Association-Dissociation Phenomena of d-Amino Acid Oxidase

The association-dissociation equilibria of D-amino acid oxidase have been examined by light scattering under a variety of conditions.Under all conditions the value of molecular weight extrapolated to zero protein concentration appears to be in the neighborhood of 90,000.As the minimum molecular weight, based on flavin adenine dinucleotide content, is 45,500, the enzyme is thus concluded to have 2 molecules of FAD in its monomeric form.The light scattering results show that, under all conditions examined, a concentration-dependent equilibrium exists which is comprised of dimers and still higher molecular weight species.The extent of equilibrium polymer formation is decreased as the pH and temperature are lowered and as the concentration of chloride ions is increased.The extent of polymer formation is also affected by whether the enzyme is in the form of the holoenzyme, the benzoate complex, or the apoprotein.Concentration-dependent polymerization increases in the order apoprotein, benzoate complex, holoenzyme.At pH values close to the optimum for catalytic activity (pH 8.5), the effect of temperature on the apparent molecular weight indicates a transition from one equilibrium to another with a midpoint temperature of 12-15".Considerable confusion has arisen in the literature concerning the molecular weight of n-amino acid oxidase.The pure crystalline enzyme, isolated by Massey, Palmer, and Bennett (l), was found to contain 1 molecule of the prosthetic group flavin adenine dinucleotide per unit molecular weight of 45,500.An investigation of this enzyme with ultracentrifugal techniques by Charlwood, Palmer, and Bennett (2) showed that in solution the enzyme existed as an equilibrium mixture of aggregates in which molecular weight species 2 and 4 times this unit molecular weight were the predominant species.Evidence was also obtained in this study of the presence of even higher molecular weight aggregates.