γ-lyase were determined with substrates in γ-elimination reactions as well as the inhibition of the enzyme in the γ-elimination of L-methionine by amino acids with different structure. The data indicate an important contribution of the sulfur atom and methylene groups to the efficiency of binding of substrates and inhibitors. The rate constants of the enzyme-catalyzed exchange of C-α- and C-β-protons with deuterium were determined, as well as the kinetic isotope effect of the deuterium label in the C-α-position of inhibitors on the rate of exchange of their β-protons. Neither stereoselectivity in the β-proton exchange nor noticeable α-isotope effect on the exchange rates of β-protons was found. The ionic and tautomeric composition of the external Schiff base of methionine γ-lyase was determined. Spectral characteristics (absorption and circular dichroism spectra) of complexes with substrates and inhibitors were determined. The spectral and kinetic data indicate that deamination of aminocrotonate should be the rate-determining stage of the enzymatic reaction.
Novel indolocarbazole derivative 12-(alpha-L-arabinopyranosyl)indolo[2,3-alpha]pyrrolo[3,4-c]carbazole-5,7-dione (AIC) demonstrated high potency (at submicromolar concentrations) against the NCI panel of human tumor cell lines and transplanted tumors in vivo. In search of tentative targets for AIC, we found that the drug formed high affinity intercalative complexes with d(AT)(20), d(GC)(20) and calf thymus DNA (binding constants (1.6x10(6)) M(-1)< or =K(a)< or =(3.3x10(6)) M(-1)). The drug intercalated preferentially into GC pairs of the duplex. Importantly, the concentrations at which AIC formed the intercalative complexes with DNA (C< or =1 microM) were identical to the concentrations that triggered p53-dependent gene reporter transactivation, the replication block, the inhibition of topoisomerase I-mediated DNA relaxation and death of HCT116 human colon carcinoma cells. We conclude that the formation of high affinity intercalative complexes with DNA is an important factor for anticancer efficacy of AIC.
Camptothecin (CPT) and its clinically important antitumor derivative topotecan (Tpt) were traditionally described as unique antitumor compounds exhibiting no affinity toward DNA alone or DNA topoisomerase I (top1) alone but interacting with both the enzyme and the DNA within the so-called ternary cleavable complexes. We present here the first experimental data on the molecular structure and geometry of Tpt-DNA complexes in solution. Tpt interacts with DNA within the DNA minor groove and demonstrates the preferential binding to GC-rich DNA. The flow linear dichroism (FLD) spectra show that the Tpt binds DNA only in lactone form and its chromophore forms the angle nearly 55°with the DNA long axis. Induced circular dichroism (CD) data independently confirm conclusions about Tpt preferable orientation drawn from the FLD experiments. The Raman spectroscopy data confirm the FLD and CD results and further demonstrate direct interactions of Tpt lactone ring with dG. The capability of Tpt to bind DNA in the minor groove of GC-rich DNA regions must be taken into account when considering molecular structure of ternary cleavable complexes of CPTs, DNA, and top1 in solution.
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