Proc. Nat. Acad. Sci. USA Vol. 69, No. 12, pp. 3723-3726, December 1972

Alkylamine-Dependent Amino-Acid Oxidation by Lysine Monooxygenase- Fragmented Substrate of Oxygenase (Pseudomonas/enzyme/aminefamide/flavoprotein)

SHOZO YAMAMOTO, TAKASHI YAMAUCHI, AND OSAMU HAYAISHI

Department of Medical Chemistry, Kyoto University Faculty of Medicine, Kyoto, Japan

Contributed by Osamu Hayaishi, October 10, 1972d

ABSTRACT Lysine monooxygenase catalyzes the cIxy- a significant role in the catalysis of the enzyme. It would be of a rre- genative decarboxylation of L-lysine and produces coi heefoeto investigate the reaction of the enzyme sponding acid amide. L-Alanine was inactive as substriate. However, when propylamine was present, oxidation,Ibut in the presence of both a-monoamino acids and alkylamines- not oxygenation, of alanine was demonstrated with the of various carbon chain lengths-the two fragments of the oxygenase. Alanine was converted to pyruvate, with the normal substrate. liberation of ammonia and hydrogen peroxide, but proyI~yl- amine remained unchanged. Other a-monoamino accids MATERIALS AND METHODS were also oxidized in the presence of alkylamines wi various carbon chain lengths. The highest oxidase actiiV~it Lysine monooxygenase was purified and assayed as described was observed when the total chain length of both anm Lio (1). Pyruvate was determined with the aid of rabbit-muscle acid and amine was nearly identical with that of lysiine, lactate dehydrogenase Type II (Sigma) (4) or as its hydrazone Available evidence indicates that the amine-depend,lent (5). Oxygen consumption was followed by an oxygen electrode 5ine amino-acid oxidase activity is associated with the lys (1). As the solubility of oxygen in water decreases when the oxygenase activity. concentration of salt is raised, the approximate oxygen con- Lysine monooxygenase, a flavoprotein isolated and crystalliized centration at various concentrations of alanine and propyl- from a pseudomonad, catalyzes the oxygenation of L-lysi mie, amine was determined with protocatechuate 3,4-dioxygenase an a,e-diamino hexanoic acid, and produces an acid-arniide (6). The dioxygenase was kindly provided by Dr. M. Fujiwara concomitant with decarboxylation (Eq. 1) (1, 2). With I L- of this laboratory. Ammonia was measured by beef liver glu- ornithine, a diamino acid shorter by one carbon atom, h(ow- tamic dehydrogenase Type II (B36hringer) (7). Propionalde- ever, the reaction catalyzed by the enzyme is not oxygenateion, hyde was determined by yeast aldehyde dehydrogenase Grade but is oxidation of the amino acid to produce an a-keto awcid II (Sigma) (8). The trans-isomer of the 2,4-dinitrophenyl- (Eq. 2) (3). Thus, the enzyme functions either as an oxygen Lase hydrazone of pyruvate was prepared by the method of Katsuki et al. (9). Thin-layer chromatography of the 2,4-dinitrophenyl- CH2--NH2 CH2-NH2 hydrazone of pyruvate was performed with solvents (a) ethyl OH, OH2 acetate saturated with 0.1 M NaHCOO,-methyl alcohol 5:1 and the upper layer of n-butyl alcohol-ethyl alcohol-0.1 OH, (b) OH2 + 002 + H2O M NaHCOs 10: 3: 10 (10). A solvent of isopropyl ether-ligroin OUH, 1: 1 was used for the 2,4-dinitrophenylhydrazone of propional- 0-H2 C.H-NH2 dehyde (11).

00 03 sEnz Enz L-Aia 3mmrd CHr-NH2 CH2--NH2 L-Aia % Prop Prop L -Prop i -L-Ala I.-Enz OH2 N0.2 + 02 + H2O ---l + NH2 + H202 [2] w

&-NH2 11 0.I ~__OH OOH (C 0 if~ ~ ~ -(B) or as an oxidase, and the a-amino acid portion of the substrate TIME is oxygenated or oxidized depending on the carbon chain Fig. 1. Oxygen consumption by lysine monooxygenase in length of a substrate acw-diamino acid: 06 and 07, oxygenated; the presence of L-alanine ar~d propylamine. The reaction mixture, 05 and 08, oxidized; 04 and 09, inactive (3). a-Amino acids, in a total volume of 2.2 ml, contained iL-alanine (0.75 mmol), however, are inactive as substrates unless the co-amino group propylamine (0.4 mmol), borate buffer, pH 9.9 (0.22 mmol), is the co-amino group in the of present, implicating binding and the enzyme (0.28 Mg). L-Alanine (L-Ala), propylamine to substrate the enzyme (3). (Prop), and the enzyme (Enz) were added as indicated by arrows.

These results indicate a structural feature of the substrate Oxygen consumption was followed by an oxygen electrode at

with two amino groups at the a- and co-positions, each playing 240.

3723 Downloaded by guest on September 28, 2021 3724 Biochemistry: Yamamoto et al. Proc. Nat. Acad. Sci. USA 69 (1972)

0.4 0.4

E -23E a 10a O0.2 / 'a0 I0.

