Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A simple to use, robust, quantitative, and extremely sensitive colorimetric assay for alkaline phosphatase (EC 3.1.3.1), designed to be used as a detection system in diagnostic assays employing antibodies or gene probes, is described. This technology is based on the novel principle of prosthetogenesis, according to which a purpose-designed substrate (a prosthetogen) for a primary analyte-linked enzyme label is hydrolyzed to produce a prosthetic group for a detector enzyme system. The prosthetogen employed here is a derivative of FAD which is phosphorylated at the 3'-position of the ribose ring (FADP), the label enzyme is alkaline phosphatase, and the detector is a D-amino-acid oxidase/horseradish peroxidase-coupled system. Essentially each turnover of every molecule of alkaline phosphatase produces a molecule of D-amino-acid oxidase for detection. Thus enormous amplification of the initial signal is achieved in short time periods because of the relatively high turnover number of alkaline phosphatase for FADP. The system can be formatted as a stable, preformed, freeze-dried preparation containing all analytical components, which is reconstituted simply by addition of buffer solution. This methodology can quantitate less than 0.1 amol of alkaline phosphatase in 30 min at 25 degrees C using microtiter plates.
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PMID:Amplified assay of alkaline phosphatase using flavin-adenine dinucleotide phosphate as substrate. 136 Jul 71

Antiserum against homogeneous hog kidney D-amino-acid oxidase (D-amino-acid: oxygen oxidoreductase (deaminating), EC 1.4.3.3) was elicited in rabbits, and monospecific antibodies were prepared by affinity chromatography. The antibodies inhibited up to 90% of hog D-amino-acid oxidase activity, and 100% of the enzyme could be immunoprecipitated. The antibodies inhibited both holoenzyme and reconstituted apoprotein to a similar degree, indicating that they did not interfere with the FAD-binding site of the protein. The antibodies inhibited D-amino-acid oxidase activity from other mammalian species to a similar degree, while the enzyme activities from birds, amphibians, fishes and yeast were inhibited and immunoprecipitated to lower extents. In immunoblotting experiments, after SDS-polyacrylamide gel electrophoresis, the antibodies recognized a single band of about 40 kDa in all the species analyzed, and the entity of the signal was inversely related to the phylogenetic distance from mammals. The antibodies did not inhibit D-alanine dehydrogenase activity from Escherichia coli, but gave positive bands in immunoblotting.
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PMID:Immunochemical properties of D-amino-acid oxidase. 244 78

The flavoprotein D-amino-acid oxidase was purified to homogeneity from the yeast Rhodotorula gracilis by a highly reproducible procedure. The amino acid composition of the protein was determined; the protein monomer had a molecular mass of 39 kDa and contained one molecule of FAD. The ratio between A274/A455 was about 8.2. D-Amino-acid oxidase from yeast showed typical flavin spectral perturbations on binding of the competitive inhibitor benzoate and was reduced by D-alanine under anaerobiosis. The enzyme reacted readily with sulfite to form a covalent reversible adduct and stabilized the red anionic form of the flavin semiquinone on photoreduction in the presence of 5-deazariboflavin; the 3,4-dihydro-FAD form was not detectable after reduction with sodium borohydride. Thus D-amino-acid oxidase from yeast exhibited most of the general properties of the dehydrogenase/oxidase class of flavoproteins; at the same time, the enzyme showed some peculiar features with respect to the same protein from pig kidney.
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PMID:Properties of D-amino-acid oxidase from Rhodotorula gracilis. 256 32

Protein dynamics of D-amino-acid oxidase in the picosecond region was investigated by measuring time-resolved fluorescence of the bound coenzyme, FAD. The observed nonexponential fluorescence decay curves were analyzed with four-exponential decay functions. The fluorescence lifetimes at the best fit were 26.6 +/- 0.7 ps, 44.0 +/- 4.2 ps, 177 +/- 11 ps, and 2.28 +/- 0.21 ns at 20 degrees C and 25.2 +/- 3.0 ps, 50.3 +/- 8.7 ps, 228 +/- 27 ps, and 2.75 +/- 0.33 ns at 5 degrees C. Component fractions with the shortest lifetime, ca. 26 ps, were always negative and close to -1. The other fluorescent components of the lifetimes, ca. 47 ps, 200 ps, and 2.6 ns, with positive fractions were assigned to different forms of the enzyme including the dimer, the monomer, and free FAD dissociated from the enzyme. Measurements of the time-resolved fluorescence spectra revealed that the maximum wavelengths of the spectra shifted toward shorter wavelength by 65 nm at 20 degrees C and 36 nm at 5 degrees C within 100 ps after pulsed excitation. The remarkable blue shift was not observed in free FAD. The first spectra immediately after the excitation of the enzyme exhibited maximum wavelengths of 584 nm at 20 degrees C and 557 nm at 5 degrees C. The fluorescence spectra obtained at times later than 100 ps are in good agreement with the one obtained under steady-state excitation of D-amino-acid oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Picosecond-resolved fluorescence spectra of D-amino-acid oxidase. A new fluorescent species of the coenzyme. 256 89

