D-Amino Acid Oxidase I. SPECTROPHOTOMETRIC STUDIES* (Received for publication, March 22, 1967)

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1 THE JOURNAL OF BOLOGCAL CHEWSTRY Vol. 242, No. 17, ssue of September 10, pp ,1967 Printed in U.S.A. D-Amino Acid Oxidase. SPECTROPHOTOMETRC STUDES* MARGARET L. FONDA AND BRUCE M. ANDERSON From the Department of Biochemistry, University of Tennessee, Knoxville, Tennessee $7916 (Received for publication, March 22, 1967) SUMMARY The D-amino acid oxidase-catalyzed conversion of D- phenylglycine to benzoylformic acid can be followed spectrophotometrically by measuring the increase in optical density at 252 rnp which accompanies this reaction. Various properties of this D-phenylglycine reaction were studied, and conditions are given for the application of this reaction as a spectrophotometric assay of D-amino acid oxidase. Several previously established properties of D-amino acid oxidase were redetermined with the spectrophotometric method, and the results obtained were in good agreement with those previously observed with other assay methods. The spectrophotometric method was used to study the binding of pyridine carboxylic acid derivatives to the enzyme. The three isomeric pyridine carboxylic acids and N-methylnicotinic acid effectively inhibit D-amino acid oxldase, and the inhibition observed was in each case competitive with respect to substrate. nhibitor dissociation constants are listed for the derivatives studied. The ease of removal of flavin adenine dinucleotide from D- amino acid oxidase (n-amino acid:02 oxidoreductase (deaminating), EC ) and the stability of the resulting apoenzyme have encouraged many researchers to use this enzyme to study flavin-protein interactions. One can investigate the formation of catalytically active holoenzyme by recombining FAD with the isolated apoprotein. Recent studies (1,2) of this recombina- tion process have indicated t.hat conformational changes of the protein are of importance in the formation of the catalytically active form of the enzyme. Time-dependent changes such as these, as well as dissociation of FAD from the holoenzyme, induced by dilution (3), can contribute to the lag phase usually encountered in studies of the catalytic activity of this enzyme. n order to study the changes in activity that occur in the early * Contribution 37 from the Department of Biochemistry, The University of Tennessee. This work was supported by Research Grant GB 5503 from the National Science Foundation. time period of these reactions, methods allowing convenient measurement at low enzyme concentrations are needed. The recognition of this need prompted Dixon and Kleppe (3) to apply oxygen electrode techniques to the study of n-amino acid oxidase. t was therefore of interest to develop a spectrophotometric method for assaying this enzyme, which would also allow early time measurements as well as provide a means of assay when low enzyme concentrations must be used. EXPERMENTAL PROCEDURE Benzoylformic acid, N-methylnicotinic acid hydrochloride, and D- (-) a-phenylglycine were obtained from the Aldrich Chemical Company. DL-phenylglycine, pyridine-2-carboxylic acid, and pyridine-4-carboxylic acid were purchased from Eastman Organic Chemicals, and nicotinic acid from Matheson, Coleman, and Bell. FAD (disodium salt), Grade, and n-alanine were obtained from Sigma. Catalase (lyophilized) and crystalline D- amino acid oxidase (elect.rophoretically purified) were obtained from Worthington. The apoenzyme form of n-amino acid oxidase was prepared according to the method of Massey and Curti (l), and stock solutions of 1.2 to 1.4 mg per ml in 0.1 M sodium pyrophosphate, ph 8.5, were stored at 4. The stock solutions were diluted with 0.05 M sodium pyrophosphate, ph 8.5, to 0.3 mg per ml prior to use. Stock solutions of holoenzyme were stored at 4 in 0.05 M sodium pyrophosphate, ph 8.5, at a concentration of 1.2 mg per ml. These solutions were likewise diluted to 0.3 mg per ml with 0.05 M sodium pyrophosphate, ph 8.5, prior to use. Stock solutions of substrates, inhibitors, and FAD were all prepared in 0.05 M sodium pyrophosphate and adjusted to the appropriate ph for the reactions to be studied. The velocity measurements reported in these studies for n-amino acid oxidase-catalyzed reactions are not typical initial velocities but, rather, linear rates attained after the initial lag phase of these reactions. Spectrophotometric measurements were carried out in temperature-controlled cell compartments of a Zeiss PM& spectrophotometer or a Gilford model 2000 recording spectrophotometer, with l-cm light path cuvettes used in all studies. Measurements of ph were made at 25 with a Radiometer ph meter, PHM4b, with a G-200-B glass electrode.

