The pH variation of the kinetic parameters and the catalytic mechanism of malic enzyme

Abstract

The pH variation of the kinetic parameters for the oxidative decarboxylation of L-malate and decarboxylation of oxalacetate catalyzed by malic enzyme [pigeon liver] was used to gain information on the catalytic mechanism of this enzyme. With Mn2+ as the activator, an active-site residue with a pK of 5.4 must be protonated for oxalacetate decarboxylation and ionized for the oxidative decarboxylation of L-malate. With Mg2+ as the metal, this pK is 6, and, at high pH, V[velocity]/Ko for L-malate decreases when groups with pK of 7.8 and 9 are deprotonated. The group at 7.8 is a neutral acid (thought to be H2O coordinated to Mg2+), while the group at 9 is a cationic acid such as lysine. The V profile for reaction of malate shows these pK displaced outward by 1.4 pH units, since the rate-limiting step is normally NADPH release, and the chemical reaction, which is pH sensitive, is 25 times faster. NADP binding is decreased by ionization of a group with pK 9.3 or protonation of a group with pK 5.3. The pH variation of the Km for Mg shows that protonation of a group with pK 8.7 (possibly SH) decreases metal binding in the presence of malate by a factor of 1400, and in the absence of malate by a factor of 20. A catalytic mechanism is proposed in which hydride transfer is accompanied by transfer of H+ to the group with pK 5.4-6, and enolpyruvate is protonated by H2O coordinated to the Mg2+ (pK 7.8) after decarboxylation and release of CO2.