URACIL-DNA GLYCOSYLASE - INTERPRETATION OF X-RAY DATA IN THE LIGHT OFKINETIC 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 oligonu cleotides and earlier published data of X-ray analysis are compared. U DG interacts with ten ssDNA links or one chain of ribo- or deoxyribodu plex with approximately equal affinities: dNMP and NMP are minimal lig ands of glycosylase. Weak additive interactions of UDG with internucle oside phosphate groups provide for about four orders of magnitude in l igand affinity, whereas formation of even weaker van der Waals interac tions of bases with UDG results in additional 30-fold increase of affi nity which is described by the equation K-d(n) = Kd(1) [(1/e) (1/h)](n -1); (n less than or equal to 10), where K-d(1) is dissociation consta nt for dNMP or NMP, e and h are factors of affinity increase due to UD G interaction with one internucleoside phosphate group (e = 1.35) and ligand base (h increases with relative hydrophobicity of the bases), r espectively. The conclusion about formation of these contacts correlat es with the data of X-ray analysis concerning the presence in the UDG DNA-binding site of many positively charged amino acid residues and hy drophobic contacts of the enzyme with bases. It has been shown that fo rmation of pseudo-Watson-Crick bonds of UDG with dU considered as the most important element in DNA recognition is not very important at thi s stage: complex formation cannot provide for UDG specificity. UDG aff inity to DNA is provided mainly by weak nonspecific interactions (abou t six orders of magnitude), whereas introduction of dU causes only its 9-20-fold increase. UDG specificity is connected with the reaction ra te 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 con version to an optimal conformation owing to its melting, breakdown of stacking interactions, and uracil ''flipping out.''.