Kinetics and thermodynamics of nick sealing by T4 DNA ligase

摘要: T4 DNA ligase is an Mg2+-dependent and ATP-dependent enzyme that seals nicks in three steps: it covalently binds AMP, transadenylates the nick phosphate, catalyses formation of phosphodiester bond releasing AMP. In this kinetic study, we further detail reaction mechanism, showing overall ligation a superimposition two parallel processes: 'processive' ligation, which without dissociating from dsDNA, 'nonprocessive' takes part abortive adenylation cycle (covalent binding transadenylation nick, dissociation). At low concentrations ATP (<10 microM) when sealed with mismatching base pairs (e.g. five adjacent), resolves into phases, burst (approximately 0.2 min(-1)) subsequent slow 2x10(-3) min(-1)). The relative rate extent each phase depend on Mg2+. activation energies self-adenylation (16.2 kcal.mol(-1)), (0.9 nick-sealing (16.3-18.8 kcal.mol(-1)) were determined for several substrates. energy implies transfer AMP to terminal phosphate spontaneous reaction, ligase-AMP complex high-energy intermediate. To summarize current findings field, delineate mechanism catalysis.