Hog Kidney D-amino Acid Oxidase Research Articles

Kinetics and Heat-inactivation mechanisms of d-amino acid oxidase

A study was made of the stability of crude hog kidney d-amino acid oxidase ( d-AAO) under different experimental conditions of temperature, enzyme concentration, buffer, and in the presence of flavine adenine dinucleotide (FAD)_ and glycerol. A deactivation mechanism is proposed. The kinetic deactivation studies were performed in a buffer at pH over a temperature range of 277-327K (4–54°C). The activity for d-AAO at 1 U/ml at 277 K in potassium phosphate and potassium pyrophosphate buffer remains almost constant for 20 days. For d-AAO at 0·02 U/ml at 303 K (30°C) in phosphate buffer, the presence of FAD and glycerol causes the deactivation constant to decrease, while the initial activity and the half-life time increase, the latter doubling on passing from buffer alone to buffer with FAD and 10% glycerol. In the case of d-AAO at 0·2 U/ml, the stability increases considerably with respect to 0·02 U/ml. Both FAD and glycerol enhance this stabilization. For temperatures above 313 K (40°C), the deactivation of d-AAO at 0·2 U/ml showed values of the thermodynamic variables for the overall deactivation constant and for the deactivation constant due to the loss of FAD that indicate that the dissociation of FAD is the main deactivation mechanism. Finally, deactivation due to the loss of FAD is higher for d-AAO at 0·1 U/ml as compared with a concentration of 0·2 U/ml.

Occurrence of free D-aspartic acid in the circumsoesophageal ganglia of [formula omitted][formula omitted

This study reports the presence of high concentrations of free D-aspartic acid (D-Asp) in the circumoesophageal ganglia of the opisthobranch mollusc Aplysia fasciata . D-Asp was discovered using specific methods that employ Octopus D-aspartate oxidase and hog kidney D-amino acid oxidase to measure D-Asp levels. The concentration of D-Asp was 0.281 μmol/g wet tissue weight, which was 8.3% of the total free aspartic acid (D and L forms) present. No other free D-amino acids that were oxidised by D-amino acid oxidase were detected. To our knowledge the only molluscs that have previously been shown to have D-Asp within their nervous tissue are the cephalopods Octopus vulgaris , Loligo vulgaris and Sepia officinalis . In these, as in A . fasciata , no other D-amino acids were detected within the nervous tissue. The fact that free D-Asp occurs specifically in the nervous tissue of the above molluscs suggests that it may have a neurological function that is yet to be described.

Total hydrolysis of proteins with strongly reduced racemization of amino acids

A new method has been devised for the complete hydrolysis of proteins with an extremely low level of racemization of amino acids. Proteins are incubated in 10 M HCl at a low temperature to obtain partial hydrolysis. They are then incubated with pronase and finally with leucine aminopeptidase and peptidyl-d-amino-acid hydrolase from Loligo vulgaris. The proposed method ensures the total hydrolysis of either purified proteins contained in a crude homogenate of animal or vegetable tissue. In both cases, the racemization of amino acids (expressed as rate of d form / d + l form × 100) was lower than 0.015% for aspartic acid and lower than 0.01% for other amino acids. d-Amino acids released from peptides or proteins were estimated with enzymatic methods based on the use of octopus d-aspartate oxidase or hog kidney d-amino acid oxidase; with these enzymes, 0.05 nmol of a d-amino acid was determined in the presence of up to 20 μmol of a mixture of l-amino acids (ratio % d/d + l= 0.00025). The method allows the determination of d-amino acids either in tissues in which they are present in high concentrations (as human cataract lenses, tooth enamel, etc.) or in those with low enantiomer content (as brain, erythrocytes, etc.). Using the method described, we hydrolyzed several synthetic peptides consisting of d- and l-amino acids and determined the amount of d-amino acids. In addition, we totally hydrolyzed all the nuclear proteins of human cataractous lenses. The amount of d-aspartic acid was 0.026 μmol/mg in lenses of woman aged between 71 and 76 years and 0.0256 μmol/mg in lenses of men aged between 55 and 72 years. The d-aspartic acid measured corresponds to about 12% with respect to total aspartic acid.

