Kinetic properties of NAD malic enzyme from cauliflower.

The kinetic characteristics of NAD malic enzyme purified to homogeneity from cauliflower florets have been examined. Free NAD+ is the active form of this coenzyme. Double-reciprocal plots of data obtained by varying NAD+ and malate2- at a saturating concentration of Mg2+ or by varying Mg2+ and NAD+ at a saturating level of malate2-are of intersecting type. This indicates that NAD malic enzyme obeys a sequential mechanism. Analysis of these sets of data suggests that each of these substrate pairs binds randomly to the enzyme. However, each substrate binds tighter when others are already present on the enzyme. NAD malic enzyme cannot decarboxylate malate2- in the absence of either Mg2+ or NAD+. Arrhenius plots of the NAD-linked reaction are concave downward, indicating the existence of two rate-determining steps with activation energies of 26.5 and 14.2 kcal/mol, respectively. In addition to Mg2+, the enzyme can also use Mn2+ and Co2+. Using Co2+ in place of Mg2+ does not change Vmax or Km, malate2- but the Km for metal and NAD+ are greatly decreased. At pH 7.0 and above, Mn2+ isotherms and malate2- curves with Mn2+ are nonlinear and appear to be composed of two separate saturation curves. NAD malic enzyme is completely and irreversibly inactivated by N-ethylmaleimide. The enzyme is also irreversibly inactivated approximately 50% by KCNO.