Substrate Of D-amino Acid Oxidase Research Articles

Peptidoglycan-Targeted [18F]3,3,3-Trifluoro-d-alanine Tracer for Imaging Bacterial Infection.

Imaging is increasingly used to detect and monitor bacterial infection. Both anatomic (X-rays, computed tomography, ultrasound, and MRI) and nuclear medicine ([111In]-WBC SPECT, [18F]FDG PET) techniques are used in clinical practice but lack specificity for the causative microorganisms themselves. To meet this challenge, many groups have developed imaging methods that target pathogen-specific metabolism, including PET tracers integrated into the bacterial cell wall. We have previously reported the d-amino acid derived PET radiotracers d-methyl-[11C]-methionine, d-[3-11C]-alanine, and d-[3-11C]-alanine-d-alanine, which showed robust bacterial accumulation in vitro and in vivo. Given the clinical importance of radionuclide half-life, in the current study, we developed [18F]3,3,3-trifluoro-d-alanine (d-[18F]-CF3-ala), a fluorine-18 labeled tracer. We tested the hypothesis that d-[18F]-CF3-ala would be incorporated into bacterial peptidoglycan given its structural similarity to d-alanine itself. NMR analysis showed that the fluorine-19 parent amino acid d-[19F]-CF3-ala was stable in human and mouse serum. d-[19F]-CF3-ala was also a poor substrate for d-amino acid oxidase, the enzyme largely responsible for mammalian d-amino acid metabolism and a likely contributor to background signals using d-amino acid derived PET tracers. In addition, d-[19F]-CF3-ala showed robust incorporation into Escherichia coli peptidoglycan, as detected by HPLC/mass spectrometry. Based on these promising results, we developed a radiosynthesis of d-[18F]-CF3-ala via displacement of a bromo-precursor with [18F]fluoride followed by chiral stationary phase HPLC. Unexpectedly, the accumulation of d-[18F]-CF3-ala by bacteria in vitro was highest for Gram-negative pathogens in particular E. coli. In a murine model of acute bacterial infection, d-[18F]-CF3-ala could distinguish live from heat-killed E. coli, with low background signals. These results indicate the viability of [18F]-modified d-amino acids for infection imaging and indicate that improved specificity for bacterial metabolism can improve tracer performance.

Chemogenetic emulation of intraneuronal oxidative stress affects synaptic plasticity

Overproduction of reactive oxygen species (ROS) and oxidative cell damage are commonly associated with most brain pathologies [1, 2]. Dysregulation of redox homeostasis in the aging brain is thought to be responsible for impaired synaptic transmission and plasticity, leading to reduced neuronal computational capacity and learning and memory deficits. Studying the contribution of oxidative stress to the development of diseases, such as age-related dementia and Alzheimer’s disease, is complex due to the lack of methods for modeling isolated oxidative damage in individual cell types [3]. We introduce a chemogenetic approach utilizing D-amino acid oxidase (DAAO) from yeast to produce hydrogen peroxide intraneuronally, which is one of the most stable ROS [4]. H2O2 generation was evaluated in primary cultured neurons and acute mouse brain slices through the utilization of a genetically encoded fluorescent biosensor, HyPer7, to validate the methodology [5]. The changes in the fluorescence signal of HyPer7 after treating neurons that expressed DAAO with D-Norvaline (D-Nva), a substrate for DAAO, confirmed the targeted production of H2O2 through chemogenetics. Using electrophysiological recordings in acute brain slices, we demonstrated that intraneuronal oxidative stress induced by chemogenetics did not affect basal synaptic transmission and the probability of neurotransmitter release from presynaptic terminals. However, it diminished long-term potentiation (LTP) at the single-cell level. Astrocytes have the ability to metabolize d-amino acids, rendering the proposed approach ineffective in vivo experiments. Consequently, in vivo testing of the tool was necessary for validation. To achieve this, an optical setup for exciting and detecting the HyPer7 signal was developed and implanted into the mouse brain via optical fibers. By using this approach, we were able to demonstrate the generation of H2O2 in DAAO-expressing neurons in vivo, upon intraperitoneal administration of D-amino acids. The results demonstrate that using a DAAO-based chemogenetic tool, along with electrophysiological recordings, clarifies numerous unanswered queries regarding the part of ROS-dependent signaling in typical brain activities and the impact of oxidative stress on the development of cognitive aging and preliminary neurodegenerative stages. The suggested method is valuable for detecting initial indicators of neuronal oxidative stress. Additionally, it can be used for evaluating probable antioxidants that can effectively combat neuronal oxidative harm.

