D-amino Acid Oxidase Reaction Research Articles

Sensitive d-amino acid biosensor based on oxidase/peroxidase system mediated by pentacyanoferrate-bound polymer

A sensitive d-amino acid oxidase (DAAO)/peroxidase (POD) bienzyme biosensor is constructed, in which pentacyanoferrate-bound poly(1-vinylimidazole) polymer (PVI[Fe(CN)5]) is selected as a mediator. Reductive current of PVI[Fe(CN)5] related to the H2O2 concentration generated in the DAAO reaction was measured at −0.1V vs. Ag|AgCl with DAAO/POD/PVI[Fe(CN)5]-modified electrode. The result revealed that PVI[Fe(CN)5] is suitable as a mediator for this bienzyme system due to its appropriate formal potential and its extremely low reactivity against DAAO. The stability of DAAO was improved by adding free flavin adenine dinucleotide and the electrode composition was optimized for the detection of d-alanine. Nafion and ascorbate oxidase-immobilized films worked successfully to prevent severe interference from uric acid and ascorbic acid. The low detection limits of d-alanine (2μM) and d-serine (2μM) imply its possibility for the determination of extremely low concentration of d-amino acids in physiological fluids. The proposed bienzyme biosensor is proved to be capable of detecting d-amino acids in urine.

On the reaction of d-amino acid oxidase with dioxygen: O2 diffusion pathways and enhancement of reactivity

Evidence is accumulating that oxygen access in proteins is guided and controlled. We also have recently described channels that might allow access of oxygen to pockets at the active site of the flavoprotein D-amino acid oxidase (DAAO) that have a high affinity for dioxygen and are in close proximity to the flavin. With the goal of enhancing the reactivity of DAAO with oxygen, we have performed site-saturation mutagenesis at three positions that flank the putative oxygen channels and high-affinity sites. The most interesting variants at positions 50, 201 and 225 were identified by a screening procedure at low oxygen concentration. The biochemical properties of these variants have been studied and compared with those of wild-type DAAO, with emphasis on the reactivity of the reduced enzyme species with dioxygen. The substitutions at positions 50 and 225 do not enhance this reaction, but mainly affect the protein conformation and stability. However, the T201L variant shows an up to a threefold increase in the rate constant for reaction of O(2) with reduced flavin, together with a fivefold decrease in the K(m) for dioxygen. This effect was not observed when a valine is located at position 201, and is thus attributed to a specific alteration in the micro-environment of one high-affinity site for dioxygen (site B) close to the flavin that plays an important role in the storage of oxygen. The increase in O(2) reactivity observed for T201L DAAO is of great interest for designing new flavoenzymes for biotechnological applications.

On The Mechanism of D-Amino Acid Oxidase: STRUCTURE/LINEAR FREE ENERGY CORRELATIONS AND DEUTERIUM KINETIC ISOTOPE EFFECTS USING SUBSTITUTED PHENYLGLYCINES

