title: Physical and Kinetic Properties and Regulation of the NAD Malic Enzyme Purified from Leaves of Crassula argentea.
abstract: The NAD malic enzyme has been purified to near homogeneity from the leaves of Crassula argentea Thunb. The enzyme has two subunits, one of 59,000 daltons, and one of 62,000 daltons. In native gels stained for activity, the enzyme appears to exist in the dimeric, tetrameric, and predominantly the octameric forms.The enzyme uses either Mg(2+) or Mn(2+) as the required divalent cation, and utilizes NADP at a rate less than 20% of that with NAD. With Mn(2+) the K(m) for malate(2-) is lower than with Mg(2+), but V(max) is lower than with Mg(2+). In the forward (malate-decarboxylating) direction with NAD, the kinetic parameters are essentially like those observed for the enzyme from C(3) plants. In the reverse reaction, run with Mn(2+), the activity is 1.5% of that in the forward reaction. The equilibrium constant is 1.1 x 10(-3) molar.The kinetic mechanism of the reaction, at least in the forward direction, is sequential, with apparently random binding of all reaction components. Product inhibition patterns confirm this.The enzyme displays a strong hysteretic lag, which is shortened by high enzyme concentrations, high substrate concentrations, and the presence of the product NADH.The enzyme is activated by coenzyme A with K(a) = 4 micromolar. AMP also shows competitive activation, with K(a) = 24 micromolar. The activation by coenzyme A and AMP is additive, implying separate sites for their binding. Phosphoenolpyruvate activates the reaction at low (micromolar) concentrations, but higher concentrations of phosphoenolpyruvate cause deactivation. Fumarate(2-) is a strong activator, with K(a) = 0.3 millimolar. Fructose-1,6-bisphosphate activates the enzyme, but its most pronounced effect is in shortening the lag. Citrate is a competitive inhibitor of malate, with K(i) = 4.9 millimolar.
authors: Department of Biochemistry, University of California, Riverside, California 92521.
authors: R T
authors: RT
authors: Wedding
authors: M K
authors: MK
authors: Black