Chemogenetic emulation of intraneuronal oxidative stress affects synaptic plasticity

Oxidative stress, a state of disrupted redox signaling, reactive oxygen species (ROS) overproduction, and oxidative cell damage, accompanies numerous brain pathologies, including aging-related dementia and Alzheimer's disease, the most common neurodegenerative disorder of the elderly population. However, a causative role of neuronal oxidative stress in the development of aging-related cognitive decline and neurodegeneration remains elusive because of the lack of approaches for modeling isolated oxidative injury in the brain. Here, we present a chemogenetic approach based on the yeast flavoprotein d-amino acid oxidase (DAAO) for the generation of intraneuronal hydrogen peroxide (H2O2). To validate this chemogenetic tool, DAAO and HyPer7, an ultrasensitive genetically encoded H2O2 biosensor, were targeted to neurons. Changes in the fluorescence of HyPer7 upon treatment of neurons expressing DAAO with d-norvaline (D-Nva), a DAAO substrate, confirmed chemogenetically induced production of intraneuornal H2O2. Then, using the verified chemogenetic tool, we emulated isolated intraneuronal oxidative stress in acute brain slices and, using electrophysiological recordings, revealed that it does not alter basal synaptic transmission and the probability of neurotransmitter release from presynaptic terminals but reduces long-term potentiation (LTP). Moreover, treating neurons expressing DAAO with D-Nva via the patch pipette also decreases LTP. This observation indicates that isolated oxidative stress affects synaptic plasticity at single cell level. Our results broaden the toolset for studying normal redox regulation in the brain and elucidating the role of oxidative stress to the pathogenesis of cognitive aging and the early stages of aging-related neurodegenerative diseases. The proposed approach is useful for identification of early markers of neuronal oxidative stress and may be used in screens of potential antioxidants effective against neuronal oxidative injury.

Coumarin derivatives as inhibitors of d-amino acid oxidase and monoamine oxidase

d-Amino acid oxidase (DAAO) oxidises d-amino acids to ultimately produce the corresponding α-keto acids. The DAAO substrate, d-serine, is a co-agonist at NMDA receptors, while NMDA receptor hypo-function has been implicated in the pathophysiology of schizophrenia. Through the modulation of d-serine levels, the inhibition of DAAO represents a strategy to increase NMDA receptor function, and thus a potential treatment for schizophrenia. Literature reports that 3-hydroxycoumarin is a potent inhibitor of DAAO and represents an ideal lead for the development of novel DAAO inhibitors. Based on this, the present study investigated DAAO inhibition by a series of synthetic and commercially available coumarin derivatives. Due to structural similarity to coumarin, a synthetic series of 3,4-dihydroisoquinolin-1(2H)-one derivatives has also been included in this study. The results show that among 37 compound evaluated, four inhibit porcine kidney DAAO with IC50 < 10 µM. The most potent inhibitors are 3,7-dihydroxycoumarin and 6,7-dihydroxycoumarin with an IC50 values of 0.167 and 0.224 µM, respectively. These values are an improvement on that of the reference DAAO inhibitor, 3-methylpyrazole-5-carboxylic acid (IC50 = 1.88 µM). Coumarin compounds are also known to inhibit the monoamine oxidase (MAO) enzymes, which are well established targets for the treatment of depression and Parkinson’s disease. As DAAO and MAO are flavoenzymes, off-target inhibition may occur. The series were thus evaluated as potential MAO inhibitors, and a number of high potency inhibitors were identified. Seven compounds inhibit the recombinant human MAOs with IC50 < 0.1 µM, with the most potent MAO-A and MAO-B inhibitors exhibiting IC50 values of 0.033 and 0.012 µM, respectively. This is significantly more potent than the reference inhibitors, curcumin, isatin and toloxatone. This study concludes that active DAAO and MAO inhibitors may serve as novel leads for the design of compounds that may find future application in the treatment of neuropsychiatric (e.g. schizophrenia, depression) and neurodegenerative disorders (e.g. Parkinson’s disease).

