Abstract
We report a continuous hyperchromicity assay (CHA) for monitoring and characterizing enzyme activities associated with DNA processing. We use this assay to determine kinetic and thermodynamic parameters for a repair enzyme that targets nucleic acid substrates containing a specific base lesion. This optically based kinetics assay exploits the free-energy differences between a lesion-containing DNA duplex substrate and the enzyme-catalyzed, lesion-excised product, which contains at least one hydrolyzed phosphodiester bond. We apply the assay to the bifunctional formamidopyrimidine glycosylase (Fpg) repair enzyme (E) that recognizes an 8-oxodG lesion within a 13-mer duplex substrate (S). Base excision/elimination yields a gapped duplex product (P) that dissociates to produce the diagnostic hyperchromicity signal. Analysis of the kinetic data at 25°C yields a
K
m
of 46.6 nM for the E·S interaction, and a
k
cat
of 1.65 min
−1
for conversion of the ES complex into P. The temperature dependence reveals a free energy (Δ
G
b
) of −10.0 kcal·mol
−1
for the binding step (E + S ↔ ES) that is enthalpy-driven (Δ
H
b
= −16.4 kcal·mol
−1
). The activation barrier (Δ
G
‡
) of 19.6 kcal·mol
−1
for the chemical step (ES ↔ P) also is enthalpic in nature (Δ
H
‡
= 19.2 kcal·mol
−1
). Formation of the transition state complex from the reactants (E + S ↔ ES
‡
), a pathway that reflects Fpg catalytic specificity (
k
cat
/
K
m
) toward excision of the 8-oxodG lesion, exhibits an overall activation free energy (Δ
G
T
‡
) of 9.6 kcal·mol
−1
. These parameters characterize the driving forces that dictate Fpg enzyme efficiency and specificity and elucidate the energy landscape for lesion recognition and repair.