Association-Dissociation Phenomena of d-Amino Acid Oxidase

From the ¹ From the Institute of Biological Chemistry, the Consiglio Nazionale delle Ricerche Center of Molecular Biology, University of Rome; and the Regina Elena Institute for Cancer Research, Rome, Italy

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°.

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