Flavoenzyme D-amino Acid Oxidase Research Articles

Complex formation between anionic semiquinoid form of a flavoenzyme D-amino acid oxidase and ligands. Stabilizing mechanism of anionic semiquinoid flavoenzyme.

Picolinate binds to the anionic semiquinoid form of D-amino acid oxidase (DAO), and the complex formed has a broad absorption band in the long-wavelength region extending beyond 800 nm, which is reminiscent of a charge transfer interaction. The binding has a stoichiometry of 1:1 with respect to the enzyme. The dissociation constant at 25 degrees C was 30 microM at pH 7.0. The pH dependence (pH 7.0-8.3) of the dissociation constant indicates that one proton is associated with the complex formation, and suggests that picolinate able to bind to the anionic semiquinoid enzyme is in the cationic form protonated at the nitrogen atom. By adding dithionite to the oxidized DAO solution containing pyruvate and various amines, a similar anionic semiquinoid DAO complex having a broad long-wavelength absorption band, appeared. Resonance Raman spectra with excitation at 623.8 nm of the anionic semiquinoid DAO complex formed in the presence of pyruvate and methylamine indicate that the complex consists of the anionic semiquinoid DAO and N-methyl-alpha-iminopropionate produced from pyruvate and methylamine, and that the imino group must be protonated. This supports the proposal that the presence of a positively charged group in the vicinity of flavin is required for the stabilization of the anionic semiquinoid flavin. The results also suggest that the broad absorption band is derived from the charge transfer interaction between the anionic semiquinoid flavin and the imino acid, in which the flavin C(4a)-N(5) locus and the locus containing (Formula: see text) of the amino acid are important for the interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

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, ...).

Self-association mode of a flavoenzyme D-amino acid oxidase from hog kidney. I. Analysis of apparent weight-average molecular weight data for the apoenzyme in terms of models.

The self-association of D-amino acid oxidase apoenzyme in 0.1 M sodium pyrophosphate, pH 8.3, at 25 degrees C was studied by low-angle laser light scattering. The concentration (c) dependence of the apparent weight-average molecular weight (Mwapp) was determined over a wide concentration range of 0.04 to 6.1 mg/ml. The extrapolated Mwapp value, to zero enzyme concentration, corresponded to the Mr value of the monomer. The self-association mode of the apoenzyme was systematically explored with nonlinear least-squares analysis of the Mwapp versus c data. The simplest model that fitted the data well was a model of isodesmic indefinite self-association of the monomer with the isodesmic association constant of 0.467 +/- 0.034 liter/g. The monomer-dimer model proposed previously, but only in a low enzyme concentration range of less than 0.9 mg/ml at 5-20 degrees C (Henn, S. W., and Ackers, G. K. (1969) Biochemistry 8, 3829-3838), did not fit the Mwapp versus c data either in the limited low concentration range or in the whole concentration range examined at 25 degrees C. To test the validity of the chosen model, the observed sedimentation boundary profiles were compared with the idealized boundary profiles calculated for the better-fit models. The profile calculated with the model of the isodesmic indefinite self-association mechanism was qualitatively consistent with the observed ones. The utility of the nonlinear least-squares procedure for analyzing self-associating systems was demonstrated.

Histochemical staining of cells containing flavoenzyme D-amino acid oxidase based on its enzymatic activity: Application of a coupled peroxidation method.

A histochemical method for the demonstration of D-amino acid oxidase activity in rat kidney, liver and brain is described. The method is a peroxidase-coupled procedure, which is based on the intensifying effect of nickel ions on 3, 3′-diaminobenzidine-based product formation with peroxidase. The substrates used were D-proline and D-alanine, of which the former showed more intense staining. The histochemical reaction was suppressed by benzoate, a competitive inhibitor. There was no staining on omission of a substrate or peroxidase from the incubation medium. The method was specific for D-amino acid oxidase. The oxidase in kidney and brain showed resistance to the fixatives used (e. g. 8% glutaraldehyde), while the enzyme in hepatocytes did not. The reaction product was black, electron dense, stable and strictly confined to cells containing the oxidase. The method was successfully applied to the histochemical identification of the type of cell containing the oxidase at both the levels of light and electron microscopy. The oxidase activity was shown to be present in hepatocytes in the liver, proximal tubule cells in the kidney, and only astrocytes, including Bergmann glial cells, in the brain. In the midbrain, the oxidase was almost exclusively restricted to the tegmentum.

Gel chromatographic evidence for the participation of the higher polymers in the self-association system of a flavoenzyme D-amino acid oxidase.

The self-association of subunits of D-amino acid oxidase holoenzyme was studied by high-speed gel filtration with a short column of TSK-GEL G3000 SW in 0.1 M sodium pyrophosphate (pH 8.3) at 25 degrees C. Over the range of the peak concentrations of 0.009-4.45 mg/ml in the presence of FAD the apparent Stokes radii increased with an increase of the concentrations and did not level off. The largest value obtained in this study was 61.5 A. This would correspond to that calculated for the hexamer with linear subunit arrangement which has the largest Stokes radius among the various arrangements. These results provide the first gel chromatographic evidence that the higher polymers greater than the dimer participate in the self-associating system of the enzyme.