Kinetic properties of rat kidney d-amino acid oxidase associated with peroxisomes

1. 1. In contrast to hog kidney d-amino acid oxidase, the v vs s plots of d-amino acid oxidase in homogenized rat kidney did not have the form of a rectangular hyperbola, and showed an apparent negative cooperativity. 2. 2. After subcellular fractionation of rat kidney, both of the oxidases in the supernatant fraction and the peroxisomal fraction showed Michaelis-Menten type kinetics. The K m values for d-alanine and d-proline of the peroxisomal fraction were significantly lower than those of the supernatant fraction. 3. 3. The partially purified enzyme from the peroxisomal fraction showed the same kinetic properties as the supernatant fraction. 4. 4. These facts suggest that the two types of rat kidney d-amino acid oxidase were originally identical and that some interaction between the enzyme and peroxisomes is physiologically important for the function of the enzyme.

Inhibition of glutamate-aspartate transaminase by β-methylene- DL-aspartate

β-Methylene- DL-aspartate, a new β,γ-unsaturated amino acid, is an irreversible inhibitor of soluble pig heart glutamate-aspartate transaminase ( K i − 3 mM with respect to the L-form; limiting rate constant for inactivation ∼ 0.4 min −1). The new amino acid is the most specific inhibitor of glutamate-aspartate transaminase thus far studied. It does not inactivate pig heart glutamate-alanine transaminase, soluble rat kidney glutamine transaminase K, γ-aminobutyrate transaminase (from Pseudomonas fluorescens), glutamate decarboxylase ( Escherichia coli), snake venom L-amino acid oxidase, or hog kidney D-amino acid oxidase. In addition, the following enzymes were not inhibited by β-methylene- DL-aspartate in rat tissue homogenates: γ-aminobutyrate transaminase (brain), tyrosine transaminase (liver), glutamine transaminase L (liver), asparagine transaminase (liver), ornithine transaminase (liver) or branch-chain transaminase(s) (kidney). Intraperitoneal injection of β-methylene- DL-aspartate into mice decreased kidney and liver glutamate-aspartate transaminase activities but had no effect on liver glutamate-alanine transaminase activity.

The kinetic mechanism of D-amino acid oxidase with D-alpha-aminobutyrate as substrate. Effect of enzyme concentration on the kinetics.

The kinetic mechanism of hog kidney D-amino acid oxidase with D-alpha-aminobutyrate as substrate has been examined in detail using a combination of steady state and rapid reaction methods. At concentrations of D-alpha-aminobutyrate below 0.5 mM, the rapid reaction and steady state results are consistent with the mechanism previously proposed for D-alanine (Massey, V., and Gibson, Q. H. (1964) Fed. Proc. 23, 18-29; Porter, D. J. T., Voet, J. G., and Bright, H. J. (1977) J. Biol. Chem. 252, 4464-4473). Both flavin reduction by D-alpha-aminobutyrate and reoxidation are quite rapid. Release of product from the oxidized enzyme has been measured directly and matches the turnover number at infinite concentrations of both substrates. Substitution of deuterium for the alpha-hydrogen decreases the rate of reduction 1.4-fold, without any effect on the apparent Kd. Computer simulations show that the kinetic isotope effects on the reductive half-reaction with D-alanine reported by Porter et al. (see above reference) can be explained using a two-step model with a kinetic isotope effect of 1.75 on the limiting rate of reduction. The effect of enzyme concentration on the kinetics has been examined in some detail. With D-alanine as substrate, increasing the enzyme concentration over the range 29 nM to 17 microM resulted in less than a 2-fold decrease in the turnover number. The Kd for benzoate binding also decreased marginally with increasing enzyme concentration. The effect of enzyme concentration is consistent with a decrease in the rate of release of ligands from the oxidized enzyme as the enzyme concentration is increased.

Thermodynamic characterization of hog kidney D-amino acid oxidase apoenzyme in concentrated guanidine hydrochloride solution. Preferential interaction with the solvent components and the molecular weight of the monomeric unit.

