Purification and characterization of fumarase from Corynebacterium glutamicum

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2006-05, Vol.70 (5), p.1102-1109
• Main Authors: Genda, T.(Yamaguchi Univ. (Japan). Faculty of Education), Watabe, S, Ozaki, H
• Format: Article
• Language: English
• Subjects: ACIDE FUMARIQUE; ACIDO FUMARICO; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Benzoates - chemistry; Biological and medical sciences; Brevibacterium flavum; CORYNEBACTERIUM; Corynebacterium glutamicum; Corynebacterium glutamicum - enzymology; Enzyme Stability; equilibrium constant; fumarase; Fumarate Hydratase - antagonists & inhibitors; Fumarate Hydratase - chemistry; Fumarate Hydratase - isolation & purification; FUMARIC ACID; Fundamental and applied biological sciences. Psychology; homotetramer; Hydrogen-Ion Concentration; LIASAS; LYASE; LYASES; Molecular Weight; Protein Subunits - antagonists & inhibitors; Protein Subunits - chemistry; Protein Subunits - isolation & purification; PURIFICACION; PURIFICATION; Substrate Specificity; Sulfhydryl Compounds - chemistry; Tartrates - chemistry
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.70.1102

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 (Ksub(eq)) for the enzyme reaction were determined at pH 6.0, 7.0, and 8.0, resulting in Ksub(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.