Stereospecificity of thermostable ornithine 5-aminotransferase for the hydrogen transfer in the L- and D-ornithine transamination.
The thermostable ornithine 5-aminotransferase of a thermophile, Bacillus sp. YM-2, is unique in acting on both enantiomers of ornithine, although less effectively on the D-enantiomer. We studied the stereospecificity of the enzyme for the hydrogen abstraction from C-5 of the substrate moiety and the addition and removal of the hydrogen at C-4' of the cofactor (pyridoxal phosphate and pyridoxamine phosphate) moiety of the external Schiff base intermediate in the transamination of L- and D-ornithine. L- and D-[5-3H]ornithines were prepared by incubation of L- and D-ornithines with the enzyme in 3H2O, respectively. When the L-[5-3H]ornithine was incubated with L-ornithine 5-aminotransferase of a mesophile, Bacillus sphaericus, which catalyzes the stereospecific abstraction of pro-S hydrogen from C-5 of L-ornithine, most of the tritium was released into the solvent. The D-[5-3H]ornithine also reacted with the enzyme of B. sphaericus in the presence or absence of the amino acid racemase of Pseudomonas putida. Tritium was released only in the presence of the racemase, which catalyzes the racemization of ornithine but does not act on C-5 of ornithine. These results show that the Bacillus sp. YM-2 ornithine 5-aminotransferase stereospecifically abstracts the pro-S hydrogen from C-5 of L- and D-ornithine. When the apo form of the enzyme was incubated with pyridoxamine 5'-phosphate that was stereospecifically tritiated at C-4' and 2-oxoglutarate in the presence of L-ornithine or D-ornithine, tritium was released exclusively from (4'S)-[4'-3H]pyridoxamine. Therefore, addition and abstraction of hydrogen at C-4' of the cofactor moiety stereospecifically occur on the si face of the external Schiff base intermediate in the overall transamination catalyzed by Bacillus sp. YM-2 ornithine 5-aminotransferase irrespective of the C-2 configuration of the amino donor.
Jhee
K H
KH
Laboratory of Biofunctional Molecules, Kyoto University, Japan.
Yoshimura
T
T
Esaki
N
N
Soda
K
K
D000591:Amino Acid Isomerases / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D004795:Enzyme Stability; D006358:Hot Temperature; D009682:Magnetic Resonance Spectroscopy; D008961:Models, Structural; D009952:Ornithine / Q000737:chemistry; D009953:Ornithine-Oxo-Acid Transaminase / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D016958:Pseudomonas putida / Q000201:enzymology; D013237:Stereoisomerism; D013379:Substrate Specificity; D013816:Thermodynamics; D014316:Tritium
D014316:Tritium; D009952:Ornithine; D009953:Ornithine-Oxo-Acid Transaminase; D000591:Amino Acid Isomerases