D-Serine as a Key Building Block: Enzymatic Process Development and Smart Applications within the Cascade Enzymatic Concept

An efficient enzymatic method catalyzed by an enzyme from the d-threonine aldolase (DTA) family was developed for d-serine production at industrial scale. This process was used for the synthesis of two valuable ketoses, l-erythrulose and d-fructose, within the cascade enzymatic concept involving two other enzymes. Indeed, d-serine was used as a substrate of d-amino acid oxidase (DAAO) for the in situ generation of the corresponding α-keto acid, hydroxypyruvic acid (HPA), a key donor substrate of transketolase (TK). This enzyme catalyzed the irreversible transfer of the ketol group from HPA to an aldehyde acceptor to form a (3S)-ketose by stereoselective carbon–carbon bond formation. The compatibility of all enzymes and substrates allowed a sequential three-step enzymatic process to be performed without purification of the intermediates. This strategy was validated with two TK aldehyde substrates to finally obtain the corresponding (3S)-ketoses with high control of the stereoselectivity and excellent aldehyde conversion rates.

Construction of a D-amino acid oxidase reactor based on magnetic nanoparticles modified by a reactive polymer and its application in screening enzyme inhibitors.

Developing facile and high-throughput methods for exploring pharmacological inhibitors of D-amino acid oxidase (DAAO) has triggered increasing interest. In this work, DAAO was immobilized on the magnetic nanoparticles, which were modified by a biocompatible reactive polymer, poly(glycidyl methacrylate) (PGMA) via an atom transfer radical polymerization technique. Interestingly, the enzyme immobilization process was greatly promoted with the assistance of a lithium perchlorate catalyst. Meanwhile, a new amino acid ionic liquid (AAIL) was successfully synthesized and employed as the efficient chiral ligand in a chiral ligand exchange capillary electrophoresis (CLE-CE) system for chiral separation of amino acids (AAs) and quantitation of methionine, which was selected as the substrate of DAAO. Then, the apparent Michaelis-Menten constants in the enzyme system were determined with the proposed CLE-CE method. The prepared DAAO-PGMA-Fe3O4 nanoparticles exhibited excellent reusability and good stability. Moreover, the enzyme reactor was successfully applied in screening DAAO inhibitors. These results demonstrated that the enzyme could be efficiently immobilized on the polymer-grafted magnetic nanoparticles and that the obtained enzyme reactor has great potential in screening enzyme inhibitors, further offering new insight into monitoring the relevant diseases.

D -Amino Acid Oxidase–Nanoparticle System: A Potential Novel Approach for Cancer Enzymatic Therapy

The authors propose a new magnetic nanoparticle-enzyme system for cancer therapy capable of targeting the enzyme and consequently decreasing the adverse effects, meanwhile improving the patient's life quality. The authors have functionalized Fe3O4 nanoparticles with 3-amino-propyltriethoxysilane (APTES) and conjugated it to yeast D-amino acid oxidase (DAAO) by coupling this with glutaraldehyde. The authors have tested the Fe3O4-APTES-DAAO system on three tumor cell lines. Exposed cells show, at the electron microscope level, nanoparticles on the surface of the plasma membrane and inside endocytic vesicles. Fe3O4-APTES-DAAO caused a substantial decrease of cell viability greatly augmented when D-alanine, a DAAO substrate, was added. Fe3O4-APTES-DAAO was demonstrated to be more effective than free DAAO, confirming the validity of the system in cancer therapy.

