title: Variation in the steady state kinetic parameters of wild type and mutant T5 5'-3'-exonuclease with pH. Protonation of Lys-83 is critical for DNA binding.
abstract: T5 5'-3'-exonuclease is a member of a family of homologous 5'-nucleases essential for DNA replication and repair. We have measured the variation of the steady state parameters of the enzyme with pH. The log of the association constant of the enzyme and substrate is pH-independent between pH 5 and 7, but at higher pH, it decreases (gradient -0.91 +/- 0.1) with increasing pH. The log of the turnover number increases (gradient 0.9 +/- 0.01) with increasing pH until a pH-independent plateau is reached. The T5 5'-3'-exonuclease-catalyzed reaction requires the protonation of a single residue for substrate binding, whereas kcat depends on a single deprotonation as demonstrated by the bell-shaped dependence of log (kcat/Km) on pH. To investigate the role of a conserved lysine (Lys-83), the pH profile of log (kcat/Km) of a K83A mutant was determined and found to increase with pH (gradient 1.01 +/- 0. 01) until a pH-independent plateau is reached. We therefore conclude that protonation of Lys-83 in the wild type protein facilitates DNA binding. The origin of the pH dependence of the kcat parameter of the wild type enzyme is discussed.
authors.affiliation: Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom.
authors.forename: T J
authors.initials: TJ
authors.lastname: Pickering
authors.forename: S
authors.initials: S
authors.lastname: Garforth
authors.forename: J R
authors.initials: JR
authors.lastname: Sayers
authors.forename: J A
authors.initials: JA
authors.lastname: Grasby
mesh_terms: D001435:Bacteriophages / Q000201:enzymology; D002384:Catalysis; D004268:DNA-Binding Proteins / Q000737:chemistry; D003854:Deoxyribonucleotides / Q000737:chemistry; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D013379:Substrate Specificity
chemical_list: D004268:DNA-Binding Proteins; D003854:Deoxyribonucleotides; D005090:Exodeoxyribonucleases; C079044:T5 D15 exonuclease; D008239:Lysine