Structural and catalytic insights into HoLaMa， a derivative of Klenow DNA polymerase lacking the proofreading domain.

We report here on the stability and catalytic properties of the HoLaMa DNA polymerase， a Klenow sub-fragment lacking the 3'-5' exonuclease domain. HoLaMa was overexpressed in Escherichia coli， and the enzyme was purified by means of standard chromatographic techniques. High-resolution NMR experiments revealed that HoLaMa is properly folded at pH 8.0 and 20°C. In addition， urea induced a cooperative folding to unfolding transition of HoLaMa， possessing an overall thermodynamic stability and a transition midpoint featuring ΔG and CM equal to (15.7 ± 1.9) kJ/mol and (3.5 ± 0.6) M， respectively. When the catalytic performances of HoLaMa were compared to those featured by the Klenow enzyme， we did observe a 10-fold lower catalytic efficiency by the HoLaMa enzyme. Surprisingly， HoLaMa and Klenow DNA polymerases possess markedly different sensitivities in competitive inhibition assays performed to test the effect of single dNTPs.