This paper describes the physical characterization of the monomeric unit of hog kidney D-amino acid oxidase apoenzyme in 6 M guanidine hydrochloride (GuHCl) solution by means of differential refractometry, densimetry, light scattering, equilibrium sedimentation, and high-speed gel filtration chromatography. In 6 M GuHCl solution, the oxidase interacts preferentially with GuHCl: the values of the preferential interaction parameter are 0.11 +/- 0.03 (S.D.) g/g of protein by densimetry and 0.14 +/- 0.04 g/g of protein by refractometry. The volume change, delta V, of the oxidase on transfer from the native to the denatured state is -350 ml/mol. The molecular weight of the monomeric apoenzyme is 39,600 +/- 1,700 by light scattering and 38,000 +/- 1,200 by high-speed equilibrium sedimentation. The values of the molecular weight estimated by the empirical methods, i.e., sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and high-speed gel filtration chromatography in 6 M GuHCl, agree well with those obtained by the thermodynamic methods mentioned above. These results confirm definitely that the complex of the apoenzyme with SDS normally behaves in the same manner as those of standard proteins in SDS-gel electrophoresis. This is also supported in this study by the analysis of the electrophoretic data at several gel concentrations by Ferguson plots. The molecular weight of quasi-D-amino acid oxidase apoenzyme was also examined by the empirical methods.

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.

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.

Immobilization and characterization of D-amino acid oxidase.

Optimal conditions with respect to pH, concentration of glutaraldehyde and enzyme, and order of addition of enzyme and crosslinking reagent were established for the immobilization of hog kidney D-amino acid oxidase to an attapulgite support. Yields of 40 to 70% were generally attained although when low concentrations of enzyme were used yields were consistently greater than 100%. It is suggested that this is due to a dimer leads to monomer shift at low protein concentrations. The stability of soluble D-amino acid oxidase was dependent on the buffer in which it was stored (pyrophosphate-phosphate greater than borate greater than Tris). Stability of immobilized enzyme was less than soluble in pyrophosphate-phosphate buffer, but storage in the presence of FAD improved stability. In addition, treatment of stored, immobilized enzyme with FAD before assay restored some of its activity. The immobilized D-amino acid oxidase was less stable to heat (50 degrees C) than the soluble enzyme from pH 6 to 8 but was more stable above and below these values. Apparent Km values for D-alanine, D-valine, and D-tryptophan decreased for the immobilized enzyme compared to the soluble.

Carboxylic and polyethyleneimine-bound FAD derivatives. Synthesis, coenzymic properties, conformational and enzyme-coenzyme interaction studies.

Alkylation at N-1 of the FAD adenine ring with 3,4-epoxybutanoic acid gave 1-(2-hydroxy-3-carboxypropyl)-FAD. Dimroth rearrangement of the latter afforded N6-(2-hydroxy-3-carboxypropyl)-FAD. Coupling of the two carboxylic FAD derivatives to polyethyleneimine gave the corresponding soluble macromolecular FAD analogues. Fluorescence and circular dichroism determinations at neutral pH showed for the N-1 carboxylic derivative an open conformation, while for the corresponding N6 derivative a stacked one as for FAD. All the synthesized derivatives have been shown to reactivate Aspergillus niger glucose oxidase and hog kidney d-amino acid oxidase apoenzymes, affording holoenzymes whose activity relative to that of the corresponding natural holoenzymes ranged from 3 to 15% for the N-1 derivatives and from 20 to 97% for the N6 derivatives. Investigations by ultraviolet difference spectra on the interaction with apo-d-amino acid oxidase showed substantial similarities between the N6 carboxylic derivative and FAD in the binding to the apoenzyme, while a completely different behaviour was observed for the N-1 carboxylic derivative. The d-amino acid oxidase apoenzyme, the holoenzyme and the glutaraldehyde-treated holoenzyme were entrapped each in cellulose triacetate fibres together with the polyethyleneimine N6 derivative and beef liver catalase; the operational stability of the three recycling systems with dl-alanine as substrate was shown to be respectively 6, 5 and 12 times higher than the one found for the entrapped natural holoenzyme system.

Association-dissociation of the flavoprotein hog kidney D-amino acid oxidase. Determination of the monomer-dimer equilibrium constant and the energetics of subunit association.

The enzyme concentration dependence of spectrophotometric titrations of hog kidney D-amino acid oxidase [EC 1.4.3.3] with p-aminobenzoate was studied. The monomer-dimer equilibrium constant of the oxidized holoenzyme at 25 degrees C was estimated to be 7 X 10(5)M-1 at pH 7.5 and 4X 10(6)M-1 at pH 8.3. The energetics of subunit association are discussed.

Effect of pH on the interaction of benzoate and D-amino acid oxidase.

