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 Streptococcus 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 eukaryotic 5' nucleases. The mutations, but not the fusion to the C-terminal end of the wild-type, reduced the exonuclease activity. The residual exonuclease activity of the mutant proteins has been kinetically studied, together with potential alterations in metal binding at the active site. Comparison of the catalytic rate and dissociation constant of the D10G, E114G, and E88K 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 exonucleolytic reaction, being secondarily involved in both catalysis and DNA binding, and (iii) a nonessential function of Glu(88) for the exonuclease activity of Spn pol I. Moreover, the pattern of metal activation of the mutant proteins indicates that none of the three residues is a metal-ligand at the active site. These findings and those previously obtained with D190A mutant of Spn pol I are discussed in relation to structural and mutational data for related 5' nucleases.
Amblar
M
M
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas, Velázquez 144, 28006 Madrid, Spain.
de Lacoba
M G
MG
Corrales
M A
MA
Lopez
P
P
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004256:DNA Polymerase I / Q000737:chemistry* / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry; D006639:Histidine / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D015636:Magnesium Chloride / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013296:Streptococcus pneumoniae / Q000201:enzymology*
D004277:DNA, Single-Stranded; D008024:Ligands; D011993:Recombinant Fusion Proteins; D015636:Magnesium Chloride; D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D004247:DNA; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V