title: Characterization of a fully active N-terminal 37-kDa polypeptide obtained by limited tryptic cleavage of pig kidney D-amino acid oxidase.
abstract: In order to obtain further information on the structure of D-amino acid oxidase (EC 1.4.3.3), limited proteolysis experiments have been carried out on its apo-, holo-, and holoenzyme-benzoate forms. The enzyme is unsensitive to 10% (w/w) chymotrypsin, while incubation with 10% (w/w) trypsin, under nondenaturating conditions, produces inactivation and proteolysis patterns which are different for the three forms of enzyme analyzed. These results confirm the previously reported conformational changes which occur upon binding of coenzyme to the apoprotein, and of benzoate to holoenzyme. The stable 37.0-kDa polypeptide, obtained from the apo- and holoenzyme-benzoate complex upon cleavage of a C-terminal 2.0-kDa fragment, retains full catalytic activity with unaltered kinetic parameters, and the coenzyme binding properties of the native enzyme. These results are in agreement with the tentative localization of the FAD-binding domain in the N-terminal region of the enzyme, and with the hypothesis that the function of the C-terminal region of D-amino acid oxidase could be related to the import of the enzyme into the peroxisomes, as suggested by Gould et al. (Gould, S. J., Keller, G. A., and Subramani, S. (1988) J. Cell. Biol. 107, 897-905).
authors.affiliation: Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Italy.
authors.forename: G T
authors.initials: GT
authors.lastname: Tarelli
authors.forename: M A
authors.initials: MA
authors.lastname: Vanoni
authors.forename: A
authors.initials: A
authors.lastname: Negri
authors.forename: B
authors.initials: B
authors.lastname: Curti
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003605:D-Amino-Acid Oxidase / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine; D014357:Trypsin / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D003605:D-Amino-Acid Oxidase; D014357:Trypsin