| Abstract: |
The pH dependences of the internal equilibrium (Kc1) and rate constants for site-specific DNA strand cleavage (kc1) and resealing (kr) catalyzed by Vaccinia DNA topoisomerase I have been investigated using single-turnover conditions in the pH range 4.6–9.8 at 20 °C. The pH dependence of the rate constant for strand cleavage (kc1) shows a bell-shaped profile with apparent pKa values of 6.3 ± 0.2 and 8.4 ± 0.2, suggesting base catalysis of the attack of the active site Tyr-274 on the phosphodiester phosphorus, and acid catalysis of the expulsion of the 5′-deoxyribose oxygen. A low pKa (i.e., 6.3) for Tyr-274 in the free enzyme is ruled out by NMR titration from pH 5.1 to 8.8 monitoring the C-ζ chemical shift of [ζ-13C]- tyrosine-enriched topoisomerase. The dependence of the internal equilibrium constant (Kc1) on pH reveals very similar pKa values as Kc1 (5.8 ± 0.2 and 8.6 ± 0.2). However, kr is found to be independent of pH. The differing response of Kc1 and Kr to pH rules out a simple two-state internal cleavage equilibrium and suggests that a conformational change occurs following formation of the covalent complex which retains the correct protonation state for strand religation. A conformation step is further indicated by a 4.6-fold “thio effect” on Kc1 upon substitution of the nonbridging oxygen atom of the attacked phosphoryl group by sulfur [Stivers, J. T., Shuman, S., & Mildvan, A. S. (1994) Biochemistry 33, 327], and the absence of such an effect on kr, (kr phos/kr thio = 0.9±0.2), indicating the rates of cleavage and religation to be limited by covalent chemistry and a conformational step, respectively. The rate constant of this conformational change in the direction of religation agrees with the average rate constant for supercoil release from plasmid substrates, suggesting this conformational change to be a part of the topoisomerization step. Although the general acid and general base catalysts have not yet been identified, the quantitative roles of these and other residues in catalysis are discussed. © 1994, American Chemical Society. All rights reserved. |