The kinetic mechanism of the reaction of D-amino acid oxidase (EC 1.4.3.3) from Trigonopsis variabilis with [alpha-1H]- and [alpha-2H]phenylglycine has been determined. The pH dependence of Vmax is compatible with pKa values of approximately 8.1 and >9.5, the former of which is attributed to a base which should be deprotonated for efficient catalysis. The deuterium isotope effect on turnover is approximately 3.9, and the solvent isotope effect approximately 1.6. The reductive half-reaction is biphasic, the first, fast phase, k2, corresponding to substrate dehydrogenation/enzyme flavin reduction and the second to conversion/release of product. Enzyme flavin reduction consists in an approach to equilibrium involving a finite rate for k-2, the reversal of k2. k2 is 28.8 and 4.6 s-1 for [alpha-1H]- and [alpha-2H]phenylglycine, respectively, yielding a primary deuterium isotope effect approximately 6. The solvent deuterium isotope effect on the apparent rate of reduction for [alpha-1H]- and [alpha-2H]phenylglycine is approximately 2.8 and approximately 5. The rates for k-2 are 4.2 and 0.9 s-1 for [alpha-1H]- and [alpha-2H]phenylglycine, respectively, and the corresponding isotope effect is approximately 4.7. The isotope effect on alpha-H and the solvent one thus behave multiplicatively consistent with a highly concerted process and a symmetric transition state. The k2 and k-2 values for phenylglycines carrying the para substituents F, Cl, Br, CH3, OH, NO2 and OCH3 have been determined. There is a linear correlation of k2 with the substituent volume VM and with sigma+; k-2 correlates best with sigma or sigma+ while steric parameters have little influence. This is consistent with the transition state being structurally similar to the product. The Bronsted plot of DeltaG versus DeltaG0 allows the estimation of the intrinsic DeltaG0 as approximately 58 kJ.M-1. From the linear free energy correlations, the relation of DeltaG versus DeltaG0 and according to the theory of Marcus it is concluded that there is little if any development of charge in the transition state. This, together with the recently solved three-dimensional structure of D-amino acid oxidase from pig kidney (Mattevi, A., Vanoni, M.A., Todone, F., Rizzi, M., Teplyakov, A., Coda, A., Bolognesi, M., and Curti, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7496-7501), argues against a carbanion mechanism in its classical formulation. Our data are compatible with transfer of a hydride from the substrate alphaC-H to the oxidized flavin N(5) position, although, clearly, they cannot prove it.

PH and Secondary Kinetic Isotope Effects on the Reaction of d-Amino Acid Oxidase with Nitroalkane Anions: Evidence for Direct Attack on the Flavin by Carbanions

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTpH and Secondary Kinetic Isotope Effects on the Reaction of d-Amino Acid Oxidase with Nitroalkane Anions: Evidence for Direct Attack on the Flavin by CarbanionsKevin A. Kurtz and Paul F. FitzpatrickView Author Information Departments of Chemistry and Biochemistry and Biophysics Texas A&M University College Station, Texas 77843-2128 Cite this: J. Am. Chem. Soc. 1997, 119, 5, 1155–1156Publication Date (Web):February 5, 1997Publication History Received9 August 1996Published online5 February 1997Published inissue 1 February 1997https://doi.org/10.1021/ja962783nCopyright © 1997 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views644Altmetric-Citations23LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (100 KB) Get e-AlertsSUBJECTS:Anions,Flavins,Isotope effects,Monomers,Peptides and proteins Get e-Alerts

Properties and chemical modification of D -amino acid oxidase from Trigonopsis variabilis

The basic properties of purified d-amino acid oxidase from the yeast Trigonopsis variabilis were investigated. The pH optimum of activity was between pH 8.5 and 9.0, and the native molecular masses of holo- and apo-enzyme were determined to be 170 kDa; higher aggregates corresponded to molecular masses of 320 and 570 kDa. The apparent Vmax and Km values for different substrates varied between 3.7 to 185 U/mg and 0.2 to 17.3 mM, respectively. The reaction of d-amino acid oxidase with sulfite was followed by the typical spectral modifications of the FAD resembling the reduced enzyme; a Kd of 30 μM was calculated for the N(5)-adduct. The red anionic flavin radical of the enzyme was stable; benzoate had no influence on the spectral properties. A complete loss of enzyme activity was observed after chemical modification by the histidine-specific reagent diethyl pyrocarbonate. The inactivation showed pseudo-first-order kinetics, with a second-order rate constant of 13.6 M–1 min–1 at pH 6.0 and 20°C. The addition of a substrate under anoxic conditions led to a substantial protection from inactivation, which indicates a localization of the modified residues close to the active site. The pKa of the reacting group was determined to be 7.7, and the rate of inactivation reached a limiting value of 0.031 min–1.

Ultrastructural localization of catalase and D-amino acid oxidase in 'normal' fetal mouse liver.

In the hepatocytes of 'normal' fetal mice from mothers which were carriers of muscular dysgenesis, catalase and D-amino acid oxidase (DAAO) positive as well as negative peroxisomes were observed. DAAO reaction product was occasionally localized in patches around cell membranes and DAAO-positive peroxisomes were frequently observed near mitochondria.

Reaction of enzyme-bound 5-deazaflavin with peroxides. Pyrimidine ring contraction via an epoxide intermediate.

