title: The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.
abstract: We have used site-directed mutagenesis to change amino acid side chains that have been shown crystallographically to be in close proximity to a DNA 3' terminus bound at the 3'-5' exonuclease active site of Klenow fragment. Exonuclease assays of the resulting mutant proteins indicate that the largest effects on exonuclease activity result from mutations in a group of carboxylate side chains (Asp355, Asp424 and Asp501) anchoring two divalent metal ions that are essential for exonuclease activity. Another carboxylate (Glu357) within this cluster seems to be less important as a metal ligand, but may play a separate role in catalysis of the exonuclease reaction. A second group of residues (Leu361, Phe473 and Tyr497), located around the terminal base and ribose positions, plays a secondary role, ensuring correct positioning of the substrate in the active site and perhaps also facilitating melting of a duplex DNA substrate by interacting with the frayed 3' terminus. The pH-dependence of the 3'-5' exonuclease reaction is consistent with a mechanism in which nucleophilic attack on the terminal phosphodiester bond is initiated by a hydroxide ion coordinated to one of the enzyme-bound metal ions.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06510.
authors.forename: V
authors.initials: V
authors.lastname: Derbyshire
authors.forename: N D
authors.initials: ND
authors.lastname: Grindley
authors.forename: C M
authors.initials: CM
authors.lastname: Joyce
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation
chemical_list: D010446:Peptide Fragments; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V