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.

DNA synthesis from diphosphate substrates by DNA polymerases

“…In Pol β, these two steps have previously been proposed to have similar rate constants (15), and the calculated activation energies for the forward and reverse reactions utilizing the high-energy substrates (dNTPs and pyrophosphate) differ by as little as 15 kJ/mol (4). In the case of the thermophilic Taq and Klentaq1 enzymes, the activation energy of the forward reaction varies widely, ranging from 90 to 125 kJ/mol, depending on the experimental conditions and likely the identity of the reacting nucleotides (8,16), whereas the activation energy of pyrophosphorolysis has not been, to our knowledge, experimentally determined.…”

“…To further compare the kinetics of DNA synthesis on long templates from dNDPs and dNTPs, we used a singe-stranded DNA (ssDNA) M13mp18 plasmid template of 7,249 nt (8). Bsu (at 37°C), Bst, and Taq (at 60°C) polymerases showed robust DNA synthesis from dNDPs ( Fig.…”

Temperature Effect on Polymerase Fidelity

“…Intriguingly, PIPI and MBP-PIPB exhibit characteristically distinct temperature profiles for replication activity: PIPI reaches peak polymerase activity at 37 o C, similar to KleExo-(left panel in Figure 2a). As observed previously, the optimal temperature of enzyme activity can differ from the temperature to which the host organism is adapted to 31,32 . On the other hand, polymerase activity of MBP-PIPB is optimal at 20 o C and becomes inactivated above 30 o C. Combined, our results show that the temperature profiles are distinct for the two psychrophilic DNA polymerases, suggesting a different physiological requirement for their catalytic rates and structural robustness.…”

“…As we were unable to obtain a soluble version of PIPB after protease digestion of the fusion tag, 30,31 . Intriguingly, PIPI and MBP-PIPB exhibit characteristically distinct temperature profiles for replication activity: PIPI reaches peak polymerase activity at 37 o C, similar to KleExo-(left panel in Figure 2a).…”

Investigation of the Binding Behavior between the S-heterocyclic Aromatic Palladium(II) Complex and Human Serum Albumin: Spectroscopic Approach

“…The molar enthalpy of HSA in the absence of Pd(II) complex, Hå, can be calculated from the Gibbs-Helmholtz equation as follows (34,35):…”