Properties of meso - a , € - Diaminopimelate D - Dehydrogenase from Bacillus sphaericus

meso-a,eDiaminopimelate D-dehydrogenase, which has been purified to homogeneity from the extract of Bacillus sphaericus IF0 3626, 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 mso-a,diaminopimelate, lanthionine is deaminated by the enzyme to a far lesser extent. NADP+ is the exclusive cofactor. The pH optima were at about 10.6 for the deamination of meso-a,€-diaminopimelate and at 7.6 for its amination. L and D isomers of a,ediaminopimelate and meso-a,$-diaminoadipate competitively inhibit the oxidation of mso-a,diaminopimelate. Initial velocity and product inhibition studies show that the reductive amination proceeds through a sequential ordered ternary-binary mechanism. NADPH binds iirst to the enzyme followed by L-a-amino-eketopimelate and ammonia, and the products are released in the order of meso-a,€-diaminopimelate and NADP+. The Michaelis constants are as follows: meso-a,e-diaminopimelate (2.6 m), NADP’ (83 p~), NADPH (0.2 m), La-amino-eketopimelate (0.24 m), and ammonia (12.6 m ~ ) . 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.