Complex Of D-amino Acid Oxidase Research Articles

Commentary on “Crystallization of Michaelis complex of d-amino acid oxidase’ by K. Yagi and T. Ozawa Biochim. Biophys. Acta 60 (1962) 200–201

Commentary on “Crystallization of Michaelis complex of d-amino acid oxidase’ by K. Yagi and T. Ozawa Biochim. Biophys. Acta 60 (1962) 200–201

Picosecond laser fluorometry of FAD of D-amino acid oxidase-benzoate complex.

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.

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.

Spectroscopic demonstration of an initial stage of the complex of D-amino acid oxidase and its substrate D-α-aminobutyric acid

D-Amino acid oxidase [D-amino acid: O 2 oxidoreductase (deaminating), EC 1.4.3.3] was anaerobically mixed with its substrate D-α-aminobutyric acid at −10°C and pa H ∗ 7 which were apart from their maxima for the enzymatic reaction. By an ordinary self-recording spectrophotometer, the absorption spectrum of an initial stage of the complex could be observed. The spectrum was in principle similar to that of the complex of this enzyme with benzoate, the enzyme-substrate complex model. The spectroscopic observation revealed that this species is in an equilibrium with the purple intermediate, a strong charge transfer complex between the enzyme and its substrate neutral D-amino acid.

Exchange of free and bound coenzyme of flavin enzymes studied with [ 14C]FAD

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 β- d-glucose oxidase (β- 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°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 β- d-glucose oxidase, however, the exchange occurred very slowly at 25°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.

Change in the charge on flavin-adenine dinucleotide by forming a model of the enzyme-substrate complex of D-amino acid oxidase.

Change in the charge on flavin-adenine dinucleotide by forming a model of the enzyme-substrate complex of D-amino acid oxidase.

Change in the charge on flavin-adenine dinucleotide by forming a model of the enzyme-substrate complex of d-amino acid oxidase

Change in the charge on flavin-adenine dinucleotide by forming a model of the enzyme-substrate complex of d-amino acid oxidase

Complex formation of apoenzyme, coenzyme and substrate of d-amino acid oxidase V. Change in conformation of the protein by forming a model of enzyme-substrate complex

1. 1. The conformations of the apoenzyme, the holoenzyme and a model of enzyme-substrate complex of d-amino acid oxidase ( d-amino acid: O 2 oxidoreductase (deaminating), EC 1.4.3.3), were determined by using β-function obtained from the combination of hydrodynamic properties, together with the results of light-scattering and of optical-rotatory-dispersion measurements. 2. 2. The results indicate that remarkable changes in conformation of the molecule (from random ellipsoid to rigid sphere) occur with the change from apoenzyme to enzyme-substrate model. 3. 3. The above changes are discussed in connection with the mechanism of the enzymic catalysis.

Crystallization of Michaelis complex of d-amino acid oxidase

Crystallization of Michaelis complex of d-amino acid oxidase