The kinetic and equilibrium dissociation constants of the reversible binding of benzoate to hog kidney D-amino acid oxidase (DAAO) were studied at 19 degrees C over the pH range 5.3-10.5 by means of a stopped-flow apparatus and spectrophotometric titrations. A simple bimolecular reaction of the form second order-first order was observed; a two-step reaction was seen. Analysis of the pH dependence of the bimolecular rate constants and equilibrium dissociation constants is consistent with three ionizable groups which are important for benzoate binding. The pK values of the enzyme-related ionization are 6.3, 9.2, and 9.6. Analysis of the change in extinction coefficient at 360 nm indicates the pK of 9.6 can be assigned to the 3-imino group of the enzyme-bound flavin. The effect of benzoate on the apparent pK for the ionization of the 3-imino group of the enzyme-bound Fad has been reexamined. The presence of benzoate causes an apparent shift of this ionization from a pK value of 9.6 to 10.7.

Interaction between D-amino acid oxidase and small molecules.

1. From the standpoint of monomer-dimer equilibrium of hog kidney D-amino acid oxidase [EC 1.4.3.3] and the interaction between the enzyme and small molecules, the effect of pH on the binding of p-aminobenzoate to the monomer and dimer of the enzyme was studied by kinetic methods and spectrophotometric titration. 2. The maximum binding number of p-aminobenzoate to the dimer is two molecules, and there is no interaction between the two active sites of the dimer (i.e., no cooperativity) over the range of pH from 6.5 to 10. 3. The affinity of the dimer for p-aminobenzoate is several times higher than that of the monomer at pH 6.5-10, and consequently p-aminobenzoate induces dimerization in the equilibrium state of D-amino acid oxidase. The interaction energy of two subunits of the dimer is stabilized by the binding of p-aminobenzoate by 1-2 kcal/mole over the pH range studied. 4. The binding sites of the quasi-substrate, p-aminobenzoate, in the dimer and the intersubunit binding site of the dimer are clearly different, because p-aminobenzoate induces dimerization of the enzyme. 5. The pK values of ionizing groups in the free monomer and the free dimer which participate in the binding of the competitive inhibitor, p-aminobenzoate, are approximately the same, 8.7, as determined from the pH dependence of the affinity of the inhibitor for the enzyme. Furthermore, no pK for the enzyme-inhibitor complex in the pH range 6.5-10 was observed. 6. There is no interaction between the two ionizing groups of the dimer during protonation-deprotonation, because a theoretical equation involving no cooperativity between the two ionizing groups in the dimer explains the results well.

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.

Affinity Labeling of D-Amino Acid Oxidase with an Acetylenic Substrate<xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

The acetylenic substrate, D-2-amino-4-pentynoic acid (D-propargylglycine), was oxidatively deaminated by hog kidney D-amino acid oxidase[EC 1.4.3.3], with accompanying inactivation of the enzyme. The flavin which was extracted by hot methanol from the inactivated enzyme was identical with authentic FAD by thin-layer chromatography and circular dichroism. The excitation spectrum of emission at 520 nm of the released flavin was very similar to the absorption spectrum of oxidized FAD. The released flavin was reduced by potassium borohydride. The apoenzyme prepared after propargylglycine treatment did not show restored D-amino acid oxidase activity on adding exogenous FAD. The absorption spectrum of this inactivated apoenzyme showed absorption peaks at 279 and 317 nm, and a shoulder at about 290 nm. These results strongly indicate that the inactivation reaction is a dynamic affinity labeling with D-propargylglycine which produces irreversible inactivation of the enzyme by a covalent modification of an amino acid residue at the active site.

Hydroxy-L-proline as a substrate for HOG kidney D-amino acid oxidase

Summary In contrast to earlier findings, hydroxy-L-proline is oxidized by hog kidney D-amino acid oxidase, with a V max comparable to that of L-proline but with a somewhat higher K. Kinetic constants and pH-dependency data are in reported both for hydroxy-L-proline and allohydroxy-D-proline.

Studies on the metabolism of D- and L-isomers of 3,4-dihydroxyphenylalanine (DOPA). VI. Metabolism of D-DOPA in rat kidney.

