IDENTIFICATION OF ASP(258) AS THE METAL COORDINATE OF PIGEON LIVER MALIC ENZYME BY SITE-SPECIFIC MUTAGENESIS

Pigeon liver malic enzyme was inactivated by ferrous sulfate in the presence of ascorbate. Manganese and some other divalent metal ions provided complete protection of the enzyme against the Fe2+-induced inactivation. The inactivated enzyme was subsequently cleaved by the Fe2+-ascorbate system at Asp(258)-Ile(259), which was presumably the Mn2+-binding site of the enzyme [Wei, C. H., Chou, W. Y., Huang, S. M., Lin, C. C., and Chang, G. G. (1994) Biochemistry 33, 7931-7936]. For identification of Asp(258) the putative metal-binding site of the enzyme, we prepared four mutant enzymes substituted at Asp(258) with glutamate (D258E), asparagine (D258N), lysine (D258K), or alanine (D258A), respectively. These mutant proteins were recombinantly expressed in a bacterial expression system (pET-15b) with a stretch of histidine residues attached at the N-terminus and were successfully purified to apparent homogeneity by a single Ni-chelated affinity column. Among the four mutants, only D258E possessed 0.8% residual activity after purification; all other purified mutants had <0.0001% residual activity in catalyzing the oxidative decarboxylation of L-malate. The D258E mutant was susceptible to inactivation by the Fe2+-ascorbate system, albeit with much slower inactivation rate, and was protected by the Mn2+ to a lesser extent as compared to the wild-type enzyme. None of the mutants were cleaved by the Fe2+-ascorbate system under conditions that cleaved the natural or wild-type enzyme at Asp(258). The apparent K-mNADP and K-mMal values of D258E mutant did not change very much as compared with those of the wild-type enzyme; the K-mMn and K-dMn values, on the other hand, increased approximately 1600-fold and 3000-fold, respectively, which resulted in the specific constant (k(cat)/K-mMn) Of the D258E mutant being decreased by approximately 40 000-fold, and the catalytic efficiency [k(cat)/(KmNADPKmMalKdMn)] of D258E was only 0.000 52% of that of the WT. These results conclusively indicated the catalytic role of metal ion in the malic enzyme catalyzed reaction and that Asp(258), the metal coordinate for pigeon liver malic enzyme. The amino acid sequence around this aspartate residue or in an equivalent site of other malic enzymes is highly conserved. We propose that this aspartate residue is critical for the catalysis in all malic enzymes.