The presence of arginine in the active center of D-amino-acid oxidase is well documented although its role has been differently interpreted as being part of the substrate-binding site or the positively charged residue near the N1-C2 = O locus of the flavin coenzyme. To have a better insight into the role of the guanidinium group in D-amino-acid oxidase we have carried out inactivation studies using phenylglyoxal as an arginine-directed reagent. Loss of catalytic activity followed pseudo-first-order kinetics for the apoprotein whereas the holoenzyme showed a biphasic inactivation pattern. Benzoate had no effect on holoenzyme inactivation by phenylglyoxal and the coenzyme analog 8-mercapto-FAD did not provide any additional protection in comparison to the native coenzyme. Spectroscopic experiments indicated that the modified protein is unable to undergo catalysis owing to the loss of coenzyme-binding ability. Analyses of time-dependent activity loss versus arginine modification or [14C]phenylglyoxal incorporation showed the presence of one arginine essential for catalysis. The protection exerted by the coenzyme is consistent with the involvement of an active-site arginine in the correct binding of FAD to the protein moiety. Comparative analyses of CNBr fragments obtained from apoenzyme, holoenzyme and the 8-mercapto derivative of D-amino-acid oxidase after reaction with phenylglyoxal did not provide unequivocal identification of the essential arginine residue within the primary structure of the enzyme. However, they suggest that it might be localized in the N-terminal portion of the polypeptide chain and point to a role of phenylglyoxal-modifiable arginine in binding to the adenylate/pyrophosphate moiety of the flavin coenzyme.
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PMID:Phenylglyoxal modification of arginines in mammalian D-amino-acid oxidase. 288 28

D-Amino-acid oxidase is a flavoprotein using FAD as cofactor. The enzyme has been immobilized in the presence of FAD on a non-porous matrix: chitosan. This support is covalently bound to the enzyme with glutaraldehyde as cross-linking reagent. It is characterized by a good mechanical resistance to mechanical stirring. The enzymatic assays have been performed in batch reactor with D-phenylglycine as substrate by a spectrophotometric method which is based on the variation of the absorbance at 252 or 280 nm. The behaviour of the biocatalysts has been studied during repeated assays of 1 h at 25 degrees C in the absence of exogenous FAD. The experimental results have been compared with those obtained with the soluble enzyme tested in the presence or in the absence of FAD. The dependence of D-amino-acid oxidase on FAD concentration has been studied. Immobilized enzyme on chitosan appears to be less sensitive to the association-dissociation equilibrium of FAD. This property and the capacity of the enzyme to polymerize spontaneously in solution according to the experimental conditions have been established. The fact that the enzyme can exist in various oligomeric forms is of major importance because its catalytic expression is dependent of this phenomenon. The polymerization is known to be responsible for a decrease of the maximal rate V of the enzyme. It has also been shown that in the same way this decrease was accompanied by an improvement of the affinity of enzyme for substrates. Furthermore, the value of the dissociation constant of the apoenzyme-FAD complex is significantly smaller as the degree of polymerization is high. The conclusion is that the dissociation of the cofactor can be avoided if the immobilization step is carried out at high concentration of enzyme which is favourable to its polymerization.
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PMID:[Influence of polymerization of D-amino acid oxidase on the behavior of the enzyme immobilized on chitosan by covalent fixation]. 289 90

We measured the redox potentials of frozen inactivated L-amino-acid oxidase (L-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.2) and inhibitor-bound (anthranilic acid) enzyme, and compared these redox properties to those of active L-amino-acid oxidase and benzoate-bound D-amino-acid oxidase (EC 1.4.3.3), respectively. The redox properties of the inactive enzyme are similar to the properties of free flavin; the potential is within 0.015 V of free flavin and no radical stabilization is seen. This corresponds to the loss of most interactions between apoprotein and flavin. In contrast, the anthranilic acid lowers the amount of radical stabilized from 85% to 35%. The potentials are still 0.150 V positive of free flavin, indicating that in the presence of inhibitor, many flavin-protein interactions remain intact. The difference between this behavior and that of D-amino-acid oxidase bound to benzoate, where the amount of radical declined from 95% to 5%, is explained on the basis of the relative tightness of binding of apoprotein to FAD. D-Amino-acid oxidase apoprotein has a relatively low Ka (10(6)) for FAD, and benzoate has a relatively high Ka (10(5)) for the enzyme. Therefore, the binding of benzoate increases the tightness of FAD binding to apo-D-amino-acid oxidase (10(11)), indicating significant changes in flavin-protein interactions. In contrast, apo-L-amino-acid oxidase binds flavin tightly (the Ka is greater than 10(7)) and the enzyme binds to anthranilate much less tightly, with a Ka of 10(3). The L-amino-acid oxidase apoprotein binding to FAD is tight initially, and the binding of anthranilate changes it only slightly.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The effects of reversible freezing inactivation and inhibitor binding on redox properties of L-amino-acid oxidase. 380 94

