Self-association mode of a flavoenzyme D-amino acid oxidase from hog kidney. II. Stoichiometry of holoenzyme association and energetics of subunit association.

The self-association pattern of D-amino acid oxidase holoenzyme in 0.1 M sodium pyrophosphate, pH 8.3, at 25 degrees C was examined by the low-angle laser light-scattering method. As to the results of nonlinear least-squares analysis of the apparent weight-average molecular weight (Mwapp) versus protein concentration (c) data, the following three models fitted equally well the data over the concentration range of 0.03-11.4 mg/ml: 1) the model of isodesmic indefinite self-association of the monomer where the dimerization constant differs from the isodesmic association constant, 2) the model which involves the dimerization of the monomer and isodesmic indefinite self-association of the dimer, and 3) the model which involves the trimerization of the monomer and isodesmic indefinite self-association of the trimer. In a more limited concentration range (0.3-11.4 mg/ml), a model of isodesmic indefinite self-association of the stable dimer where the dimer does not dissociate into the monomers cannot be excluded from the above three models. Measurements with the concentration range lowered to 0.03 mg/ml enabled us to exclude unequivocally the model involving such a stable dimer and to extrapolate the Mwapp data to the Mr of the monomer at infinite dilution as in the case of the apoenzyme. The observed sedimentation boundary profiles were qualitatively consistent with the idealized boundary profiles calculated with the model which involves the dimerization of the monomer and isodesmic indefinite self-association of the dimer, so this model is the most probable of the models examined. These results provide the first evidence that the association mode of the holoenzyme is different from that of the apoenzyme, i.e. isodesmic indefinite self-association of the monomer (Tojo, H., Horiike, K., Shiga, K., Nishina, Y., Watari, H., and Yamano, T. (1985) J. Biol. Chem. 260, 12607-12614). The overall linkage scheme, between binding of coenzyme FAD and subunit association, was considered, and the overall free energy change in each process in the scheme was calculated. The total stabilization energies of the intersubunit interaction in the holoenzyme relative to the apoenzyme were found to be -2.2 kcal/mol at the dimerization step and -0.5 kcal/mol at the step of the addition of the dimer to any 2i-mer (i = 1,2, ...).