Kinetic and Structural Properties of NADP malic Enzyme from Sugarcane Leaves

A.A. IGLESIAS, C.S. ANDREO

PLANT PHYSIOLOGY.

Año: 1990 vol. 92 p. 66 - 72

0032-0889

Oligomeric structure and kinetic properties of NADP-malic enzyme,purified from sugarcane (Saccharam officinarum L.) leaves,were determined at either pH 7.0 and 8.0. Size exclusion chromatographyshowed the existence of an equilibrium between thedimeric and the tetrameric forms. At pH 7.0 the enzyme wasfound preferentially as a 125 kilodalton homodimer, whereas thetetramer was the major form found at pH 8.0. Although free formsof L-malate, NADP+, and Mg2+ were determined as the true substratesand cofactors for the enzyme at the two conditions, thekinetic properties of the malic enzyme were quite different dependingon pH. Higher affinity for L-malate (Km = 58 micromolar),but also inhibition by high substrate (K, = 4.95 millimolar) wereobserved at pH 7.0. L-Malate saturation isotherms at pH 8.0followed hyperbolic kinetics (Km = 120 micromolar). At both pHconditions, activity response to NADP+ exhibited Michaelis-Mentenbehavior with Km values of 7.1 and 4.6 micromolar at pH 7.0and 8.0, respectively. Negative cooperativity detected in thebinding of Mg2+ suggested the presence of at least two Mg2+-binding sites with different affinity. The K, values for Mg2+ obtainedat pH 7.0 (9 and 750 micromolar) were significantly higherthan those calculated at pH 8.0 (1 and 84 micromolar). The resultssuggest that changes in pH and Mg2+ levels could be importantfor the physiological regulation of NADP-malic enzyme.