Stivers J T, Shuman S, Mildvan A S
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Biochemistry. 1994 Dec 27;33(51):15449-58. doi: 10.1021/bi00255a027.
The pH dependences of the internal equilibrium (Kcl) and rate constants for site-specific DNA strand cleavage (kcl) 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 degrees C. The pH dependence of the rate constant for strand cleavage (kcl) 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-zeta chemical shift of [zeta-13C]-tyrosine-enriched topoisomerase. The dependence of the internal equilibrium constant (Kcl) on pH reveals very similar pKa values as kcl (5.8 +/- 0.2 and 8.6 +/- 0.2). However, kr is found to be independent of pH. The differing response of kcl 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 kcl 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, (krphos/krthio = 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.
在20℃下，利用单周转条件研究了pH值在4.6 - 9.8范围内，痘苗病毒DNA拓扑异构酶I催化的位点特异性DNA链切割（kcl）和重新封闭（kr）的内部平衡（Kcl）以及速率常数的pH依赖性。链切割速率常数（kcl）的pH依赖性呈现钟形曲线，表观pKa值为6.3±0.2和8.4±0.2，这表明活性位点Tyr-274对磷酸二酯磷的攻击存在碱催化，以及5'-脱氧核糖氧的排出存在酸催化。通过监测[ζ-13C]-酪氨酸富集的拓扑异构酶的C-ζ化学位移，从pH 5.1至8.8进行NMR滴定，排除了游离酶中Tyr-274的低pKa（即6.3）。内部平衡常数（Kcl）对pH的依赖性显示出与kcl非常相似的pKa值（5.8±0.2和8.6±0.2）。然而，发现kr与pH无关。kcl和kr对pH的不同响应排除了简单的双态内部切割平衡，并表明在形成共价复合物后发生了构象变化，该复合物保留了用于链重新连接的正确质子化状态。通过将被攻击的磷酰基的非桥连氧原子替换为硫，对kcl产生4.6倍的“硫效应”[斯蒂弗斯，J. T.，舒曼，S.，&米尔德万，A. S.（1994年）《生物化学》33，327]，以及对kr不存在这种效应（krphos/krthio = 0.9±0.2），进一步表明了构象步骤，这表明切割和重新连接的速率分别受共价化学和构象步骤的限制。在重新连接方向上这种构象变化的速率常数与从质粒底物释放超螺旋的平均速率常数一致，表明这种构象变化是拓扑异构化步骤的一部分。尽管尚未确定一般酸和一般碱催化剂，但讨论了这些以及其他残基在催化中的定量作用。
