Properties of crystalline L-ornithine: alpha-ketoglutarate delta-aminotransferase from Bacillus sphaericus.
The distribution of bacterial L-ornithine: alpha-ketoglutarate delta-aminotransferase (L-ornithine:2-oxo-acid aminotransferase [EC 2.6.1.13]) was investigated, and Bacillus sphaericus (IFO 3525) was found to have the highest activity of the enzyme, which was inducibly formed by addition of L-ornithine or L-arginine to the medium. L-Ornithine:alpha-ketoglutarate delta-aminotransferase, purified to homogeneity and crystallized from B. sphaericus, had a molecular weight of about 80,000 and consisted of two subunits identical in molecular weight (41,000) and in amino-terminal residue (threonine). The enzyme exhibited absorption maxima at 278,343, and 425 nm and contained 1 mol of pyridoxal 5'-phosphate per mol of enzyme. The formyl group of pyridoxal 5'-phosphate was bound through an aldimine linkage to the epsilon-amino group of a lysine residue of the protein. The enzyme-bound pyridoxal 5'-phosphate, absorbing at 425 nm, was released by incubation with phenylhydrazine to yield the catalytically inactive form. The inactive enzyme, which was reactivated by addition of pyridoxal 5'-phosphate, still had a 343-nm peak and contained 1 mol of a vitamin B6 compound. The holoenzyme showed positive circular dichroic bands at 340 and 425 nm, whereas the inactive form had no band at 425 nm. The enzyme was highly specific for L-ornithine and alpha-ketoglutarate and catalyzed delta-transamination between them to produce L-glutamate and L-glutamate-gamma-semialdehyde, which as spontaneously converted to delta 1-pyrroline-5-carboxylate. The enzyme activity was significantly affected by nonsubstrate amino acids, amines, and carbonyl reagents.
D000596:Amino Acids / Q000032:analysis; D001120:Arginine; D001407:Bacillus / Q000201:enzymology*; D003460:Crystallization; D004790:Enzyme Induction; D008970:Molecular Weight; D009952:Ornithine; D009953:Ornithine-Oxo-Acid Transaminase / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000032:analysis; D013057:Spectrum Analysis; D013379:Substrate Specificity; D000637:Transaminases / Q000378:metabolism*
D000596:Amino Acids; D011732:Pyridoxal Phosphate; D001120:Arginine; D009952:Ornithine; D000637:Transaminases; D009953:Ornithine-Oxo-Acid Transaminase