Selective Oxidative Stress in Cell Nuclei by Nuclear-Targeted D-Amino Acid Oxidase

The effects of nuclear-localized oxidative stress on both nuclear antioxidant systems, and the processes that they regulate, are not clearly understood. Here, we targeted a hydrogen peroxide (H(2)O(2))-producing enzyme, D-amino acid oxidase (DAAO), to the nucleus (NLS-DAAO) and used this to generate H(2)O(2) in the nuclei of cells. On addition of N-acetyl-D-alanine (NADA), a substrate of DAAO, to NLS-DAAO-transfected HeLa cells, a twofold increase in ROS production relative to untreated, transfected control was observed. Staining of cellular thiols confirmed that NLS-DAAO-induced ROS selectively modified the nuclear thiol pool, whereas the cytoplasmic pool remained unchanged. Furthermore, NLS-DAAO/NADA-induced ROS caused significant oxidation of the nuclear GSH pool, as measured by nuclear protein S-glutathionylation (Pr-SSG), but under the same conditions, nuclear Trx1 redox state was not altered significantly. NF-kappaB reporter activity was diminished by NLS-DAAO/NADA-stimulated nuclear oxidation. We conclude that nuclear GSH is more susceptible to localized oxidation than is nuclear Trx1. Furthermore, the attenuation of NF-kappaB reporter activity in the absence of nuclear Trx1 oxidation suggests that critical nuclear redox proteins are subject to control by S-glutathionylation during oxidative stress in the nucleus.

Studies on the association of 2-thiazolidinecarboxylic acid and antimony potassium tartrate: chiral recognition and prediction of absolute configuration by electrospray ionization mass spectrometry.

Optically active 2-thiazolidinecarboxylic acid (2-THC), a substrate for D-amino acid oxidase in animal kidney, is known to undergo racemization quickly in solution. The association of (+)- and (-)-2-THC with antimony potassium tartrate K(2)[Sb(2)(L or D-tart)(2)] was studied by electrospray ionization mass spectrometry (ESI-MS). We observed that relative intensities of associated ions in acetonitrile/water solution were changing as the racemization progressed. For [Sb(2)(L-tart)(2)](2-), the intensities of the associated ions increased as (+)-2-THC underwent racemization to a (-)-isomer; on the other hand, the intensity of the associated ion decreased as (-)-2-THC underwent racemization to a (+)-isomer. In the case of [Sb(2)(D-tart)(2)](2-), an opposite effect on the intensities of the associated ions was observed. The change in the intensities of associated ions can be used for chiral recognition of (+)-2-THC and (-)-2THC. Stereochemical models of the association of the optical isomers with [Sb(2)(L- or D-tart)(2)](2-) were constructed from the consideration of both hydrogen bonding of NH-O functions and HSAB (hard and soft acids and bases) interaction of S and Sb atoms. Comparison of the stereochemical models with the ESI-MS results enabled us to predict the absolute configurations of the 2-THC isomers.

内耳における神経情報伝達

Fast excitatory synaptic transmission in the inner ear has been proposed to be mediated by an excitatory amino acid, possibly L-glutamate, and its receptors. Previous studies have revealed that certain types of ionotropic glutamate receptors are expressed in various combinations of subtypes in the cochlea and vestibule. Other studies have suggested that some glutamate receptors may have non-neural functions, such as cell differentiation and migration. Recently the glutamate receptor a subfamily has been identified, although little is known about its physiological characteristics thus far. Since 1997 it has been known that the 61 subunit is highly expressed in hair cells of auditory and vestibular systems, which suggests that the 61 subunit may play an important functional role in these systems. The expression of NMDA receptor mRNA in spiral ganglion cells has also been demonstrated, suggesting involvement of the NMDA receptor in neurotransmission in the inner ear. In search of coagonists of the NMDA receptor other than glycine, D-serine has been found to be present in mammalian central nervous systems and is regarded as a possible neuro-modulator of the NMDA receptor. We have demonstrated the gene expression of D-amino acid oxidase (DAO) in the rat temporal bone using RT-PCR analysis. This observation suggests that D-serine, which is the substrate of DAO, may exist in the inner ear and may modulate the neurotransmission of NMDA receptor subunit NR1.

