posted on 2016-09-09, 00:00authored byJulius Rentergent, Max D. Driscoll, Sam Hay
Extracting kinetic parameters from
DNA polymerase-catalyzed processive
polymerization data using traditional initial-rate analysis has proven
to be problematic for multiple reasons. The first substrate, DNA template,
is a heterogeneous polymer and binds tightly to DNA polymerase. Further,
the affinity and speed of incorporation of the second substrate, deoxynucleoside
triphosphate (dNTP), vary greatly depending on the nature of the templating
base and surrounding sequence. Here, we present a mathematical model
consisting of the DNA template-binding step and a Michaelis–Menten-type
nucleotide incorporation step acting on a DNA template with a finite
length. The model was numerically integrated and globally fitted to
experimental reaction time courses. The time courses were determined
by monitoring the processive synthesis of oligonucleotides of lengths
between 50 and 120 nucleotides by DNA polymerase I (Klenow fragment
exo–) using the fluorophore PicoGreen. For processive
polymerization, we were able to estimate an enzyme–template
association rate k1 of 7.4 μM–1 s–1, a disassociation rate k–1 of 0.07 s–1, and
a Kd of 10 nM, and the steady-state parameters
for correct dNTP incorporation give kcat values of 2.5–3.3 s–1 and Km values of 0.51–0.86 μM. From the analysis
of time courses measured between 5 and 25 °C, an activation energy
for kcat of 82 kJ mol–1 was calculated, and it was found that up to 73% of Klenow fragment
becomes inactivated or involved in unproductive binding at lower temperatures.
Finally, a solvent deuterium kinetic isotope effect (KIE) of 3.0–3.2
was observed under processive synthesis conditions, which suggests
that either the intrinsic KIE is unusually high, at least 30–40,
or previous findings, showing that the phosphoryl transfer step occurs
rapidly and is flanked by two slow conformational changes, need to
be re-evaluated. We suggest that the numerical integration of rate
equations provides a high level of flexibility and generally produces
superior results compared to those of initial-rate analysis in the
study of DNA polymerase kinetics and, by extension, other complex
enzyme systems.