The metabolism of the D-isomer of 3, 4-dihydroxyphenylalanine (D-DOPA) in rat kidney was investigated. After incubation of 9.05×10-3м D-DOPA with rat kidney homogenate, 3, 4-dihydroxyphenyruvate (DHPP) was detected as the main metabolite, whereas at a lower concentration of D-DOPA (5.22×10-4м), dopamine was the dominant product. It was shown in vitro that D-DOPA is changed to L-DOPA through two steps and decarboxylated immediately to dopamine in rat kidney. The first step is an oxidative deamination of D-DOPA to DHPP by D-amino acid oxidase. This step was inhibited by D-alanine and benzoic acid wherein these substances act as a substrate and inhibitor, respectively. L-Alanine had almost no effect. The Km value of D-DOPA for purified hog kidney D-amino acid oxidase was 24 mм. The second step is conversion of DHPP to L-DOPA by transaminase. This step was accelerated by aspartate, glutamate, tyrosine, tryptophan and phenylalanine. The latter two were particularly effective as amino donor. L-DOPA was identified by thin-layer chromatography, paper electrophoresis and reverse isotope dilution method. The transaminase activity was observed both in the 9000 g supernatant and precipitate fractions from rat kidney. The rat kidney slice had greater activity than the liver slice in metabolizing D-DOPA to dopamine. This difference is attributable to the content of D-amino acid oxidase in these organs and may give an explanation of the fact that D-DOPA is metabolized in vivo almost exclusively in the kidney.

Structure and function of D-amino acid oxidase. II. Terminal structure and amino acid composition of hog kidney D-amino acid oxidase.

Gross structure of hog kidney D-amino acid oxidase [EG 1.4.3.3] has been characterized by analyzing both N- and G-terminal residues and amino acid composition. The enzyme has been found to be composed of two methionine chains and two FAD per 100,000 g., and to be fairly rich in tyrosine and tryptophan residues. The C-terminal residue of these methionine chains has been identified to be leucine from the results of hydrazinolysis and carboxypeptidase A digestion. The partial specific volume of the enzyme calculated from the amino acid composition was 0.73 ml./g. which fairly agreed with the value of 0.742 ml./g. of the enzyme-benzoate complex previously measured by the usual pycnometer method. The results presented here and in the previous paper were likely to suggest that the enzyme is made up of two identical polypeptide chains.

Structure and function of D-amino acid oxidase. I. Further purification of hog kidney D-amino acid oxidase and its hydrodynamic and optical rotatory properties.

1. Methods for the preparation of the pure benzoate complex, holoenzyme, and apoenzyme of D-amino acid oxidase [EC 1.4.3.3] are described. Chromatography with Sephadex G-75 at pH 8.3 is a satisfactory procedure for further purification of the crystalline benzoate complex prepared by the previous method. Crystalline enzyme is prepared from the gel-eluate by repeated expulsion of benzoate from the complex with an excess amount of D-alanine. The cleavage of the holoenzyme into apoenzyme and FAD is performed on a TEAE-cellulose column equilibrated at pH 5.3. 2. The physico-chemical properties of three species of the oxidase prepared by the method described above are as follows: For the ben-zoate complex, s020,ω=6.3×1013sec.−1; D20,ω=5.3×l0−7cm.2sec.−1; [η] = dl.g.−1; V¯=0.742 ml.g.−1. For the holoenzyme, s020, ω=7.1 × 10−13 sec.−1; D20, ω= 5.8×10−7cm2. sec.−1; [η]=0.026 dl. g.−1. For the apoenzyme, s020, ω= 3.6×10−13sec.−1; D20, ω=6.2×l0−7 cm2. sec.−1. From these results, the molecular weights of both the holoenzyme and the benzoate complex are calculated to be 103,000–134,000 and that of the apoenzyme to be about 55,000. These results are not inconsistent with the minimal molecular weight calculated from the amount of bound FAD, provided the holoenzyme and benzoate complex exist in a dimeric form. It is concluded that the enzyme is a basic protein bearing one molecule of FAD per 50,000 g. of protein. 3. The optical rotatory properties of the apoenzyme in M/60 pyro-phosphate buffer (pH 8.3) at 25°C are found to be [α]25D= −47°; λc=23 mμ; a0=−240; and b0=−72. These values indicate that it contains about I2% helices. This content is confirmed by the measurement of amplitude of the trough at 233 mμ. No significant differences are found in the helical contents of the apoenzyme, the holoenzyme and its benzoate complex.