2 3958 D-Amino Acid Ozidase. Vol Wo. 17.ooB Wavelength FG. 1. The formation of benzoylformic acid from n-phenylglycine as catalyzed by n-amino acid oxidase. The reaction mixture contained 5 X 1W6 M n-phenylglycine, 5 X 10-t M sodium pyrophosphate, ph 7.9, and 15 pg of n-amino acid oxidase (holoenzyme), in a total volume of 3 ml , the spectrum of 5 X lo-6 M benzoylformic acid in 5 X lo+ M sodium pyrophosphate, ph 7.9, with the same amount of enzyme added. RESULTS imp) J 1 The search for a spectrophotometric assay for n-amino acid oxidase was started with a study of aromatic amino acids as substrat.es, since it was assumed that oxidative deamination, which resulted in the formation of a keto group conjugated with an aromatic system, would be associated with a measurable spectral change in the ultraviolet region. n-phenylglycine was found to serve as a substrate for the enzyme and to provide the necessary spectral change required when converted to benzoylformic acid. The spectral changes observed in the n-amino acid oxidase-catalyzed conversion of n-phenylglycine to benzoylformic acid are shown in Fig. 1. At this low concentration of D-phenylglycine, the reaction proceeds slowly at room temperature; however, the gradual development of the 252rnp maximum of benzoylformic acid can be demonstrated. At higher subst.rate concentrations, the reaction proceeds more rapidly, and appreciable changes in absorbance at 252 mp are observed within minutes after the reaction is started. The effect of D- phenylglycine concentration on the velocity of the enzyme reaction was studied in this manner. The data obtained at two different temperatures, plotted according to Lineweaver and Burk (4), are shown in Fig. 2. These experiments represent preliminary studies of substrate concentration effects carried out before the effects of ph and ionic strength on the reaction had been determined. Higher enzyme concentrations were used in these studies so that the data obtained could be more adequately compared with those from reactions measured manometrically. Under the conditions described in Fig. 2, the K, for n-phenylgjyzri;7yas calculated to be 6.7 X 10v3 M at 25 and 4.5 X 10M3. The effect of substrate concentration on the rate of the reaction was also measured manometrically at 37 under i conditions very similar to those used in the spectrophotometric study. Reaction mixtures contained 0.05 M sodium pyrophosphate, ph 8.3, o-phenylglycine, 30 pg of catalase, and 50 pg of o-amino acid oxidase, in a total volume of 2.8 ml. The substrate was varied from 8 X 10P4 M to 5 X 1O-3 M. The reactions were initiat.ed by the addition of enzyme, and oxygen uptake was measured with a Gilson model G-14 differential respirometer. A linear relationship was obtained between the reciprocal of the velocities and the reciprocal of the substrate concentration over the concentration range studied, and the K, value calculated from these data was 4.7 X lop3 M. This value agrees well with that obt.ained from measurements made spectrophotometrically. t has been demonstrated recently (5) that ph effects on D- amino acid oxidase can vary markedly with the substrate used. t was of interest therefore to investigate the effects of ph on the o-phenylglycine reaction. Reactions were carried out at 25 as described in Fig. 2, with the exception of the variation in the ph of the pyrophosphate buffer employed. K, and,,, values were calculated from double reciprocal plots of data obtained at 11 different ph values from ph 7.0 to ph 9.5. The ionic strength in all reaction mixtures was maintained constant at 0.4 M by varying the concentration of pyrophosphate buffer used. The results of these studies are shown in Fig. 3. A ph optimum of approximately 7.9 was observed for t.he maximum velocities, while K, values decreased steadily with increasing ph. Subsequent studies of t.he enzyme with o-phenylglycine were carried out. predominantly at ph 8.5 to take advantage of the lower K, value for the substrate. n-phenylglycine does absorb somewhat at 252 rnp, which necessitates the subtraction of a base line absorbance in order to follow the appearance of benzoylformic acid. Studies carried out at substrate saturation would require much too high a blank at lower ph values where the K, values /[D-Phenylglycine] (M-l x 10m3 ) FG. 2. The effect of n-phenylglycine concentration on rates of oxidation. n-phenylglycine concentrations varied from 2.5 X lo-+ to 3.5 X O-3 M. Reaction mixtures contained 5 X 10m2 M sodium pyrophosphate, ph 8.3, varying substrate, and 15 fig of holoenzyme, in a total volume of 1 ml. Reactions were initiated by the addition of enzyme.

