Uracil-DNA glycosylase: Interpretation of X-ray data in the light of kinetic and thermodynamic studies

Data of thermodynamic and kinetic analyses of uracil-DNA glycosylase (UDG) [EC 3.2.2.3] interaction with single-and double-stranded oligonucleotides and earlier published data of X-ray analysis are compared. UDG interacts with ten ssDNA links or one chain of ribo-or deoxyriboduplex with approximately equal affinities: dNMP and NMP are minimal ligands of glycosylase. Weak additive interactions of UDG with inter-nucleoside phosphate groups provide for about four orders of magnitude in ligand affinity, whereas formation of even weaker van der Waals interactions of bases with UDG results in additional 30-fold increase of affinity which is described by the equation K_d(n) = K_d(1) [(1/e) (1/h)]^n-1; (n ≤ 10), where K_d(1) is dissociation constant for dNMP or NMP, e and h are factors of affinity increase due to UDG interaction with one internucleoside phosphate group (e = 1.35) and ligand base (h increases with relative hydrophobicity of the bases), respectively. The conclusion about formation of these contacts correlates with the data of X-ray analysis concerning the presence in the UDG DNA-binding site of many positively charged amino acid residues and hydrophobic contacts of the enzyme with bases. It has been shown that formation of pseudo-Watson-Crick bonds of UDG with dU considered as the most important element in DNA recognition is not very important at this stage: complex formation cannot provide for UDG specificity. UDG affinity to DNA is provided mainly by weak nonspecific interactions (about six orders of magnitude), whereas introduction of dU causes only its 9-20-fold increase. UDG specificity is connected with the reaction rate increase by 3-4 orders of magnitude upon DNA transition to dU-DNA. The increase in k_cat is the result of an easier enzymatic dU-DNA conversion to an optimal conformation owing to its melting, breakdown of stacking interactions, and uracil "flipping out.".