Mechanism of pigeon liver malic enzyme: modification of essential carboxyl groups

The maximum velocity of the reaction catalyzed by the pigeon liver malic enzyme depends on the ionization of a functional group of pKa 6.7. This pKa value is independent of temperature within the range 30 degrees-49 degrees C, suggesting the ionization of a carboxyl group. The enzyme activity is inactivated by N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward reagent K) at pH 6.0 and 25 degrees C. N-Methylhydroxamine regenerates the enzymatic activity whereas glycine ethyl ester does not. The addition of Mn2+, NADP+, and L-malate to the incubation mixture decreases the inactivation rate, suggesting that the reaction takes place in the active center. The binding capacities of the modified enzyme with NADP+, L-malate, pyruvate, and Mn2+ are not impaired. The kinetic and chemical evidence indicates that the inactivation is due to the modification of a carboxyl group which may be from glutamyl or aspartyl residues of the enzyme. This carboxyl group might function as a general acid-base catalyst. A detailed mechanism in terms of the exact amino acid residues involved is proposed.