Limited proteolysis and X-ray crystallography reveal the origin of substrate specificity and of the rate-limiting product release during oxidation of D-amino acids catalyzed by mammalian D-amino acid oxidase.

Limited proteolysis of D-amino acid oxidase holoenzyme with trypsin cleaves the protein at Arg 221 and near the C-terminus, producing stable 25, 13.4, and 2 kDa polypeptides [Torri-Tarelli, G., Vanoni, M. A., Negri, A., & Curti, B. (1990) J. Biol. Chem. 265, 21242-21246]. The 25 and 13.4 kDa polypeptides remain associated to form a nicked D-amino acid oxidase species. This nicked protein form maintains the ability to bind FAD, but exhibits altered catalytic efficiency toward the oxidation of various D-amino acids when compared to native DAAO. Changes in substrate specificity were first monitored by measuring the activity in the presence of different amino acid substrates at various times during proteolysis. Three amino acid substrates were then selected for further analysis of the properties of the nicked D-amino acid oxidase species produced by limited tryptic proteolysis: D-serine, D-arginine, and D-alanine. The three D-amino acids represented limiting cases of the observed changes of enzyme activity on nicking: loss of activity, increase of activity, and minor activity changes, respectively. D-serine was found to be no longer a substrate of D-amino acid oxidase. D-arginine exhibited a 2.5-fold increased apparent maximum velocity although its Km value increased 2-fold with the nicked enzyme in comparison to the native species. D-alanine was oxidized 1.5-fold faster by the nicked D-amino acid oxidase at infinite substrate concentration, and its Km value increased approximately 4-fold. The Kd for benzoate, which was determined kinetically with D-alanine as the enzyme substrate, increased 17-fold in the nicked species. Primary deuterium kinetic isotope effects on V and V/K during the oxidation of D-alanine were also measured. (D)V/K increased from 1.4 +/- 0.2 to 1.8 +/- 0.3 on nicking, while (D)V increased from 1.04 +/- 0.1 to 2.53 +/- 0.5. All the observed changes of the values of the kinetic parameters and of the observed isotope effects are consistent with the hypothesis that nicking of D-amino acid oxidase at position 221 decreases the strength of binding of both substrates and products to the enzyme active site. The information obtained by limited tryptic proteolysis nicely complements that gathered from the analysis of the three-dimensional structure of D-amino acid oxidase in complex with benzoate, which was recently determined [Mattevi, A., Vanoni, M. A., Todone, F., Rizzi, M., Teplyakov, A., Coda, A., Bolognesi, M., & Curti, B. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 7496-7501]. Arginine 221 is part of the 216-228 loop that covers the active site and contributes residues to substrate binding and catalysis. The limited proteolysis data support the hypothesis that this loop acts as a lid on the active site and controls both substrate specificity and the rate of turnover of D-amino acid oxidase.

Transfection of the DAAO gene and subsequent induction of cytotoxic oxidative stress by D-alanine in 9L cells

D-amino acid oxidase (DAAO) can catalyze the dehydrogenation of D-amino acids, such as D-alanine, to the corresponding amino acids and is then reoxidized by molecular oxygen to yield hydrogen peroxide, a reactive oxygen species, which reacts with DNA, lipids and protein, inducing cell death. This study investigated whether rat glioma 9L cells infected with the recombinant retrovirus containing the DAAO cDNA fragment can be induced in order to undergo cytotoxic oxidative stress by D-alanine. The recombinant retroviral vector, plzrus-DAAO-FLAG-GFP (pl-Dfg), was constructed and used to transfect packaged phoenix cells. The supernatant containing recombinant retroviral particles from the transfected phoenix cells was harvested and utilized to infect target 9L cells. The cytotoxic oxidative stress of infected 9L cells was induced by the DAAO substrate, D-alanine. The plasmid pl-Dfg was successfully constructed. The high titer retroviral supernatant was obtained from the transfected phoenix cells. Infected 9L cells were less viable after exposure to D-alanine compared to the control group. Anti-apoptotic proteins significantly inhibited cell death. The DAAO/D-alanine system has a potential utility for gene therapy and may be an effective strategy for the treatment of brain cancer and other malignant tumors.

