Purification and characterization of peroxisomal L-pipecolic acid oxidase from monkey liver.

L-Pipecolic acid oxidase has been purified to near homogeneity from Rhesus monkey liver. The protein, a yellow monomer, has a molecular weight of 46,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 8.9. It contains a covalently bound flavin with absorption maxima at 457 and 383 nm and a shoulder at 480 nm. The purified enzyme is most reactive toward L-pipecolic acid, with lesser reactivities toward L-proline and sarcosine. The enzyme has no significant reactivity toward the D-enantiomer of pipecolic acid or toward any other amino acid tested. Benzoic acid is a competitive inhibitor of the enzyme with a Ki of 750 microM. The Km of the purified enzyme is 3.7 mM for L-pipecolic acid. With less purified preparations, the reaction product is alpha-aminodipic acid. The purified enzyme, however, produces an intermediate which reacts with ortho-aminobenzaldehyde to form an alpha-aminoadipic acid semialdehyde adduct. Thus, the formation of alpha-aminoadipic acid requires at least two enzymes.