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1. The holoenzyme of D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3] was found to combine with 1-anilinonaphthalene-8-sulfonate without liberation of its coenzyme, FAD. No energy transfer interaction was found to occur between the bound dye and FAD of the holoenzyme. On the other hand, when the apoenzyme was bound to the dye and then to FAD, energy transfer interaction between the bound dye and bound FAD was observed. In both cases, the dye competes with the substrate, D-alanine. It is concluded that the dye bound to the holoenzyme is oriented in such a special manner that the mutual orientation factor between the dye and FAD becomes very small in magnitude. 2. When the apoenzyme combined with the dye, the monomer-dimer equilibrium of the apoenzyme shifted towards the dimer. On the other hand, 4-monobenzoylamido-4'-aminostilbene-2,2'-disulfonate combined with the apoenzyme to induce monomerization.

1. D-Amino acid oxidase (D-amino acid: oxygen oxidoreductase (deaminating), EC 1.4.3.3) apoenzyme, holoenzyme and the enzyme-benzoate complex were active and stable when immobilized to aminoalkyl or carboxyalkyl agarose, or to cyanogen bromide-activated agarose. The immobilized enzyme-benzoate complex could be converted into the holo- and apoenzyme without being liberated from the agarose. 2. The apparent Michaelis constant and substrate specificity of the immobilized enzyme were similar to those of the free enzyme. The optimum pH of the reaction was shifted towards acidic side by 1.0-2.0 pH units from that of the free enzyme. 3. With increasing number of methylene groups of the 'spacer' from 3 to 5, molecular activity of the immobilized enzyme increased, while the apparent Miachaelis constant decreased.

The absorption spectrum of D-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3) was significantly perturbed by various alcohols; typical fine structures were observed in the visible absorption bands, accompanied by blue shifts of the peaks. Both fluorescence intensity and fluorescence polarization were increased upon the addition of alcohols, indicating that the coenzyme is not liberated from the apoenzyme but the hydrophobicity of the environment of the enzyme-bound flavin is increased. Upon the addition of alcohols, the circular dichroism of the enzyme was markedly modified in the visible and near-ultraviolet regions, while that of the apoenzyme in the near- and far-ultraviolet regions was scarcely modified, indicating a change in the interaction between the flavin coenzyme and protein. Both the apparent maximal velocity and the apparent Michaelis constant of the enzyme were increased by the addition of alcohols. The presence of alcohols tends to dissociate the dimer of this enzyme into the monomer, but the dissociation does not fully explain the increase in the maximal velocity of the enzyme by alcohols, because the increase in the maximal velocity caused by alcohols is larger than that expected from the dissociation. Since the rate of formation of the purple intermediate was decreased by alcohols in both the dimer and the monomer, the increase in the maximal velocity could be ascribed to an increase in the rate of dissociation of the enzyme-product complex. This increase could be ascribed to the protein conformational change, which is probably provoked by combination of alcohols with the enzyme at a locus other than that for substrate binding.

1. The fluorescence polarization, P, of FAD increased on complex formation with the apoenzyme of D-amino acid oxidase [D-amino acid: O2 ocidoreductase (deaminating), EC 1.4.3.3]. The time course of the increase was monophasic. The values of P were extimated to be 0.04, 0.4, and 0.4 for FAD, the enzyme and the enzyme-benzoate complex, respectively. 2. The value of P of the enzyme is dependent on its concentration, indicating that the degrees of dissociation of FAD in the monomer and dimer are different. The dissociation constant was calculated to be 7 times 10-minus 7 M for the monomeric form of the enzyme. This value is far larger than the value for the dimeric form of the enzyme, 1 times 10-minus 8 M, calculated from equilibrium dialysis data. 3. Changes in fluorescence polarization of the enzyme due to changes in solution pH or temperature can be explained in terms of the monomer-dimer equilibrium.

1. Sarcosine was oxidized by D-amino-acid oxidase (D-amino-acid: O2 oxidoreductase (deaminating), EC 1.4.3.3) to yield methylamine and glyoxylic acid. A seriies of N-alkyl glycines were also oxidized by this enzyme. 2. N-Acetyl- and N-Phenylglycine inhibited the oxidase by competing with the substrate, while N-methyl-N-acetylglycine did not bind to the enzyme. This suggests the requirement of at least one unsubstituted hydrogen atom at the amino group ofglycine for binding. 3. The primary step in the reaction was the release of a proton from the substrate, indicating the formation of a substituted imino acid, which was spontaneously hydrolyzed to glyoxylic acid acid and an amine.

1. Progesterone inhibited D-amino acid oxidase (D-amino acid : O2 oxidoreductase (deaminating), EC 1.4.3.3) in competition with its substrate, D-alanine. Binding of progesterone brought about the increase in both fluorescence intensity and fluorescence polarization of FAD, which indicates that the environment surrounding FAD chromophore is modified due to a conformational change in the apoenzyme. 2. Ethinyl estradiol, testosterone, testosterone propionate, corticosterone and aldosterone also inhibited the enzyme slightly in the same manner. Their binding also produced a slight increase in FAD fluorescence without decreasing the fluorescence polarization. 3. Cholesterol did not inhibit the enzyme, though it increased the fluorescence polarization of FAD. This indicates the binding of cholesterol with the enzyme at a site other than the substrate binding site.

