Biochemical analysis of point mutations in the 5 '-3 ' exonuclease of DNA polymerase I of Streptococcus pneumoniae - Functional and structural implications

To define the active site of the 5 ' -3 ' exonucleolytic domain of the Stre ptococcus pneumoniae DNA polymerase I (Spn pol I), we have constructed His- tagged Spn pol I fusion protein and introduced mutations at residues Asp(10 ), Glu(88), and Glu(114), which are conserved among all prokaryotic and euk aryotic 5 ' nucleases, The mutations, but not the fusion to the C-terminal end of the wild-type, reduced the exonuclease activity. The residual exonuc lease activity of the mutant proteins has been kinetically studied, togethe r with potential alterations in metal binding at the active site. Compariso n of the catalytic rate and dissociation constant of the D10G, E114G, and E 88K mutants and the control fusion protein support: (i) a critical function of Asp(10) in the catalytic event, (ii) a role of Glu(114) in the exonucle olytic reaction, being secondarily involved in both catalysis and DNA bindi ng, and (iii) a nonessential function of Glu(88) for the exonuclease activi ty of Spn pol I. Moreover, the pattern of metal activation of the mutant pr oteins indicates that none of the three residues is a metal-ligand at the a ctive site. These findings and those previously obtained with D190A mutant of Spn pol I are discussed in relation to structural and mutational data fo r related 5 ' nucleases.