3 ssue of September 10, 1967 M. L. Fonda and B. M. Anderson are high. Since spontaneous dissociation of FAD from the holoenzyme form of n-amino acid oxidase was noted at low enzyme concentrations (3), a small amount of FAD was routinely added to reaction mixtures when the holoenzyme was used. For the most part, n-amino acids have been demonstrated to be rather poor inhibitors of n-amino acid oxidase (6). t was of interest therefore to investigate the possibility of using DLphenylglycine as a substrate in the spectrophotometric assay. These studies were carried out in 3-ml reaction mixtures containing 0.05 M sodium pyrophosphate, ph 8.5, 3.6 X 10M6 M FAD, 15 pg of n-amino acid oxidase, and nn-phenylglycine in concentrations varying from 1.16 X lo+ ivr to 4.64 X 10m3 M. The reaction was initiated by the addition of the holoenzyme. Rates were obtained for seven different substrate concentrations in the concentration range indicated, and the double reciprocal plot of these data was linear. When the substrate concentration term used in this plotting method was calculated on the basis of the amount of n-phenylglycine present in the reaction mixtures, the K, and V,,, values determined were identical to those obtained when pure n-phenylglycine was used as substrate under the same conditions. Various properties of n-amino acid oxidase that have been well established through previous manometric studies of the enzyme were redetermined with the spectrophotometric method. For example, benzoic acid can be shown to be a competitive inhibitor with respect to n-phenylglycine in these reactions (Fig. 4). The inhibitor dissociation constants for benzoic acid at the two concentrations used were calculated from the data of Fig. 4 to be 1.15 X 10F5 M and 1.19 X 10M5 M, respectively. The binding of FAD to the apoenzyme form of n-amino acid oxidase was also studied spectrophotometrically with D-phenylglycine as substrate. The reaction mixtures for these studies contained 0.05 M sodium pyrophosphate, ph 8.5, M D- phenylglycine, 15 pg of apoenzyme, and FAD in varying amounts, from 1.43 X lo- M to 7.18 X 10-T M, in a total volume of 3 ml. The reaction velocities were measured at 25 by following the increase in optical density at 252 rnp. Six concentrations of FAD in the concentration range indicated above were used. From the linear double reciprocal plot obtained, a K, value for FAD was calculated to be 3.7 x 10-T M. At ph 8.5, M n-phenylglycine was used whenever the condition of substrate saturation was necessary. At concentrations of the substrate greater than 0.02 M, reaction rates begin to fall off. Massey and Curti () demonstrated a time-dependent refor- 24O.6 20 i m; 16 - x t 6 PH FG. 3. The effect of ph on vm,, and Km values obtained in the oxidation of n-phenylglycine. 0, V,,,; 0, Km. P /D-Phenylglycine] (M- x 10w2) FG. 4. Competitive inhibition by benzoic acid of the n-phenylglycine reactions at 25. n-phenylglycine concentration was varied from 2 X lo+m to 6 X 10w3~. Reaction mixtures contained 5 X 10e2 M sodium pyrophosphate, ph 8.5, 3.6 X 10-B M FAD, 15 pg n-amino acid oxidase (holoenzyme), substrate, and inhibitor as indicated, in a total volume of 3 ml. Line 1, no inhibitor; Line 2,1.33 X lo+ M benzoic acid; Line 3,2 X 1OW M benzoic acid..48k O 12 Minutes FG. 5. The formation of active holoenzyme from apoenzyme and FAD at The reaction mixture contained 1.5 X 1OP M n-phenylglycine, 5 X 1OP M sodium pyrophosphate, ph 8.5, 1.2 X lo+ M FAD, and 7.5 pg of apoenzyme, in a total volume of 3 ml. mation of active holoenzyme, the second stage of which proceeded with a rate constant in the range of to mine1 at This activation step was studied spectrophotometrically with n-phenylglycine as substrate, and the results obtained are shown in Fig. 5. The reaction proceeds through a lag phase with a constant rate being reached at about 9 min. Tangents drawn to the curve at various times during the lag phase were replotted as a function of time and agreed well with the first order kinetics reported for this process by Massey and Curti (1). The first order rate constant obtained in this study was min-. t is of interest to note that only 7.5 pg of apoenzyme were needed to carry out this experiment. The validity of this type of an experiment would lie in the ability of the method used to ac-

