VACCINIA DNA TOPOISOMERASE-I - KINETIC EVIDENCE FOR GENERAL ACID-BASECATALYSIS AND A CONFORMATIONAL STEP

The pH dependences of the internal equilibrium (K-cl) and rate constan ts for site-specific DNA strand cleavage (k(cl)) 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 degrees C. T he pH dependence of the rate constant for strand cleavage (k(cl)) show s 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 sit e 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 Ty r-274 in the free enzyme is ruled out by NMR titration from pH 5.1 to 8.8 monitoring the C-xi chemical shift of [xi-C-13]-tyrosine-enriched topoisomerase. The dependence of the internal equilibrium constant (K- cl) on pH reveals very similar pK(a) values as k(cl) (5.8 +/- 0.2 and 8.6 +/- 6.2). However, k(r) is found to be independent of pH. The diff ering response of k(cl) and k(r) to pH rules out a simple two-state in ternal cleavage equilibrium and suggests that a conformational change occurs following formation of the covalent complex which retains the c orrect protonation state for strand religation. A conformation step is further indicated by a 4.6-fold ''thio effect'' on k(cl) upon substit ution of the nonbridging oxygen atom of the attacked phosphoryl group by sulfur [Stivers, J. T., Shuman, S., and Mildvan, A. S. (1994) Bioch emistry 33, 327], and the absence of such an effect on k(r), (k(r)(pho s)/k(r)(thio) = 0.9 +/- 0.2), indicating the rates of cleavage and rel igation 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 supe rcoil release from plasmid substrates, suggesting this conformational change to be a part of the topoisomerization step. Although the genera l acid and general base catalysts have not yet been identified, the qu antitative roles of these and other residues in catalysis are discusse d.