In situ kinetic measurements of d-amino acid oxidase in rat liver with respect to its substrate specificity

D-Amino acid oxidase activity was demonstrated in peroxisomes of rat liver using unfixed cryostat sections and a histochemical technique using cerium ions as capture reagent for hydrogen peroxide and diaminobenzidine, cobalt ions and exogenous hydrogen peroxide to visualize the final reaction product for light microscopical analysis. Cytophotometric analysis of liver sections revealed similar zero-order reaction velocities of D-amino acid oxidase with activity twice as high in periportal areas as in pericentral areas of liver lobuli when using either D-proline or D,L-thiazolidine-2-carboxylic acid as substrates. On the other hand, a 4-5 times higher KM value was found for D-proline than for D,L-thiazolidine-2-carboxylic acid. The KM values in periportal and pericentral areas were similar for each substrate. These findings support the suggestion that the physiological substrate for D-amino acid oxidase may be D,L-thiazolidine-2-carboxylic acid, the adduct of cysteamine and glyoxylic acid. D-Amino acid oxidase may play a role in vivo in the production of oxalate which may participate in metabolic control processes as an intracellular messenger molecule.

Thiazolidine-2-carboxylic acid, an adduct of cysteamine and glyoxylate, as a substrate for D-amino acid oxidase.

A mixture of cysteamine and glyoxylate, proposed by Hamilton et al. to form the physiological substrate of hog kidney D-amino acid oxidase (Hamilton, G. A., Buckthal, D. J., Mortensen, R. M., and Zerby, K. W. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2625-2629), was confirmed to act as a good substrate for the pure enzyme. As proposed by those workers, it was shown that the actual substrate is thiazolidine-2-carboxylic acid, formed from cysteamine and glyoxylate with a second order rate constant of 84 min-1 M-1 at 37 degrees C, pH 7.5. Steady state kinetic analyses reveal that thiazolidine-2-carboxylic acid is a better substrate at pH 8.5 than at pH 7.5. At both pH values, the catalytic turnover number is similar to that obtained with D-proline. D-Amino acid oxidase is rapidly reduced by thiazolidine-2-carboxylic acid to form a reduced enzyme-imino acid complex, as is typical with D-amino acid oxidase substrates. The product of oxidation was shown by NMR to be delta 2-thiazoline-2-carboxylic acid. Racemic thiazolidine-2-carboxylic acid is completely oxidized by the enzyme. The directly measured rate of isomerization of L-thiazolidine-2-carboxylic acid to the D-isomer was compared to the rate of oxidation of the L-isomer by D-amino acid oxidase. Their identity over the range of temperature from 2-30 degrees C established that the apparent activity with the L-amino acid can be explained quantitatively by the rapid, prior isomerization to D-thiazolidine-2-carboxylic acid.

125I-beta-iodo-D-alanine-synthesis, biodistribution and antimicrobial activity.

Methyl N-carbobenzoxy-beta-iodo-D-alaninate (1) served as an intermediate to synthesize methyl beta-iodo-D-alaninate (2) and beta-iodo-D-alanine (3). The 125I-labeled compound 1 was synthesized by the melt method and used to synthesize 125I-labeled compounds 2 and 3. Compound 3 was shown to be substrate for D-amino acid oxidase. It was also shown that compounds 2 and 3 were rapidly eliminated from normal mammalian tissues and that compound 3 inhibited the Escherichia coli growth in a dose-dependent manner at 100-500 microgram/ml while compound 2 showed no effect at 500 microgram/ml level. Therefore, it was suggested that compound 3 may serve as an abscess localizing agent.

Reactions of cysteamine and other amine metabolites with glyoxylate and oxygen catalyzed by mammalian D-amino acid oxidase.