4 3960 D-Amino Acid Oxidase. Vol. 242, No E c\ t al g 0.06 z b a t oo2* Enzyme &q/ml 1 FG. 6. The relationship between spectrophotometrically measured enzyme activity and enzyme concentration. Reaction mixtures contained 1.5 X 10e2 M D-phenylglycine, 3.3 X 1Om6 M FAD, 5 X 10-Z M sodium pyrophosphate, ph 8.5, and holoenzyme as indicated, in a total volume of 3 ml.?.- 24 / /[D-Phenylglycine] (M-i x lo-21 FG. 7. Competitive inhibition by W-methylnicotinic acid of the D-phenylglycine reactions. D-Phenylglycine concentration was varied from 3 X 1W3 M to 7 X 10e3 M. Reaction mixtures contained 5 X 1OW M sodium pyrophosphate, ph 8.5, 3.6 X 10-G M FAD, 15 pg of n-amino acid oxidase (holoenzyme), substrate, and inhibitor as indicated, in a total volume of 3 ml. Line 1, no inhibitor; Line 2, 2.67 X lo+ M W-methylnicotinic acid; Line 8, 5.33 X 1OV M Nl-methylnicotinic acid. curately reflect changes in the active form of the enzyme. For this reason, the proportionality between the measured activity and the amount of enzyme present was investigated over the enzyme concentration range involved. The linear relationship observed between 1 kg per ml of enzyme and 5 pg per ml of enzyme is shown in Fig. 6. At higher enzyme concentrations, the activity measurements began to fall off. However, the recombination experiments (Fig. 5) were carried out at 2.5 fig per ml of enzyme, where deviation from linearity in the proportionality curve was not observed. Benzoic acid derivatives markedly inhibit n-amino acid oxidase (7--g), and the substituent effects on inhibition observed raise some interesting questions concerning the mode of binding of these compounds. t was of interest, therefore, to initiate a study of pyridine carboxylic acid derivatives where quaternization of the ring nit.rogen could provide large changes in the properties of the compounds involved. For this study, inhibition of the enzyme by the three isomeric pyridine carboxylic acids was investigated. nhibition by N-methylnicotinic acid was also studied, so that a comparison could be made between effects observed with a free base and a quaternary form of one of these inhibitors. nhibition by the various pyridine carboxylic acid derivatives was studied both at constant substrate concentration and varying substrate concentrations. For example, the inhibition by N-methylnicotinic acid was shown to be competitive with respect to the substrate when studied at. varying substrate concentrations. The data obtained in these studies, plotted according to Lineweaver and Burk (4) are shown in Fig. 7. nhibition by N-methylnicotinic acid was also studied at two constant concentrations of substrate and varying inhibitor concentrations. The data obtained in this manner were plotted according to Dixon (10) and are shown in Fig. 8. This relationship is consistent with the competitive inhibition observed in the double reciprocal plot. nhibitor dissociation constants, calculated from the two types of plots used, are listed in Table. nhibition by the three pyridine carboxylic acids was studied in the same manner as described above for N-methylnicotinic acid. These compounds effectively inhibited the enzyme and, in each case, the inhibition observed was competitive with respect to 1 i h [N-methylnicotinic acid] CM x 104) FG. 8. nhibition of n-amino acid oxidase by W-methylnicotinic acid plotted as reciprocal velocity against inhibitor concentration. Reaction mixtures contained 5 X 10e2 M sodium pyrophosphate, ph 8.5, 3.6 X 1W6 M FAD, 15 pg of D-amino acid oxidase (holoenzyme), substrate, and inhibitor as indicated, in a total volume of 3 ml. Line 1, 6 X 10m3 M D-phenylglycine; Line 2, 3 X 10-s M D-phenylglycine. TABLE nhibitor dissociation constants (K) for pyridine carboxylic acid derivatives nhibitor K Linew;;;yBurk Dixon plots Pyridine-2-carboxylic acid x 10-d 3.38 X 1O-4 Pyridine-3.carboxylic acid x 10-d 1.45 X 10-d Pyridine-4-carboxylic acid X 1O x 10-h N-methylnicotinic acid x X 1O-4 dl Y