Reaction of peroxides with 5-deazaflavin bound to glucose oxidase, lactate oxidase, or D-amino acid oxidase results in the formation of 5-deazaflavin 4a, 5-epoxide. The reaction of D-amino acid oxidase with m-chloroperoxybenzoate is an exception since the reagent reacts rapidly with the protein moiety to form m-chlorobenzoate which then binds noncovalently near the unmodified coenzyme. Epoxide bound to glucose oxidase is converted to deazaFAD X X in a reaction similar to that observed previously with oxynitrilase and glycolate oxidase. With lactate oxidase the epoxide is quite stable in the absence of light. With D-amino acid oxidase, denaturation of the protein is accompanied by the release of the epoxide into solution where it decomposes in a manner similar to that observed with model epoxide compounds at neutral pH. Reaction of deazaFAD X X with phosphodiesterase and alkaline phosphatase yields deazariboflavin X X. The same compound has been formed in model studies by exposing 5-deazariboflavin 4a,5-epoxide to alkaline conditions. Structural studies indicate that this reaction involves contraction of the pyrimidine ring to yield 4-ribityl-6,7-dimethyloxazolo[ 4,5-b ]quinolin-2(4H)-one. Model reaction studies are consistent with a mechanism initiated by alkaline hydrolysis of the pyrimidine ring at position 4 followed by two additional steps which proceed at neutral pH. A similar mechanism for the enzyme reactions appears likely since analogous intermediates are detected in the glycolate oxidase and the model reactions. The results suggest that position 4 of the coenzyme in oxynitrilase, glycolate oxidase, and glucose oxidase must be accessible to solvent and that the protein moiety must facilitate the initial hydrolysis of the pyrimidine ring since the enzyme reactions occur at neutral pH. Failure to observe formation of deazaFMN X X with lactate oxidase is attributed, at least in part, to the inaccessibility of the pyrimidine ring to solvent.

The inhibition of mammalian d-amino acid oxidase by metabolites and drugs. Inferences concerning physiological function

The inhibition of d-amino acid oxidase (EC 1.4.3.3) by a large number of metabolites and drugs is reported. When the substrate is an adduct (thiazolidine-2-carboxylate) formed from cysteamine and glyoxylate, at least five different mechanisms of inhibition are possible, and examples of each are given. Effective inhibitors include (a) some adenosine containing nucleotides, in particular ADP, AMP, ADP ribose, NADH, NADPH and dephospho-CoA; (b) diuretics, especially furosemide, ethacrynic acid, and mersalyl; (c) anti-inflammatory agents, such as salicylate, indomethacin, phenylbutazone, acetylsalicylate, mefenamic, and flufenamic acids; (d) hypoglycemic, hypocalcemic, and hypolipidemic compounds, including 5-methylpyrazole-3-carboxylate, pyrrole-2-carboxylate, 5-methylthiophene-2-carboxylate, thiophene-2-carboxylate, benzoate, and nicotinate; (e) aldehydes, for example, formaldehyde, acetaldehyde, and succinate semialdehyde; (f) thiols, especially β-aminothiols such as cysteine and penicillamine; (g) tropolone, and (h) hydrogen peroxide. The rate of the reaction is also very sensitive to oxygen presure; at pH 7.4 and 25°C the K m for O 2 is 1.1 m M. These data, in conjunction with literature information concerning the biological affects of such compounds, are used to suggest possible physiological processes in which the d-amino acid oxidase reaction may be involved. Although such correlations based on circumstantial evidence are not conclusive, they suggest that d-amino acid oxidase may play a major role in the control of metabolism in animals. Some processes in which it may participate include maintenance of ion and water balance in the kidney, inflammatory response, transmission of nerve impulses, sensing of O 2 concentration, control of cell growth, and as part of an intracellular messenger system for some hormones, especially insulin.

Studies on the reaction of D-amino acid oxidase with beta-cyano-D-alanine. Observation of an intermediary stable charge transfer complex.