Pig kidney D-amino acid oxidase [D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3] catalyzes a rapid uptake of oxygen when high concentrations (50-100 mM) of glyoxylate and the following amines are present under usual assay conditions (pH 8.3): cysteamine, 2-aminoethanol, putrescine, D,L-1-amino-2-propanol, D,L-2-amino-1-propanol, 3-amino-1-propanol, D,L-octopamine, ethylenediamine, and L-cysteine ethyl ester. Notable physiological amines that do not support a rapid O2 reaction under the above conditions include histamine, serotonin, epinephrine, norepinephrine, spermidine, spermine, and cadaverine. A more detailed kinetic investigation of the reactions involving the first four reactive amines listed above indicated that the cysteamine reaction proceeds at a rapid rate even when cysteamine and glyoxylate are present at less than millimolar concentrations, but greater than millimolar concentrations are needed in the other amine reactions in order to observe a reasonable rate. At low concentrations and pH 7.4, the cysteamine-glyoxylate substrate (presumably thiazolidine-2-carboxylic acid) reacts an order of magnitude faster than any other known D-amino acid oxidase substrate. Considerable circumstantial evidence suggests that the reaction involving cysteamine is occurring physiologically, but the reactions of other amines would be occurring in the cell at a very low rate, if at all. It is proposed that the product of the enzymic reaction may be a metabolic effector that can modify the reactivity of proteins or nucleic acids by covalent attachment.

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.

Complex formation of apo-enzyme, coenzyme and substrate of d-amino acid oxidase IV. Changes in physico-chemical properties of the protein

1. 1. A molecular weight of 115 000 was found for the apo- d-amino acid oxidase, for the holo- d-amino acid oxidase and for the artificial Michaelis complex of this enzyme with benzoate. 2. 2. The differences in s 20 ,w, D 20 ,w, v, [η] and [α] D 20 of these three products all point to configurations of the protein part, viz. an increase of α-helix and decrease of random coiling in the order apo-enzyme, holo-enzyme, artificial Michaelis complex. 3. 3. It is suggested that these changes are also involved in the action of d-amino acid oxidase.

Complex formation of apo-enzyme, coenzyme and substrate of d-amino acid oxidase : III. Crystallization of artificial Michaelis complexes

“Artificial Michaelis complexes” of d-amino acid oxidase and one or other of the “substrate-substitutes” benzoate and p-nitrobenzoate have been isolated in crystalline form. The complexes contain equimolecular amounts of apo-enzyme, FAD and substrate-substitute. The substrate-substitutes were driven off by an excess of d-alanine. The properties of the crystalline benzoate complex have been studied in detail.

Complex formation of apo-enzyme, coenzyme and substrate of d-amino acid oxidase : I. Kinetic analysis using indicators

1. I. It was found that both riboflavin-5′-monosulfate (FMS) and adenosine-5′-monosulfate (AMS) compete with FAD for binding with apo- d-amino acid oxidase but not with each other, which suggests the binding of the FMN as well as the AMP moieties of the FAD molecule with the apo-protein. 2. 2. Using FMS and AMS as indicators, p-aminobenzoic acid (PABA) was found to be a competitor with the FMN part of FAD. It was considered likely that the phenylamino group affects the binding point of the FMN part of FAD with the apo-protein. The dissociation constant of the apo-protein-PABA complex calculated from the competition of PABA with FAD for the protein appeared to be 7.3·10 −3 M. In the presence of AMS, this dissociation constant decreased to 2.5·10 −3 M, i. e., the affinity of PABA to the protein increased, which indicates an interaction between the two binding sites. 3. 3. p-Chloromercuribenzoate (PCMB) was found to be a competitor of the AMP moiety of FAD. This action was attributed to the binding of the SH-reactive group of PCMB with the sulfhydryl group of the apo-protein, which suggests that the AMP moiety of FAD combined with sulfhydryl group of the apo-protein. The dissociation constant of the complex of PCMB and apo-protein was calculated to be 1.6·10 −7 M. In the presence of FMS, this constant decreased to 8.5·10 −8 M, also suggesting an interaction of the binding sites of the apo-protein.