5 ssue of September 10, 1967 M. L. Fonda and B. M. Anderson 3961 substrate. nhibitor dissociation constants for each of these of preparation and a shorter time period of measurement, and compounds, calculated from the two types of studies used, are would allow assays to be performed with a lower consumption of also listed in Table. enzyme. DSCUSSON The oxidative deamination of n-phenylglycine to benzoylformic acid is catalyzed by n-amino acid oxidase. The change in absorbance at 252 rnp which occurs in this reaction (Fig. 1) can be used as a direct spectrophotometric method for the study of this enzyme. The reaction, as catalyzed by n-amino acid oxidase, can be characterized on the basis of K, and V,,, values. Both the K, for n-phenylglycine and the maximum velocity vary considerably with ph m the ph range of 7 to 9.5 (Fig. 3). The maximum velocity goes through a ph optimum at 7.9, while the K, values fall off to a limiting value with increasing ph. Dixon and Kleppe (5) recently demonstrated that ph effects on n-amino acid oxidase reactions can vary considerably with the substrate used. The ph effects observed with n-phenylglycine are presented, therefore, not for the sake of comparison, but as an aid to the study of these react.ions. nn-phenylglycine has been used previously as a substrate for n-amino acid oxidase (11). n these studies (ll), a K, value of 2 X lop3 M, calculated on the basis of the n-phenylglycine present, was determined manometrically at 38 and at ph 8.5. The K, values measured in t,he present study agree fairly well with this value. A K, value of 4.5 X 1O-3 M was obtained at 37 at ph 8.3 (Fig. 2). From the ph profile (Fig. 3), one would expect this value to be slightly lower at ph 8.5. The spectrophotometrically measured conversion of n-phenylglycine to benzoylformic acid represents a simple, st.raightforward method for studying n-amino acid oxidase. Upon initiation of the reaction by the addition of the enzyme to the substratecontaining reaction mixture, the reaction proceeds through a lag phase and reaches a constant velocity usually within 6 min at 25. This constant velocity, which remains linear for several minutes, is taken as the velocity measurement. This linear velocity and the duration of this linear rate period are decreased at high substrate concentrations; however, a sufficiently broad substrate concentration range (below 0.02 M) is available for studying the enzyme. As previously mentioned, the change in absorbance at 252 rnp is measured as an increase over a control reaction mixture containing the complete system minus the enzyme. This blanking procedure does not create any problems at ph 8.5 in the substrate concentration range used. t would, however, cause difficulties at lower ph values at which higher concentrations of substrate would be needed for saturation of the enzyme. The n-phenylglycine reactions reported here have been studied with both the Zeiss PMQ and the Gilford model 2000 spectrophotometers without any modification of basic equipment,. The spectrophotomet.ric method for n-amino acid oxidase described here can be applied for the assay of small amounts of the enzyme. One can assay the enzyme readily at concentrations one-tenth those normally required for most manometric techniques. The reaction velocity measured at 1 pg per ml of enzyme (Fig. 6) as the change in absorbance at 252 rnp per min was Under these conditions, the linear rate was maintained for at least 5 min, an indication that the method can be used at even lower concentrations of the enzyme. t is felt that application of this spectrophotometric technique would greatly simplify assay of the enzyme by providing a shorter time period We felt it necessary to demonstrate that, with the use of the n-phenylglycine spectrophot.ometric method, one could study D- amino acid oxidase reactions and obtain results consistent with various properties of the enzyme, established previously by other methods. A K, value of 3.7 x 10-7 M was observed for FAD at 25 and