Resonance Raman study on the purple intermediates of the flavoenzyme D-amino acid oxidase

Resonance Raman (RR) spectra excited at 632.8 nm within a charge transfer absorption band were obtained for a catalytic intermediate, the purple complex of D-amino acid oxidase with D-proline or D-alanine as a substrate. The resonance enhanced Raman lines around 1605 and 1360 cm −1 in either of the complexes were suggested to be derived from vibrational modes of reduced flavin molecule. Since the highest energy band at 1692 cm −1 in the RR spectrum with D-alanine was shifted to 1675 cm −1 upon [ 15N] substitution of alanine and ammonium, this Raman line in the spectrum with D-alanine or the line at 1658 cm −1 with D-proline is assigned to the CN stretching mode of an imino acid corresponding to each amino acid. These results confirm the concept that the purple intermediate of D-amino acid oxidase consists of reduced flavin and an imino acid.

An active site-tyrosine-containing heptapeptide from D-amino acid oxidase.

The flavoenzyme D-amino acid oxidase (Eo) is rapidly chlorinated by N-chloro-D-leucine (Rudie, N.G., Porter, D.J.T., and Bright, H.J. (1980) J. Biol. Chem. 255, 498-508). We have carried out chymotryptic digestion of E0-36Cl2 and find that all of the radiolabel is located in a heptapeptide having [3.5-36Cl2]chlorotyrosine as the COOH-terminal residue. This heptapeptide, having the sequence -Asp-Leu-Glu-Arg-Gly-Ile-Tyr-, is located within a larger fragment obtained previously from cyanogen bromide cleavage of E0. These results demonstrate that the target for chlorination in E0 must be a single tyrosine residue and provide, when taken together with previous findings, the first clear evidence for the identity and location of an active site residue in the polypeptide chain of D-amino oxidase.

Coenzyme-induced subunit association of the flavoenzyme D-amino acid oxidase: a kinetic light scattering study

Hog kidney D-amino acid oxidase [EC I .4.3.3] contains a single FAD per 35 000-40 000 MW monomer [l-4], and undergoes self-association [S-12]. Many ligands, such as competitive inhibitor, product, and substrate, influence the association process of the oxidase f 11 ,13-161. The holoenzyme has a higher apparent MW than the apoenzyme at a given enzyme concentration [6,9-l 11. Recently, Yagi et al, [ 17,181 analyzed the binding property of FAD in D-amino acid oxidase according to monomerdimer equilibrium and demonstrated that the affinity of the dimer for the coenzyme FAD is about 70 times higher than that of the mono~~er. These thermodynamic studies indicate that the coenzyme induces association of the subunits in the equilibrium state of the oxidase. However, there is no direct evidence yet available concerning the reaction mechanism of FADinduced subunit association. In this communication, we demonstrate the subunit association subsequent to FAD binding to the apoenzyme and explore the ratelimiting step of this reaction by the kinetic light scattering method.

Chlorination of an active site tyrosyl residue in D-amino acid oxidase by N-chloro-D-leucine.

The chlorination of the flavoenzyme D-amino acid oxidase (EC 1.4.3.3, from hog kidney) by N-chloro-D-leucine, which specifically slows flavin reduction by a factor of 2 x 10(3), has been studied with respect to the site and mechanism of chlorination and the chlorinated enzyme has been extensively characterized. Our major results are as follows: 1. The oxidized holoenzyme (E0) incorporates 2 eq of 36Cl/flavin site from D-[N-36Cl]leucine. Evidence from amino acid and 36Cl analysis, isotopic dilution, and spectral titrations shows that the target residue is tyrosine and that this is converted to 3,5-dichlorotyrosine. The resulting decrease in basicity of this tyrosine (similar to 10(3)-fold) parallels the decrease (2 x 10(3)-fold) in the first order rate constant for flavin reduction observed with E0-Cl2 and we show that this residue satisfies all available criteria as being directly involved in the catalysis of flavin reduction. 2. Half-modified enzyme is a mixture of 0.5 E0 + 0.5 E0-Cl2 and E0-Cl is therefore chlorinated much more rapidly than E0 (kCl = 8 x 10(2) M-1S-1). Several lines of evidence show that enzyme-bound N-chloro-D-leucine is not oxidized prior to chlorination of the active site tyrosine. 3. The chlorination reaction is highly specific with respect to the structure of the chlorinating agent. Only the N-chloro derivatives of D-leucine, D-isoleucine, and D-norvaline are reactive while N-chloro derivatives of L-amino acids are tightly bound, but reversibly competitive, inhibitors which markedly perturb the electronic spectrum of enzyme-bound FAD.