α-Secondary Tritium Kinetic Isotope Effects Indicate Hydrogen Tunneling and Coupled Motion Occur in the Oxidation of l-Malate by NAD-Malic Enzyme

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Abstract
The NAD-malic enzyme from Ascaris suum catalyzes the divalent metal ion-dependent oxidative decarboxylation of l-malate to give pyruvate and CO2, with NAD+ as the oxidant. α-Secondary tritium kinetic isotope effects were measured with NAD+ or APAD+ and l-malate-2-H(D) and several different divalent metal ions. The α-secondary tritium kinetic isotope effects are slightly higher than 1 with NAD+ and l-malate as substrates, much larger than the expected inverse isotope effect for a hybridization change from sp2 to sp3. The α-secondary tritium kinetic isotope effects are reduced to values near 1 with l-malate-2-D as the substrate, regardless of the metal ion that is used. Data suggest the presence of quantum mechanical tunneling and coupled motion in the malic enzyme reaction when NAD+ and malate 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 exponent 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 α-secondary tritium kinetic isotope effects are observed, and a Swain−Schaad exponent of 3.3 is estimated, consistent with rate-limiting hydride transfer and no quantum mechanical tunneling or coupled motion. Data are discussed 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].

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