Variation in the Steady State Kinetic Parameters of Wild Type and Mutant T5 5′-3′-Exonuclease With pH

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.Organisms as diverse as bacteriophage and humans require 5Ј-nucleases to participate in, and ensure the fidelity of, DNA replication and repair. These enzymes are involved in excising damaged DNA and the removal of Okazaki fragments produced during replication. T5 5Ј-3Ј-exonuclease is a virus-encoded member of a family of homologous 5Ј-nucleases which includes the N-terminal domain of DNA polymerase I proteins (1). More distantly related eukaryotic and mammalian enzymes also exhibit sequence homology, albeit to a lesser extent, and include the human enzyme hFEN-1 1 (1). The 5Ј-nucleases catalyze the exonucleolytic hydrolysis of phosphodiester bonds in a variety of nucleic acid substrates, including single-stranded and double-stranded DNA and DNA-RNA hybrids (RNase H activity).In addition, the 5Ј-nucleases also exhibit structure-specific endonucleolytic activity, resulting in the cleavage of DNA flap and pseudo-Y structures (Fig. 1a) (2-4). This structure-specific hydrolysis of bifurcated structures is mainly observed at the junction between double-stranded and single-stranded DNA (2-4). Both exonucleolytic and endonucleolytic hydrolyses proceed with scission of the 3Ј-oxygen phosphorus bond to generate products terminating in a 3Ј-hydroxyl group and a 5Ј-phosphate monoester, respectively (5-7).The x-ray crystal structures of five members of the 5Ј-nuclease family have been reported in the absence of DNA substrates (8 -12). The structures of T4 RNase H and the 5Ј-exonuclease domain of Taq polymerase I both contained areas of disorder (10, 12), but the structure of T5 5Ј-3Ј-exonuclease revealed the presence of a hole bounded by a helical arch upon a globular domain containing the putative active site (9). The hole is large enough to accommodate single-but not doublestranded DNA. To explain substrate processing, a threading mechanism in which the single-stranded 5Ј terminus enters the arch has bee...

Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions

“…Similar stimulation of binding of DNA substrates at lowered pH or by Ca 2ϩ ions has been observed with other metallonucleases and been assigned to protonation or binding of Ca 2ϩ ions at the active site carboxylates (28). Here the protonation state of DNA-binding residues could also be a factor (29,30).…”

Comparison of the Catalytic Parameters and Reaction Specificities of a Phage and an Archaeal Flap Endonuclease

“…Basic residues at the base of the helical arch of T5 and DNA polymerase I FENs have been implicated in contacting the DNA substrate. 25,28,37 The active site of FENs lies at the base of the helical arch or clamp (Figure 2(a)). Earlier models of the T5FEN-DNA interaction threaded single-stranded DNA through the arch.…”

“…Unless otherwise indicated, a pH of 9.3 was selected for evaluation of the specificity and catalytic parameters of the FEN reactions, because both the multiple turnover number and the maximal single turnover rate of the T5FEN catalysed reaction with a magnesium ion cofactor are maximal and pH-independent at this pH value. 28,29 Substrates were synthesised using conventional methods and all contain a 5′-fluorescein (FAM) label. It has previously been demonstrated that both 5′-fluorescent and 5′-radiolabelled variants of HP5F have similar catalytic parameters and therefore the fluorophore does not interfere with the reaction.…”

Brønsted Analysis and Rate-Limiting Steps for the T5 Flap Endonuclease Catalyzed Hydrolysis of Exonucleolytic Substrates

“…In FENs a possible candidate for general acid catalysis is the positionally conserved lysine residue (K83 in T5FEN), located at the base of the helical arch protruding into the active site ,, . This residue is present in all FENs and related DNA repair nucleases such as XPG, EXO-1, and GEN-1 enzymes (Figure S4 in Supporting Information).…”

“…The D201I/D204S T5FEN mutant was constructed as described for a N-terminal truncated variant but in the context of the full-length protein. K83A T5FEN has been reported earlier − . Wild-type (WT) T5FEN and mutants were overexpressed and purified to homogeneity essentially as described previously followed by an additional chromatography step employing Q-Sepharose using buffer A (25 mM Tris-HCl (pH 8.0), 1 mM DTT, 1 mM EDTA, and 5% glycerol containing 50 mM NaCl).…”