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

Timothy J. Pickering, Scott Garforth, Jon R. Sayers, Jane A. Grasby

Research output: Contribution to journalArticle

7 Scopus citations

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 k(cat) depends on a single deprotonation as demonstrated by the bell-shaped dependence of log (k(cat)/K(m)) on pH. To investigate the role of a conserved lysine (Lys-83), the pH profile of log (k(cat)/K(m)) 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 k(cat) parameter of the wild type enzyme is discussed.

Original languageEnglish (US)
Pages (from-to)17711-17717
Number of pages7
JournalJournal of Biological Chemistry
Volume274
Issue number25
DOIs
Publication statusPublished - Jun 18 1999

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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