Transient oxidation–reduction
through hydride transfer with
tightly bound NAD coenzyme is used by a large class of sugar nucleotide
epimerases to promote configurational inversion of carbon stereocenters
in carbohydrate substrates. A requirement for the epimerases to coordinate
hydride abstraction and re-addition with substrate rotation in the
binding pocket poses a challenge for dynamical protein conformational
selection linked to enzyme catalysis. Here, we studied the thermophilic
C2 epimerase from
Thermodesulfatator atlanticus
(
Ta
CPa2E) in combination with a slow CDP-glucose
substrate (
k
cat
≈ 1.0 min
–1
; 60 °C) to explore the sensitivity of the enzymatic hydride
transfer toward environmental fluctuations affected by temperature
(20–80 °C). We determined noncompetitive primary kinetic
isotope effects (KIE) due to
2
H at the glucose C2 and showed
that a normal KIE on the
k
cat
(
D
k
cat
) reflects isotope sensitivity of
the hydrogen abstraction to enzyme-NAD
+
in a rate-limiting
transient oxidation. The
D
k
cat
peaked at 40 °C was 6.1 and decreased to 2.1 at low (20 °C)
and 3.3 at high temperature (80 °C). The temperature profiles
for
k
cat
with the
1
H and
2
H substrate showed a decrease in the rate below a dynamically
important breakpoint (∼40 °C), suggesting an equilibrium
shift to an impaired conformational landscape relevant for catalysis
in the low-temperature region. Full Marcus-like model fits of the
rate and KIE profiles provided evidence for a high-temperature reaction
via low-frequency conformational sampling associated with a broad
distribution of hydride donor–acceptor distances (long-distance
population centered at 3.31 ± 0.02 Å), only poorly suitable
for quantum mechanical tunneling. Collectively, dynamical characteristics
of
Ta
CPa2E-catalyzed hydride transfer during transient
oxidation of CDP-glucose reveal important analogies to mechanistically
simpler enzymes such as alcohol dehydrogenase and dihydrofolate reductase.
A loose-fit substrate (in
Ta
CPa2E) resembles structural
variants of these enzymes by extensive dynamical sampling to balance
conformational flexibility and catalytic efficiency.