at ph 8.5. Although most of the previously reported K, values for FAD were measured at 38, the values obtained between ph 8 and 8.5 usually fall in the range of 1.1 X 10F7 M to 4.7 x lo- M (7, 12-16). The values obtained in the present study for the inhibitor dissociation constant for benzoic acid are likewise consistent with values previously reported in the literature (8, 9, 17). The same is true for the inhibitor dissociation constant for nicotinic acid (8) obtained in the present study. The rate constant calculated for the reformation of active holoenzyme also agrees well with that reported by Massey and Curti (1). These comparisons, along with the studies concerning ph effects, substrate concentration effects, and enzyme proportionality, would indicate that the spectrophotometric method could be used for studying many different properties of the enzyme. The four pyridine carboxylic acid derivat.ives studied as inhibitors of n-amino acid oxidase were all shown to inhibit the enzyme competitively with respect to the substrate, n-phenylglycine. As mentioned above, the inhibitor dissociation constants obtained with nicotinic acid (pyridine-3-carboxylic acid) in the present study (Table ) are in good agreement with that reported previously by Klein (8). Klein (8) and Webb (18) have discussed the structural requirements for effective inhibition of n-amino acid oxidase by inhibitor molecules. The advantage of having a nitrogen atom or a positively charged atom in the proper position, relative to the carboxyl group of the inhibitor molecule, has been noted. f one considers this structural analogy to substrate molecules to be of importance in the binding of pyridine carboxylic acids, then one would expect pyridine- 2-carboxylic acid to be bound more effectively to the enzyme than are the other two pyridine carboxylic acid isomers. As shown in Table, pyridine-2-carboxylic acid was the poorest inhibitor of the three isomeric carboxylic acids and had the highest dissociation constant of the three free base forms studied. Also, on the basis of the above ment.ioned structural requirements for inhibitors, N-methylnicotinic acid would be expected to be a better inhibitor than nicotinic acid. This also was found not to be the case (Table ). The binding of pyridine carboxylic acid derivatives to n-amino acid oxidase does not appear related simply to structural analogy to substrates of the enzyme. nteractions of aromatic carboxylic acid derivat.ives with FAD in the ternary complexes formed may be exerting an influence on binding not normally encountered with substrate molecules. Other mono- and disubstituted pyridine carboxylic acid derivatives are being prepared in order to study further the sbructural requirements for effective binding of inhibitor molecules to this enzyme. REFERENCES 1. MASSEY, V., AND CURT, B., J. Biol. Chem., 241, 3417 (1966). 2. YAG, K., HARADA, M., AND KOTAK, A., Biochim. Biophys. Acta, 122, 182 (1966). 3. DXON, M., AND KLEPPE, K., Biochim. Biophys. Ada, 96, 357 (1965).

6 D-Amino Acid Oxidase. Vol. 242, No LNEWEAVER, H., AND BURK, D., J. Amer. Chem. Sot., 56, 658 (1934). 5. DXON, M., AND KLEPPE, K., Biochim. Biophys. Acta, 96, 383 (1965). 6. DXON, M., AND KLEPPE, K., Biochim. Biophys. Acta, 96, 368 (1965). 7. BARTLETT, G. R., J. Amer. Chem. Sot., 70, 1010 (1948). 8. KLEN, R. J., J. Biol. Chem., 205, 725 (1953). 9. YAG, K.. OZAWA, T., AND OKADA, K., Bioehim. Biophys. Acta, 35;102 (1959). 10. DXON. M.. Biochem. J (1953). 11. NEMS: A. h., DELucA, D. C., AND E~LLERMAN, L., Biochemistry, 5, 203 (1966). 12. WARBURG, O., AND CHRSTAN, W., Biochem. Z., 298,150 (1938). 13. STADE, W. C., AND ZAPP, J. A., JR., J. Biol. Chem., 150, 165 (1943). 14. WALAAS, E., AND WALAAS, O., Acta Chem. &and., 10, 122 (1956). 15. YAG, K., AND OZAW~, T., Biochem. Biophys. Acta, 42, 381 (1960). 16. MCCORMCK, D. B., GASSY, B. M., AND TSBRS, J. C. M., Biochim. Biophys. Acta, 69, 447 (1964). 17. FRSELL, W. R., LOWE, H. J., AND HELLERMAN, L., J. Biol. Chem., 223, 75 (1956). 18. WEBB, J. L., Enzyme and metabolic inhibitors, Vol., Academic Press, New York, 1966, p. 345.