The modus operandi of a DNA enzyme: enhancement of substrate basicity
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abstract
BACKGROUND: We wished to investigate the mode of catalytic action of a small DNA enzyme (DNAzyme), PS5.M, that, when folded into an appropriate tertiary structure, catalyzes the insertion of copper and zinc ions into porphyrins such as mesoporphyrin IX (MPIX). PS5.M, originally derived from SELEX experiments for specific DNA binders of the distorted porphyrin N-methylmesoporphyrin (NMM), had enzymatic parameters (kcat/K(m) = 39,700 M-1 min-1; kcat/kuncat = 3,700) that rivalled those of an antibody that catalyzed the same reaction. RESULTS: We used ultraviolet-visible absorption and fluorescence spectroscopy to study the mode of action of PS5.M and related catalytic DNA molecules. We found that the MPIX-DNAzyme complexes had absorption spectra more closely resembling those of the DNAzymes complexed to NMM than to the spectra of MPIX itself, whether MPIX was in its monomeric or aggregated form. pH titration experiments revealed that the DNAzyme raised the protonation pK for MPIX by 3-4 pH units. CONCLUSIONS: Our results reveal that PS5.M works by enhancing the basicity (and hence the ease of metallation) of the bound porphyrin substrate. Large changes in the porphyrin's basicity may be brought about through distortion of the planar porphinato- core of MPIX to resemble that of the naturally deformed porphyrin NMM or through stabilization by a DNA phosphate of the growing positive charge in the transition state for porphyrin protonation/metallation or through a combination of the two. The catalytic strategy of enhancing substrate basicity may also hold true for porphyrin metallation by the recently described catalytic RNA and catalytic antibody for this reaction.
