Dipole-dipole interaction stabilizes the transition state of apurinic/apyrimidinic endonuclease--abasic site interaction.
Human apurinic/apyrimidinic (AP) endonuclease (hAPE) initiates the repair of an abasic site (AP site). To gain insight into the mechanisms of damage recognition of hAPE, we conducted surface plasmon resonance spectroscopy to study the thermodynamics and kinetics of its interaction with substrate DNA containing an abasic site (AP DNA). The affinity of hAPE binding toward DNA increased as much as 6-fold after replacing a single adenine (equilibrium dissociation constant, K(D), 5.3 nm) with an AP site (K(D), 0.87 nm). The enzyme-substrate complex formation appears to be thermodynamically stabilized and favored by a large change in Gibbs free energy, DeltaG degrees (-50 kJ/mol). The latter is supported by a high negative change in enthalpy, DeltaH degrees (-43 kJ/mol) and also positive change in entropy, DeltaS degrees (24 J/(K mol)), and thus the binding process is spontaneous at all temperatures. Analysis of kinetic parameters reveals small enthalpy of activation for association, DeltaH degrees++(ass) (-17 kJ/mol), and activation energy for association (E(a), -14 kJ/mol) when compared with the enthalpy of activation for dissociation, DeltaH degrees++(diss) (26 kJ/mol), and activation energy in the reverse direction (E(d), 28 kJ/mol). Furthermore, varying concentration of KCl showed an increase in binding affinity at low concentration but complete abrogation of the binding at higher concentration, implying the importance of hydrophobic, but predominantly ionic, forces in the Michaelis-Menten complex formation. Thus, low activation energy and the enthalpy of activation, which are perhaps a result of dipole-dipole interactions, play critical roles in AP site binding of APE.
Adhikari
Sanjay
S
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057. Electronic address: sa354@georgetown.edu.
Üren
Aykut
A
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057.
Roy
Rabindra
R
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C. 20057. Electronic address: rr228@georgetown.edu.
D019921:Biotinylation; D004247:DNA / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009841:Oligonucleotides / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D055672:Static Electricity; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
D009841:Oligonucleotides; D011189:Potassium Chloride; D004247:DNA; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase