We. Karsten et al., alpha-Secondary tritium kinetic isotope effects indicate hydrogen tunneling and coupled motion occur in the oxidation of L-malate by NAD-malic enzyme, BIOCHEM, 38(14), 1999, pp. 4398-4402
The NAD-malic enzyme from Ascaris suum catalyzes the divalent metal ion-dep
endent oxidative decarboxylation of L-malate to give pyruvate and CO2, with
NAD(+) as the oxidant: alpha-Secondary tritium kinetic isotope effects wer
e measured with NAD(+) or APAD(+) and L-malate-2-H(D) and several different
divalent metal ions. The alpha-secondary tritium kinetic isotope effects a
re slightly higher than 1 with NAD(+) and L-malate as substrates, much larg
er than the expected inverse isotope effect for a hybridization change from
sp(2) to sp(3). The alpha-secondary tritium kinetic isotope effects are re
duced to values near 1 with L-malate-2-D as the substrate, regardless of th
e metal ion that is used. Data suggest the presence of quantum mechanical t
unneling and coupled motion in the malic enzyme reaction when NAD(+) and ma
late are used as substrates., Isotope effects were also measured using the
D/T method with NAD(+) and Mn2+ as the substrate pair. A Swain-Schaad expon
ent of 2.2 (less than the value of 3.26 expected for strictly semiclassical
behavior) is estimated, suggesting the presence of other slow steps along
the reaction pathway. With APAD(+) and Mn2+ as the substrate pair, inverse
alpha-secondary tritium kinetic isotope effects are observed, and a Swain-S
chaad exponent of 3.3 is estimated, consistent with rate-limiting hydride t
ransfer and no quantum mechanical tunneling or coupled motion. Data are dis
cussed: in terms of the malic enzyme mechanism and the theory developed by
Huskey for D/T isotope effects as an indicator of tunneling [Huskey, W. P.
(1991) J Phys. Org. Chem. 4, 361-366].