title: Enthalpic switch-points and temperature dependencies of DNA binding and nucleotide incorporation by Pol I DNA polymerases.
abstract: This study examines the relationship between the DNA binding thermodynamics and the enzymatic activity of the Klenow and Klentaq Pol I DNA polymerases from Escherichia coli and Thermus aquaticus. Both polymerases bind DNA with nanomolar affinity at temperatures down to at least 5°C, but have lower than 1% enzymatic activity at these lower temperatures. For both polymerases it is found that the temperature of onset of significant enzymatic activity corresponds with the temperature where the enthalpy of binding (ΔHbinding) crosses zero (TH) and becomes favorable (negative). This TH/activity upshift temperature is 15°C for Klenow and 30°C for Klentaq. The results indicate that a negative free energy of DNA binding alone is not sufficient to proceed to catalysis, but that the enthalpic versus entropic balance of binding may be a modulator of the temperature dependence of enzymatic function. Analysis of the temperature dependence of the catalytic activity of Klentaq polymerase using expanded Eyring theory yields thermodynamic patterns for ΔG(‡), ΔH(‡), and TΔS(‡) that are highly analogous to those commonly observed for direct DNA binding. Eyring analysis also finds a significant ΔCp(‡) of formation of the activated complex, which in turn indicates that the temperature of maximal activity, after which incorporation rate slows with increasing temperature, will correspond with the temperature where the activation enthalpy (ΔH(‡)) switches from positive to negative.
authors.affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.
authors.forename: Hiromi S
authors.initials: HS
authors.lastname: Brown
authors.forename: Vince J
authors.initials: VJ
authors.lastname: Licata
mesh_terms: D004256:DNA Polymerase I / Q000737:chemistry* / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D009711:Nucleotides / Q000737:chemistry*; D011485:Protein Binding; D019914:Taq Polymerase / Q000737:chemistry* / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics; D013824:Thermus / Q000737:chemistry* / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009711:Nucleotides; D019914:Taq Polymerase; D004256:DNA Polymerase I
keywords: DNA replication; Eyring; Gibbs–Helmholtz; Klenow; Taq polymerase; Thermodynamics