Kinetic and Structural Properties of NADP-Malic Enzyme from Sugarcane Leaves1

Oligomeric structure and kinetic properties of NADP-malic en- zyme, purified from sugarcane (Saccharam officinarum L.) leaves, were determined at either pH 7.0 and 8.0. Size exclusion chro- matography showed the existence of an equilibrium between the dimeric and the tetrameric forms. At pH 7.0 the enzyme was found preferentially as a 125 kilodalton homodimer, whereas the tetramer was the major form found at pH 8.0. Although free forms of L-malate, NADP+, and Mg2+ were determined as the true sub-strates and cofactors for the enzyme at the two conditions, the kinetic properties of the malic enzyme were quite different depending on pH. Higher affinity for L-malate (Km = 58 micromolar), but also inhibition by high substrate (K, = 4.95 millimolar) were observed at pH 7.0. L-Malate saturation isotherms at pH 8.0 followed hyperbolic kinetics (Km = 120 micromolar). At both pH conditions, activity response to NADP+ exhibited Michaelis-Men- ten behavior with Km values of 7.1 and 4.6 micromolar at pH 7.0 and 8.0, respectively. Negative cooperativity detected in the binding of Mg2+ suggested the presence of at least two Mg2+-binding sites with different affinity. The K, values for Mg2+ ob- tained at pH 7.0 (9 and 750 micromolar) were significantly higher than those calculated at pH 8.0 (1 and 84 micromolar). The results suggest that changes in pH and Mg2+

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