Purification and characterization of fumarase from Corynebacterium glutamicum.
Fumarase (EC 4.2.1.2) from Corynebacterium glutamicum (Brevibacterium flavum) ATCC 14067 was purified to homogeneity. Its amino-terminal sequence (residues 1 to 30) corresponded to the sequence (residues 6 to 35) of the deduced product of the fumarase gene of C. glutamicum (GenBank accession no. BAB98403). The molecular mass of the native enzyme was 200 kDa. The protein was a homotetramer, with a 50-kDa subunit molecular mass. The homotetrameric and stable properties indicated that the enzyme belongs to a family of Class II fumarase. Equilibrium constants (K(eq)) for the enzyme reaction were determined at pH 6.0, 7.0, and 8.0, resulting in K(eq)=6.4, 6.1, and 4.6 respectively in phosphate buffer and in 16, 19, and 17 in non-phosphate buffers. Among the amino acids and nucleotides tested, ATP inhibited the enzyme competitively, or in mixed-type, depending on the buffer. Substrate analogs, meso-tartrate, D-tartrate, and pyromellitate, inhibited the enzyme competitively, and D-malate in mixed-type.
Genda
Tomoko
T
Biological Institute, Faculty of Education, Yamaguchi University, Ube, Japan.
Watabe
Shoji
S
Ozaki
Hachiro
H
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D001565:Benzoates / Q000737:chemistry; D048230:Corynebacterium glutamicum / Q000201:enzymology*; D004795:Enzyme Stability; D005649:Fumarate Hydratase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013644:Tartrates / Q000737:chemistry
D001426:Bacterial Proteins; D001565:Benzoates; D021122:Protein Subunits; D013438:Sulfhydryl Compounds; D013644:Tartrates; C012018:pyromellitic acid; D005649:Fumarate Hydratase; C029768:tartaric acid