A critical evaluation of metal-promoted Klenow 3′-5′ exonuclease activity: calorimetric and kinetic analyses support a one-metal-ion mechanism

Abstract

Metal-mediated hydrolysis of phosphate esters is a common catalytic pathway in nucleic acid biochemistry. Two distinct models are principally invoked in mechanistic discussions of these reactions for magnesium-dependent nuclease activation: namely, the one-versus two-metal-ion pathways. The 3′-5′ exonuclease domain of the Klenow fragment of Escherichia coli DNA polymerase I is a paradigm for the two-metal-ion mechanism; however, this reaction model is principally based on structural and kinetics experiments employing high concentrations of transition metal analogues and high concentrations of background ammonium sulfate during doping experiments. This prompted us to re-evaluate the metal cofactor stoichiometry of the 3′-5′ exonuclease mechanism for the Klenow fragment by solution kinetics and isothermal titration calorimetry using the natural Mg2+ cofactor and salt conditions. Both solution calorimetric and kinetics experiments strongly indicate binding of only one metal ion to the exonuclease active site. Comparative studies with Mn2+ also indicate a requirement for one metal ion to effect 3′-5′ exonuclease activity.