Limited proteolysis of D-amino acid oxidase holoenzyme with trypsin cleaves the protein at Arg 221 and near the C-terminus, producing stable 25, 13.4, and 2 kDa polypeptides [Torri-Tarelli, G., Vanoni, M. A., Negri, A., and Curti, B. (1990) J. Biol. Chem. 265, 21242-21246]. The 25 and 13.4 kDa polypeptides remain associated to form a nicked D-amino acid oxidase species. This nicked protein form maintains the ability to bind FAD, but exhibits altered catalytic efficiency toward the oxidation of various D-amino acids when compared to native DAAO. Changes in substrate specificity were first monitored by measuring the activity in the presence of different amino acid substrates at various times during proteolysis. Three amino acid substrates were then selected for further analysis of the properties of the nicked D- amino acid oxidase species produced by limited tryptic proteolysis: D- serine, D-arginine, and D-alanine. The three D-amino acids represented limiting cases of the observed changes of enzyme activity on nicking: loss of activity, increase of activity, and minor activity changes, respectively. D- serine was found to be no longer a substrate of D-amino acid oxidase. D- arginine exhibited a 2.5-fold increased apparent maximum velocity although its K(m) value increased 2-fold with the nicked enzyme in comparison to the native species. D-alanine was oxidized 1.5-fold faster by the nicked D-amino acid oxidase at infinite substrate concentration, and its K(m) value increased approximately 4-fold. The K(d) for benzoate, which was determined kinetically with D-alanine as the enzyme substrate, increased 17-fold in the nicked species. Primary deuterium kinetic isotope effects on V and V/K during the oxidation of D-alanine were also measured. (D)V/K increased from 1.4 ± 0.2 to 1.8 ± 0.3 on nicking, while (D)V increased from 1.04 ± 0.1 to 2.53 ± 0.5. All the observed changes of the values of the kinetic parameters and of the observed isotope effects are consistent with the hypothesis that nicking of D-amino acid oxidase at position 221 decreases the strength of binding of both substrates and products to the enzyme active site. The information obtained by limited tryptic proteolysis nicely complements that gathered from the analysis of the three-dimensional structure of D-amino acid oxidase in complex with benzoate, which was recently determined [Mattevi, A., Vanoni, M. A., Todone, F., Rizzi, M, Teplyakov, A., Coda, A., Bolognesi, M., and Curti, B. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 7496-7501]. Arginine 221 is part of the 216-228 loop that covers the active site and contributes residues to substrate binding and catalysis. The limited proteolysis data support the hypothesis that this loop acts as a lid on the active site and controls both substrate specificity and the rate of turnover of D-amino acid oxidase.