Vaccinia DNA Topoisomerase I: Kinetic Evidence for General Acid-Base Catalysis and a Conformational Step

The pH dependences of the internal equilibrium (K_c1) and rate constants for site-specific DNA strand cleavage (k_c1) and resealing (k_r) 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 (k_c1) shows a bell-shaped profile with apparent pK_a 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 pK_a (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 (K_c1) on pH reveals very similar pK_a values as K_c1 (5.8 ± 0.2 and 8.6 ± 0.2). However, k_r is found to be independent of pH. The differing response of K_c1 and K_r 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 K_c1 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 k_r, (k_r^phos/k_r^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.