Formation of a complex of D-amino acid oxidase (D-amino acid:O2 oxidoreductase (deaminating), EC 1.4.3.3) and benzoate, an enzyme-substrate complex model, was studied by measuring the fluorescence life-time of the coenzyme FAD of the complex by using a mode-locked Nd:YAG laser and a streak camera. The value of lifetime was 60 +/- 10 ps in the monomer of the complex and it was extremely short (much less than 5 ps) in the dimer of the complex. Since the values of fluorescence lifetime of the coenzyme are 130 ps in the monomeric form of free enzyme and 40 ps in the dimeric form of free enzyme, the decrease in the lifetime upon complex formation with benzoate is slight in the monomer (reduced to one-half) whereas marked in the dimer (reduced to less than 1/10). By analyzing the fluorescence decay curve, a dissociation constant of the monomer-dimer equilibrium of the complex was evaluated to be 0.4 +/- 0.3 microM, which is much smaller than that in free enzyme. Fluorescence analysis under steady state excitation revealed that the apparent dissociation constant (K) of FAD from the enzyme was decreased by 1:1000 upon the complex formation. Relative quantum yield of the fluorescence of FAD in the complex to that of free FAD exhibited appreciable dependence on the complex concentration: greater in the monomer and less in the dimer. These results suggest that a molecular interaction between FAD and amino acid residue(s) is strengthened by the complex formation, which contributes to a remarkable conformational change in the protein moiety of the complex.

Conformational difference surrounding the coenzyme, FAD, of D-amino acid oxidase (D-amino-acid:O2 oxidoreductase (deaminating), EC 1.4.3.3) between its monomeric and dimeric forms were examined by observing fluorescence of FAD. The fluorescence lifetimes of the coenzyme was measured directly with a mode-locked Nd:YAG laser and a streak camera in picosecond region. The values of lifetime of FAD fluorescence in the monomer and dimer were 130 +/- 20 ps and 40 +/- 10 ps, respectively. The relative quantum yield of the fluorescence of FAD combined with the protein to that of free FAD depended on the concentration of the enzyme; it was higher at lower concentration. Comparing the lifetime with relative quantum yield of FAD combined with the protein, it is concluded that the fluorescence is quenched mostly by a dynamic process. These results indicate that the distance between the isoalloxazine nucleus and a quencher is nearer in the dimer than in the monomer.

The lifetime of the purple intermediate formed from D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3] and a neutral D-amino acid under anaerobic conditions was measured with a series of neutral D-amino acids. The lifetime increases with increase in the number of carbons in the side chain of the amino acid up to 4-5 atoms and then decreases with further increase in the number of carbons. This suggests that the hydrophobicity of the alkyl group of the neutral D-amino acid determines the lifetime of the purple intermediate unless a steric effect occurs.

The formation of an initial enzyme-substrate complex of D-amino acid oxidase (D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3) and its substrate, D-alpha-aminobutyric acid, was studied kinetically at lower temperature and pH than their optima. The time course of the absorbance change at 516 nm in an anaerobic reaction was not exponential, but biphasic. The ratio of the rapidly reacting component to the slowly reacting one was decreased upon lowering of the temperature. The reaction rate of the rapidly reacting component depended on substrate concentration and gave a linear Arrhenius plot in the temperature range from -10 to +15 degrees C. The reaction rate of the slowly reacting component also depended on both substrate concentration and temperature. The rapidly reacting and slowly reacting components could be assigned to the substrate binding of the dimer and monomer, respectively, of this enzyme.

The exchange of bound FAD for free FAD was studied with D-amino acid oxidase (D-amino acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3) and beta-D-glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4). For a simple measurement of the reaction rate, equimolar amounts of the enzyme and [14C]FAD were mixed. The exchange occurred very rapidly in the holoenzyme of D-amino acid oxidase at 25 degrees C, pH 8.3 (half life of the exchange: 0.8 min), but slowly in the presence of the substrate or a competitive inhibitor, benzoate. It also occurred slowly in the purple complex of D-amino acid oxidase. In the case of beta-D-glucose oxidase, however, the exchange occurred very slowly at 25 degrees C, pH 5.6, regardless of the presence of the substrate or p-chloromercuribenzoate. On the basis of these findings, the turnover of the coenzymes of flavin enzymes in mammals is discussed.

In vitro synthesis of D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3], one of the peroxisomal flavin enzymes, was performed using a rabbit reticulocyte lysate system in order to elucidate the biosynthetic pathway of the enzyme. The apparent molecular weight of the synthesized enzyme protein was the same as that of D-amino acid oxidase purified from pig kidney. On the other hand, the enzyme protein was not detectable when a wheat germ lysate system was used for the translation. Denaturation of pig kidney poly(A)+ RNA with methylmercury hydroxide prior to the translation was found to enhance the synthesis of the enzyme protein. These results suggest a tight conformational structure of the mRNA used.