The reaction of D-amino acid oxidase [EC 1.4.3.3] (DAO) from porcine kidney with beta-cyano-D-alanine (D-BCNA) was studied. DAO was found to catalyze elimination of the cyano group as well as oxidation of D-BCNA. During the course of the reaction in the presence of excess oxygen, an intermediate was observed which exhibited a characteristic absorption spectrum with a broad charge transfer band in the longer wavelength region. The CD spectrum of this intermediate resembles that of DAO-anthranilate complex. The rate of oxygen consumption in the aerobic reaction decreased with time, suggesting product inhibition due to complex formation between the enzyme and the product. Anaerobic addition of D-BCNA reduced the enzyme to its fully reduced state, the CD spectrum of which closely resembles that of the enzyme reduced by excess D-alanine. When an appropriate amount of D-BCNA was added to the enzyme under air, the charge transfer complex was observed immediately, and underwent a change to the reduced state as the oxygen was consumed. The binding strength in the charge transfer complex was found to be comparable to that in DAO-benzoate complex. The accumulating product in the oxidation of D-BCNA had a strong absorption at 285 nm. The aerobic reaction of beta-cyano-L-alanine (L-BCNA) with snake venom L-amino acid oxidase (LAO) produced the same product with an absorption at 285 nm as the reaction of DAO with D-BCNA. The product obtained in the reaction with LAO was found to form the same charge transfer complex with DAO. We tentatively identified this product as alpha-amino-beta-cyanoacrylate and the charge transfer complex as the complex of alpha-amino-alpha-cyanoacrylate with the oxidized enzyme. A hypothetical reaction pathway based on the present finding is proposed. Addition of L-BCNA to the enzyme produced an absorption spectrum very similar to that of the DAO-benzoate complex without oxidation or elimination. L-BCNA was found to be a competitive inhibitor of the oxidation of D-alanine.

Mechanistic features of the D-amino acid oxidase reaction studied by double stopped flow spectrophotometry.

Mechanistic features of the D-amino acid oxidase reaction studied by double stopped flow spectrophotometry.

Use of 5-deazaFAD to study hydrogen transfer in the D-amino acid oxidase reaction.

The apoprotein of hog kidney D-amino acid oxidase was reconstituted with 5-deazaflavin adenine dinucleotide (5-deazaFAD) to yield a protein which contains 1.5 mol of 5-deazaFAD/mol of enzyme. The deazaFAD-containing enzyme forms complexes with benzoate, 2-amino benzoate, and 4-aminobenzoate which are both qualitatively and quantitatively similar to those observed with native enzyme. The complex with 2-aminobenzoate exhibits a new long wavelength absorption band characteristic of a flavin charge-transfer complex. The reconstituted enzyme exhibits no activity when assayed by D-alanine oxidation. However, the bound chromophore can be reduced by alanine, phenylalanine, proline, methionine, and valine, but not by glutamate or aspartate, indicating the deazaFAD enzyme retains the substrate specificity of the native enzyme. Reduction of the enzyme by D-alanine exhibits a 1.6-fold deuterium isotope effect. Reoxidation of the reduced enzyme occurred in the presence of pyruvate plus ammonia, but not with pyruvate alone or ammonia alone. beta-Phenylpyruvate and alpha-ketobutyrate, but not alpha-ketoglutarate could replace pyruvate. Reduced enzyme isolated following reaction with [alpha-3H]alanine was found to contain 0.5 mol of tritium/mol of deazaFADH2. After denaturation of the tritium-labeled enzyme, the radioactivity was identified as deazaFADH2. Reaction of the reduced tritium-labeled enzyme with pyruvate plus ammonia prior to denaturation yields [alpha-3H]alanine and unlabeled deazaFAD. These results suggest that reduction and reoxidation of enzyme-bound deazaFAD involves the stereo-specific transfer of alpha-hydrogen from substrate to deazaFAD.

