Cherepanov Alexey V, de Vries Simon
Kluyver Department of Biotechnology, Delft University of Technology, The Netherlands.
Eur J Biochem. 2003 Nov;270(21):4315-25. doi: 10.1046/j.1432-1033.2003.03824.x.
T4 DNA ligase is an Mg2+-dependent and ATP-dependent enzyme that seals DNA nicks in three steps: it covalently binds AMP, transadenylates the nick phosphate, and catalyses formation of the phosphodiester bond releasing AMP. In this kinetic study, we further detail the reaction mechanism, showing that the overall ligation reaction is a superimposition of two parallel processes: a 'processive' ligation, in which the enzyme transadenylates and seals the nick without dissociating from dsDNA, and a 'nonprocessive' ligation, in which the enzyme takes part in the abortive adenylation cycle (covalent binding of AMP, transadenylation of the nick, and dissociation). At low concentrations of ATP (<10 microM) and when the DNA nick is sealed with mismatching base pairs (e.g. five adjacent), this superimposition resolves into two kinetic phases, a burst ligation (approximately 0.2 min(-1)) and a subsequent slow ligation (approximately 2x10(-3) min(-1)). The relative rate and extent of each phase depend on the concentrations of ATP and Mg2+. The activation energies of self-adenylation (16.2 kcal.mol(-1)), transadenylation of the nick (0.9 kcal.mol(-1)), and nick-sealing (16.3-18.8 kcal.mol(-1)) were determined for several DNA substrates. The low activation energy of transadenylation implies that the transfer of AMP to the terminal DNA phosphate is a spontaneous reaction, and that the T4 DNA ligase-AMP complex is a high-energy intermediate. To summarize current findings in the DNA ligation field, we delineate a kinetic mechanism of T4 DNA ligase catalysis.
T4 DNA连接酶是一种依赖镁离子和ATP的酶，它通过三个步骤封闭DNA切口：它共价结合AMP，将AMP转移到切口磷酸基团上，并催化磷酸二酯键的形成，同时释放AMP。在这项动力学研究中，我们进一步详细阐述了反应机制，表明整个连接反应是两个平行过程的叠加：一个是“持续”连接，即酶在不与双链DNA解离的情况下将AMP转移并封闭切口；另一个是“非持续”连接，即酶参与无效的腺苷化循环（AMP的共价结合、切口的腺苷化转移以及解离）。在低ATP浓度（<10 microM）且DNA切口用错配碱基对（如五个相邻碱基对）封闭时，这种叠加分解为两个动力学阶段，一个是快速连接阶段（约0.2 min⁻¹），随后是缓慢连接阶段（约2×10⁻³ min⁻¹）。每个阶段的相对速率和程度取决于ATP和镁离子的浓度。我们测定了几种DNA底物的自我腺苷化（16.2 kcal·mol⁻¹）、切口腺苷化转移（0.9 kcal·mol⁻¹）和切口封闭（16.3 - 18.8 kcal·mol⁻¹）的活化能。腺苷化转移的低活化能意味着AMP转移到末端DNA磷酸基团是一个自发反应，并且T4 DNA连接酶 - AMP复合物是一种高能中间体。为总结DNA连接领域的当前研究结果，我们描述了T4 DNA连接酶催化的动力学机制。
