Fitting of the kinetic curves by a nonlinear-regression method yielded a molecular mechanism and rate constants of its individual steps. By means of fluorescent labels of different types, simultaneous detection of conformational transitions in the protein globule and DNA substrate molecule was performed. We present here a comparative study of conformational dynamics in complexes of the ALKBH2 protein with double-stranded DNA substrates containing N1-methyladenine, N3-methylcytosine, or 1,N6-ethenoadenine. This enzyme protects genomic DNA against various alkyl lesions through a sophisticated catalytic mechanism supported by a cofactor (Fe(II)), a cosubstrate (2-oxoglutarate), and O 2. Here, we applied a stopped-flow kinetic analysis based on fluorescent spectroscopy to investigate mechanisms of conformational transformations during the removal of alkylated bases from DNA by ALKBH2, a human homolog of Escherichia coli AlkB dioxygenase. High efficiency and selectivity of enzymatic catalysis are mostly ensured by conformational dynamics of enzymes and substrates. Elucidation of physicochemical mechanisms of enzymatic processes is one of the main tasks of modern biology.
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