Properties of meso-alpha,epsilon-diaminopimelate D-dehydrogenase from Bacillus sphaericus

• Published in: The Journal of biological chemistry Vol. 255; no. 22; pp. 10599 - 10605
• Main Authors: Misono, H, Soda, K
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 25-11-1980
• Subjects: Amino Acid Oxidoreductases - metabolism; Bacillus - enzymology; Binding, Competitive; Hydrogen-Ion Concentration; Kinetics; Macromolecular Substances; Molecular Weight; NADP - pharmacology; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)70348-2

meso-alpha,epsilon-Diaminopimelate D-dehydrogenase, which has been purified to homogeneity from the extract of Bacillus sphaericus
IFO 3525, has a molecular weight of about 80,000 and consists of two subunits identical in molecular weight (approximately
40,000). The enzyme has a high substrate specificity. In addition to meso-alpha,epsilon-diaminopimelate, lanthionine is deaminated
by the enzyme to a far lesser extent. NADP+ is the exclusive cofactor. The pH optima were at about 10.5 for the deamination
of meso-alpha,epsilon-diaminopimelate and at 7.5 for its amination. L and D isomers of alpha,epsilon-diaminopimelate and meso-alpha,delta-diaminoadipate
competitively inhibit the oxidation of meso-alpha,epsilon-diaminopimelate. Initial velocity and product inhibition studies
show that the reductive amination proceeds through a sequential ordered ternary-binary mechanism. NADPH binds first to the
enzyme followed by L-alpha-amino-epsilon-ketopimelate and ammonia, and the products are released in the order of meso-alpha,epsilon-diaminopimelate
and NADP+. The Michaelis constants are as follows: meso-alpha,epsilon-diaminopimelate (2.5 mM), NADP+ (83 micro M), NADPH
(0.2 mM), L-alpha-amino-epsilon-ketopimelate (0.24 mM), and ammonia (12.5 mM). The pro-S hydrogen at C-4 of the dihydronicotinamide
ring of NADPH is transferred to the substrate; the enzyme is B-stereospecific. Fluorometric study on binding of NADPH to the
enzyme revealed that the enzyme contains two coenzyme binding sites per molecule.