Identification of Asp258 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 Fe(2+)-induced inactivation. The inactivated enzyme was subsequently cleaved by the Fe(2+)-ascorbate system at Asp258-Ile259, which was presumably the Mn(2+)-binding site of the enzyme [Wei, C. H., Chou, W. Y., Huang, S. M., Lin, C. C., & Chang, G. G. (1994) Biochemistry 33, 7793-7936]. For identification of Asp258 as the putative metal-binding site of the enzyme, we prepared four mutant enzymes substituted at Asp258 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 Fe(2+)-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 Fe(2+)-ascorbate system under conditions that cleaved the natural or wild-type enzyme at Asp258.(ABSTRACT TRUNCATED AT 250 WORDS)
Wei
C H
CH
Graduate Institutes of Life Sciences and Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Chou
W Y
WY
Chang
G G
GG
D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D010856:Columbidae; D005296:Ferrous Compounds / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008274:Magnesium / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism; D008667:Metalloproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
D000596:Amino Acids; D005296:Ferrous Compounds; D008667:Metalloproteins; D001224:Aspartic Acid; D008345:Manganese; D008291:Malate Dehydrogenase; C030574:malate dehydrogenase (decarboxylating); D008274:Magnesium; D001205:Ascorbic Acid