D-valine as a selective agent for normal human and rodent epithelial cells in culture

A nutrient medium has been developed to enable the growth of normal epithelial cells while selectively inhibiting fibroblast proliferation. In this medium, D-valine is substituted for L-valine; and only those cells containing D-amino acid oxidase can convert the D-amino acid into its essential L-enantiomer. The ability to select for cells with this enzyme has enabled us to maintain epithelial cell populations free from fibroblast overgrowth. The presence of D-amino acid oxidase has been histochemically confirmed in the epithelial cells selected from renal cell suspensions and explants. The ability to proliferate in the selective medium is transmitted to the clonal progeny of these cells. Moreover, epithelial cell proliferation in this medium indicates the presence of D-amino acid oxidase, which we have detected in tissues where it had not previously been reported—fetal human kidney, lung, and cord. Fibroblasts will not grow in the selective medium, but will proliferate normally if the product of the D-amino acid oxidase reaction is supplied.

Mechanism of enzyme action. VI. Kinetic isotope effect on d-amino acid oxidase reaction

1. 1. The rate of formation of the purple itermediate of d-amino acid oxidase [ d-amino acid:O 2 oxidoreductase (deaminating), EC 1.4.3.3] in the anaerobic reaction of this enzyme with its substrate was reduced by substitution of the α-hydrogen of the substrate for deuterium. This indicates that removal of the α-hydrogen of the substrate occurs prior to or in concert with the formation of the purple intermediate. The extent of the deuterium kinetic isotope effect differed for different substrates. 2. 2. The α-deuteration of the substrate affected neither the rate of conversion of the purple intermediate to the fully reduced enzyme nor the rate of oxidation of the purple intermediate with O 2. 3. 3. The kinetic isotope effect on the catalytic oxidation of the substrate was less than that on the formation of the purple intermediate. This indicates that the purple intermediate formation partially determines the overall rate of the enzymic reaction, but the effect on the overall rate is thought to result merely from the effect on the process of the purple intermediate formation. The kinetic isotope effect on the catalytic oxidation was increased by decreasing the enzyme concentration. This may be interpreted to mean that the process of the purple intermediate formation is more rate controlling in the monomeric enzyme than in the dimeric enzyme.

Nitroalkanes as reductive substrates for flavoprotein oxidases.

Nitroalkanes have been found to be general reductive substrates for D-amino acid oxidase, glucose oxidase and L-amino acid oxidase. These enzymes show different specificities for the structure of the nitroalkane substrate. The stoichiometry of the D-amino acid oxidase reaction is straightforward, consisting of the production of one mole each of aldehyde, nitrite and hydrogen peroxide for each mole of nitroalkane and oxygen consumed. The stoichiometry of the glucose oxidase reaction is more complex in that less than one mole of hydrogen peroxide and nitrite is produced and nitrate and traces of 1-dinitroalkane are formed. The kinetics of nitroalkane oxidation show that the nitroalkane anion is much more reactive in reducing the flavin than is the neutral substrate. The pH dependence of flavin reduction strongly suggests that proton abstraction is a necessary event in catalysis. A detailed kinetic mechanism is presented for the oxidation of nitroethane by glucose. It has been possible to trap a form of modified flavin in the reaction of D-amino acid oxidase with nitromethane from which oxidized FAD can be regenerated in aqueous solution in the presence of oxygen.

The kinetics of imino acid accumulation in the D-amino acid oxidase reaction

When the oxidation of D-phenylalanine by D-amino acid oxidase is measured in stopped-flow turnover experiments there is a lag in the formation of keto-phenylpyruvate which becomes progressively greater as the pH is raised from 6.5. Borohydride trapping experiments show that the transient accumulation of free imino acid largely accounts for the lag at pH 8.7. These results are consistent with the known hydrolytic behavior of imines.

Nuclear magnetic resonance study on d-amino acid oxidase reaction

Nuclear magnetic resonance study on d-amino acid oxidase reaction

Reaction of D-amino acid oxidase with D-lysine.

Reaction of D-amino acid oxidase with D-lysine.

Occurrence of two intermediates in D-amino acid oxidase reaction.

Occurrence of two intermediates in D-amino acid oxidase reaction.

CRYSTALLIZATION OF AN INTERMEDIATE IN THE REACTION OF D-AMINOACID OXIDASE AND LACTATE.

CRYSTALLIZATION OF AN INTERMEDIATE IN THE REACTION OF D-AMINOACID OXIDASE AND LACTATE.