D-amino acid aminotransferase of Bacillus sphaericus. Enzymologic and spectrometric properties

• Published in: The Journal of biological chemistry Vol. 250; no. 17; pp. 6983 - 6989
• Main Authors: Yonaha, K, Misono, H, Yamamoto, T, Soda, K
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 10-09-1975
• Subjects: Amino Acids; Bacillus - enzymology; Circular Dichroism; Kinetics; Macromolecular Substances; Protein Conformation; Pyridoxal Phosphate - analysis; Stereoisomerism; Structure-Activity Relationship; Transaminases - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)41029-6

D-Amino acid aminotransferase, purified to homogeneity and crystallized from Bacillus sphaericus, has a molecular weight of
about 60,000 and consists of two subunits identical in molecular weight (30,000). The enzyme exhibits absorption maxima at
280, 330, and 415 nm, which are independent of the pH (5.5 to 10.0), and contains 2 mol of pyridoxal 5'-phosphate per mol
of enzyme. One of the pyridoxal-5'-P, absorbing at 415 nm, is bound in an aldimine linkage to the epsilon-amino group of a
lysine residue of the protein, and is released by incubation with phenylhydrazine to yield the catalytically inactive form.
The inactive form, which is reactivated by addition of pyridoxal 5'phosphate, still has a 330 nm peak and contains 1 mol of
pyridoxal 5'-phosphate. Therefore, this form is regarded as a semiapoenzyme. The holoenzyme shows negative circular dichroic
bands at 330 and 415 nm. D-Amino acid aminotransferase catalyzes alpha transamination of various D-amino acids and alpha-keto
acids. D-Alanine, D-alpha-aminobutyrate and D-glutamate, and alpha-ketoglutarate, pyruvate, and alpha-ketobutyrate are the
preferred amino donors and acceptors, respectively. The enzyme activity is significantly affected by both the carbonyl and
sulfhydryl reagents. The Michaelis constants are as follows: D-alanine (1.3 and 4.2 mM with alpha-ketobutyrate and alpha-ketoglutarate,
respictively), alpha-ketobutyrate (14 mM withD-alanine), alpha-ketoglutarate (3.4 mM with D-alanine), pyridoxal 5'-phosphate
(2.3 muM) and pyridoxamine 5'-phosphate (25 muM).