NDLI: 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

Content Provider	Journal of Biological Chemistry (JBC)
Author	Pickering, Timothy J. Garforth, Scott Sayers, Jon R. Grasby, Jane A.
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, whereask 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 thek cat parameter of the wild type enzyme is discussed.
Related Links	http://www.jbc.org/content/274/25/17711.abstract
Ending Page	17717
Starting Page	17711
Page Count	7
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	25
Volume Number	274
DOI	10.1074/jbc.274.25.17711
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	1999-06-18
Access Restriction	Open
Subject Keyword	Human flap endonuclease-1 (hFEN-1) Methanococcus jannaschii flap endonuclease-1 (MjFEN-1) Pyrococcus furiosus flap endonuclease-1 (PfFEN-1) 2-(N-morpholino)ethanesulfonic acid (MES) 3-(N-morpholino)propanesulfonic acid (MOPS) N-(2-hydroxyethyl)piperazine-N′-3-propanesulfonic acid (EPPS) 2-(cyclohexylamino)ethanesulfonic acid (CHES) 3-(cyclohexylamino)propanesulfonic acid (CAPS) Bovine serum albumin (BSA) Staphylococcal nuclease (SNase) ENZYMOLOGY
Alternative Title	Variation in the Steady State Kinetic Parameters of Wild Type and Mutant T5 5′-3′-Exonuclease With pH
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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