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 pr esence of ascorbate. Manganese and some other divalent metal ions prov ided complete protection of the enzyme against the Fe2+-induced inacti vation. The inactivated enzyme was subsequently cleaved by the Fe2+-as corbate system at Asp(258)-Ile(259), which was presumably the Mn2+-bin ding 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 identif ication of Asp(258) the putative metal-binding site of the enzyme, we prepared four mutant enzymes substituted at Asp(258) with glutamate (D 258E), asparagine (D258N), lysine (D258K), or alanine (D258A), respect ively. These mutant proteins were recombinantly expressed in a bacteri al expression system (pET-15b) with a stretch of histidine residues at tached at the N-terminus and were successfully purified to apparent ho mogeneity by a single Ni-chelated affinity column. Among the four muta nts, only D258E possessed 0.8% residual activity after purification; a ll other purified mutants had <0.0001% residual activity in catalyzing the oxidative decarboxylation of L-malate. The D258E mutant was susce ptible to inactivation by the Fe2+-ascorbate system, albeit with much slower inactivation rate, and was protected by the Mn2+ to a lesser ex tent as compared to the wild-type enzyme. None of the mutants were cle aved by the Fe2+-ascorbate system under conditions that cleaved the na tural 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 thos e of the wild-type enzyme; the K-mMn and K-dMn values, on the other ha nd, increased approximately 1600-fold and 3000-fold, respectively, whi ch resulted in the specific constant (k(cat)/K-mMn) Of the D258E mutan t being decreased by approximately 40 000-fold, and the catalytic effi ciency [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), th e metal coordinate for pigeon liver malic enzyme. The amino acid seque nce around this aspartate residue or in an equivalent site of other ma lic enzymes is highly conserved. We propose that this aspartate residu e is critical for the catalysis in all malic enzymes.