STRUCTURE-ENERGY ANALYSIS OF THE ROLE OF METAL-IONS IN PHOSPHODIESTERBOND HYDROLYSIS BY DNA-POLYMERASE-I

The detailed mechanism of DNA hydrolysis by enzymes is of significant current interest. One of the most important questions in this respect is the catalytic role of metal ions such as Mg2+. While it is clear th at divalent ions play a major role in DNA hydrolysis, it is uncertain what function such cations have in hydrolysis and why two are needed i n some cases and only one in others. Experimental evaluation of the ca talytic effects of the cations is problematic, since the cations are i ntimately involved in substrate binding. This problem is explored here by using a theoretical approach to analyze and interpret the key stru ctural and biochemical experiments. Taking the X-ray structure of the exonuclease domain in the Klenow fragment of E. coli DNA polymerase I we use the empirical valence bond method to examine different feasible mechanisms for phosphodiester bond cleavage in the exonuclease site. This structure-function analysis is based on evaluating the activation free energies of different assumed mechanisms and comparing the calcu lated values to the corresponding experimentally observed activation e nergy for phosphodiester bond cleavage. Mechanisms whose calculated ac tivation energies are drastically larger than the observed activation energy are eliminated and the consistency of the corresponding conclus ion is examined in view of other available experimental facts includin g mutational and pH dependence studies. This approach indicates that p hosphodiester bond hydrolysis involves catalysis by an OH- ion from aq ueous solution around the protein, rather than a general base catalysi s by an active site residue. The catalytic effect of two divalent meta l cations in the active site is found to be primarily electrostatic. T he first cation provides a strong electrostatic stabilization to the O H- nucleophile, while the second cation provides a very large catalyti c effect by its interaction with the negative charge being transferred to the transition state during the nucleophilic attack step. The calc ulations also demonstrate that the second metal ion is not likely to b e involved in a previously proposed strain mechanism. The two-metal io n catalytic mechanism is compared to the action of a single-metal cati on active site and some general rules are discussed. Finally the relat ionship between the present computer modeling study and available expe rimental information on DNA hydrolysis is discussed, emphasizing that calculations of absolute rate constants should be, at least in princip le, more effective in eliminating incorrect mechanisms than calculatio ns of mutational effects.