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J Biol Chem, Vol. 274, Issue 25, 17711-17717, June 18, 1999

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, and Jane A. Grasby

From the Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom and the  Division of Molecular and Genetic Medicine, University of Sheffield, Sheffield S10 2JF, United Kingdom

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.

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