Structure-energy analysis of the role of metal ions in phosphodiester bond hydrolysis by DNA polymerase I
- Univ. of Southern California, Los Angeles, CA (United States)
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 Mg{sup 2+}. Experimental evaluation of the catalytic effects of the cations is problematic, since the cations are intimately involved in substrate binding. This problem is explored here by using a theoretical approach to analyze and interpret the key structural 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 approach indicates that phosphodiester bond hydrolysis involves catalysis by an OH{sup -} ion from aqueous solution around the protein, rather than a general base catalysis by an active site residue. The catalytic effect of two divalent metal cations in the active site is found to be primarily electrostatic. The first cation provides a strong electrostatic stabilization to the OH{sup -} nucleophile, while the second cation provides a very large catalytic effect by its interaction with the negative charge being transferred to the transition state during the nucleophilic attack step. The calculations also demonstrate that the second metal ion is not likely to be involved in a previously proposed strain mechanism. 35 refs., 6 figs., 1 tab.
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- FG03-94ER61945
- OSTI ID:
- 171479
- Journal Information:
- Journal of the American Chemical Society, Vol. 117, Issue 47; Other Information: PBD: 29 Nov 1995
- Country of Publication:
- United States
- Language:
- English
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