At

p p"" R9 Schematic diagram of the DNase I-DNA contacts.

_C g iiii Y175 Phosphate groups are represented by the letter P,

----deoxyribose moieties by a square, and bases by

P piiii Y211 their one letter code. A, T, C, and G correspond to adenine, thymine, cytosine and guanine,

— G C — respectively. (Adapted from Weston, S.A., Lahm, A., - - and Suck, D., J. Mol. Biol., 226, 1237-1256, 1992. With

5' 3' P1111 S206 permission.)

The width of the minor groove and the helical stiffness cannot, however, explain the rather drastic differences in cutting rates at neighboring phosphodiester bonds. It is likely that the orientation of the scissile phosphate group determines the local cleavage rates. Nuclear magnetic resonance (NMR) studies showed that cleavage of the P-O3' bonds in DNA by DNase I are accompanied by an inversion of the configuration at the phosphorous atom [17]. This is brought about by the unusual stacking-type interaction of Y76 with a deoxyribose, which suggests that the hydrolysis of the P-O3' bonds occurs through an in-line nucleophilic attack by a water molecule. From crystallo-graphic data [17,35,38] and especially site-directed mutagenesis studies [27,28,37], it is known that E39, E78, H134, D168, D212, and H252 of bovine and human DNase I are directly involved in catalysis. Figure 5.5 shows how these DNase I residues are involved in the acid-base-catalyzed hydrolysis of DNA [27]. The first step in the mechanism is the abstraction of a proton from a water molecule by H252 (step 1). The obtained hydroxyl ion subsequently attacks the phosphodiester, generating a pentacovalent transition state (steps 2 and 3). The magnesium (or zinc, manganese) ions, which are coordinated to the scissile phosphate group, E39 and D168, stabilize this pentacovalent transition state. They are also required for a correct positioning of the phosphate group relative to the enzyme. The second histine residue (H134) functions as a general acid, protonating the O3' as it leaves (steps 4-6). The strong hydrogen bonds between the N1 of H134 and E78, and between H252 and D212 increase the pKa values of the histidine residues and consequently favor the acid-base-catalyzed nucleophilic attack of water. Finally, E78 is not only hydrogen-bound to H134 but also to the phenolic OH of Y76. Thus, E78 serves as a link between a DNA-binding (Y76), and a DNA-cutting amino acid (H134).

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