Aminoethylcysteine-ketimine (2H-1,4-thiazine-5,6-dihydro-3-carboxylic acid) strongly inhibits D-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3). The inhibition is purely competitive (Ki = 3.3 X 10(-7) M). Aminoethylcysteine-ketimine modifies the visible spectrum of the enzyme: the absorption maxima of bound FAD shift from 375-455 nm to 385-445 nm with a definite shoulder at 465 nm; the appearance of a large absorption band centered at 750 nm may be due to a charge-transfer complex formation. The dissociation constant for the aminoethylcysteine-ketimine-enzyme complex, calculated by a photometric procedure (4 X 10(-7) M), is in good agreement with kinetic data. The dicarboxylic analogue of this inhibitor (lanthionine-ketimine) is ineffective in D-amino-acid oxidase inhibition and does not produce any spectral modification of the enzyme. These results confirm structural requirements for D-amino-acid oxidase inhibitor reported by other researchers. Ketimine reduced forms (thiomorpholine-2-carboxylic acid and thiomorpholine-2,6-dicarboxylic acid) are chemically synthesized and checked as D-amino-acid oxidase substrates: only thiomorpholine-2-carboxylic acid is oxidized to aminoethylcysteine-ketimine (Km = 2 X 10(-4) M).
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PMID:Interaction between 1,4-thiazine derivatives and D-amino-acid oxidase. 613 40

D-Amino-acid oxidase catalyzes the oxidation of D-amino acids to imino acids. In the oxidative half-reaction, oxygen reacts with the reduced enzyme-imino acid complex to reoxidize the bound FAD. This is then followed by dissociation of the imino acid. The effects of pH and D2O on the kinetics of the oxidative half-reaction of D-amino-acid oxidase have been determined with glycine, D-alanine, and D-serine as substrates. Reaction of the reduced enzyme with oxygen requires that a group with a pKa value of about 10.5 be protonated and a group with a pKa value of 8.5 be deprotonated. The former value is not seen with D-alanine as substrate; the latter is only seen with glycine. No solvent isotope effects are seen on the V/KO2 value with D-alanine, consistent with rate-limiting electron transfer. Product release involves a pH-dependent conformational change. This is rate-limiting at all pH values with D-alanine as substrate. Significant solvent isotope effects are seen on the Vmax value with D-alanine. The proton inventory at high pH is linear, consistent with release of a single proton in the slow step; at pH 6 the solvent inventory is bowl-shaped, consistent with a solvent isotope effect on the conformation of the protein. With glycine the DV value increases to the intrinsic value at pH 10.5; this establishes that CH bond cleavage becomes rate-limiting with this substrate above pH 10.
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PMID:pH and kinetic isotope effects on the oxidative half-reaction of D-amino-acid oxidase. 791 Aug 22

The properties of D-aspartate oxidase from Octopus vulgaris (EC 1.4.3.1) have been investigated. The protein is a monomer of M(r) 37,000 containing one mol flavin/mol protein. The enzyme as isolated exists at least in two forms, one containing FAD and the other, which is catalytically inactive, probably containing 6-OH-FAD, as inferred from the absorption spectrum of the enzyme. An additional form of the enzyme, as far as the nature of the coenzyme is concerned, has been detected in the purified enzyme and shown to derive from the form originally containing FAD. The modulation of the coenzyme reactivity exerted by Octopus D-aspartate oxidase, as studied by spectrophotometric techniques, conforms to the one expected for an enzyme belonging to the oxidase class of flavoproteins. Structural investigations show similarities in both the amino-acid composition and the N-terminal amino-acid sequence to bovine D-aspartate oxidase and porcine D-amino-acid oxidase. In summary, the general properties of the enzyme from Octopus vulgaris closely resemble those of the enzyme from beef kidney. Moreover, kinetic analyses suggest that two active-site residues with pKa of 7.1 and 9.1 are critical for catalysis, and that the ionization of such residues has different effects on the catalytic activity depending whether mono- or dicarboxylic D-amino acids are used as substrate.
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PMID:Properties of the flavoenzyme D-aspartate oxidase from Octopus vulgaris. 791 43


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