alpha-Secondary tritium kinetic isotope effects indicate hydrogen tunneling and coupled motion occur in the oxidation of L-malate by NAD-malic enzyme

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].