0.5 1.0 1.5 0 0.1 0.2 0.3 ALANINECM) PROPYLAMINE(M) FIG. 2. Effect of L-alanine concentration on propylamine- FIG. 3. Effect of propylamine concentration on propylamine- dependent oxidation of L-alanine. The reaction mixture, in a dependent oxidation of L-alanine. The reaction was performed as total volume of 1.1 ml, contained L-alanine (as indicated), pro- described in Fig. 2, in the presence of L-alanine (1.5 mmol), pylamine (0.2 mmol), catalase (43 Mug), and the enzyme (7.1 propylamine (as indicated), and the enzyme (4.7 ,ug). lAg). The pH was adjusted to 9.9. After incubation at 240 for 10 min, 10 N HCl (0.2 ml) was added. The acidified reaction mix- ture was incubated at 300 for 10 min with 0.26 ml of 5 mM oxygen consumption was approximately 0.79: 1.00. Ammonia 2,4-dinitrophenylhydrazine dissolved in 2 N HC1, then 1.6 N was produced in an amount stoichiometric to pyruvate forma- NaOH (3.64 ml) was added. Absorbance at 416 nm was deter- tion (0.97: 1.00). The ratio of oxygen consumption to hydro- mined after the mixture had stood at room temperature for 10 gen peroxide accumulation was about 1.00:0.88, as judged by min. the addition of catalase. Propionaldehyde could not be de- tected in a significant quantity with aldehyde dehydrogenase. RESULTS AND DISCUSSION These observations indicate oxidation, but no oxygenation, When the oxygen consumption was followed polarographically, of L-alanine in the presence of propylamine, the latter com- L-alanine was inactive as substrate, but the addition of pro- pound presumably remaining unchanged (Eq. 3). The oxy- pylamine resulted in oxygen uptake, as shown in Fig. 1A. genative reaction would have produced acetamide concomitant With propylamine alone, no oxygen uptake was observed with decarboxylation. (Fig. 1B). The oxygen uptake observed in the presence of both CH2-NH2 CH2-NH2 L-alanine and propylamine was enzyme-catalyzed (Fig. 1C), and the rate increased in proportion to the amount of enzyme. CH2 CR2 When catalase was added after exhaustion of the oxygen in the CH3 CH3 reaction mixture, evolution of oxygen was observed, indicating + 02 + H20 + NH3 + H202 [3] the accumulation of hydrogen peroxide during the reaction. CH3 CH3 The rate of oxygen uptake was reduced by 44-48% in the CH-NH2 presence of an excess amount of catalase. These results suggest either that ialanine was oxidized to COOH COOH produce pyruvate or that propylamine was oxidized to pro- However, the oxygenation of a small portion of L-alanine can pionaldehyde. In order to identify pyruvate as its 2,4-dinitro- not be ruled out. The stoichiometric relationship between the phenylhydrazone, a solution of 2,4-dinitrophenylhydrazine oxygen consumption and the amount of products has not yet was added to the acidified reaction mixture, and the crystals been clearly established, partly because the oxygen concen- formed were collected and dissolved in 1 N Na2CO3. After re- tration in the presence of high concentrations of L-alanine and moval of insoluble materials, the solution was acidified and propylamine cannot be determined accurately. Moreover, the crystals that formed were recrystallized from acetone- sensitive assays for acetamide are not available. benzene 1:3. The reaction product thus obtained was indis- The propylamine-dependent oxidation of L-alanine was tinguishable from the authentic trans-isomer of the 2,4-di- followed by the determination of pyruvate. The reaction rate nitrophenylhydrazone of pyruvate by the following criteria: was dependent on the concentration of L-alanine (Fig. 2) and (i) thin-layer chromatography with solvents (a) and (b) as propylamine (Fig. 3), The reaction required a rather high described above. RF values of the authentic sample and the concentration of L-alanine; because of limited solubility, the reaction product were (a) 0.27 and 0.27, (b) 0.61 and 0.60; activity could not be followed up to the saturating concen- (ii) melting point, 213-215° (uncorrected); (iii) absorption tration of L-alanine. The curve was of a sigmoidal nature, a spectra in the visible region with absorption maxima at 370 result that can be compared with the case of lysine oxygena- nm in 0.1 M NaHCO3 and at 445 nm in 1 N NaOH; (iv) in- tion or ornithine oxidation (3). The optimal pH of the reaction frared absorption spectra. On the other hand, after the reaction was about 9.9. At this pH value, the specific activity was 4.0 with 2,4-dinitrophenylhydrazine, the reaction mixture was lumol/min per mg of protein with 1.36 M L-alanine and 0.18 M extracted with ethyl acetate so that any hydrazone of pro- propylamine, at 240, with an enzyme preparation that showed pionaldehyde, if present, would be transferred to the solvent; a specific activity of 10.2 with 50 mM lysine at pH 8.0 at the the extract was then examined by thin-layer chromatography. same temperature. No yellow spot was observed in the area corresponding to the Further studies on the substrate specificity of the reaction authentic 2,4-dinitrophenylhydrazone of propionaldehyde. resulted in a striking observation, as presented in Fig. 4. Other The formation of pyruvate was also demonstrated by the use alkylamines were also effective in stimulating the oxidation of of lactate dehydrogenase; the ratio of its formation to the i-alanine (C3), but propylamine (C3) was the most effective. Downloaded by guest on September 28, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Amine-Dependent Amino-Acid Oxidation by Oxygenase 3725

The following compounds with a carbon chain length of C3 did tion by alkylamine was reportedly observed in the trypsin- not show oxygen consumption in the presence of propylamine: catalyzed hydrolysis of benzoyl-iarginine ethyl ester. Inagami D-alanine, fl-alanine, -a-aminoisobutyrate, iserine, and L- and Murachi demonstrated that alkylamine activated the lactate. Other e-a-amino acids with shorter or longer carbon hydrolysis of acetylglycine ethyl ester, a smaller uncharged chains also showed oxygen consumption in the presence of substrate (13, 14). They postulated that the specificity-de- various alkylamines. Methylamine (Cl) was the most potent termining site and the catalytic site on the enzyme surface stimulator for L-a-aminobutyrate (C4) and L-norvaline (C5). were topographically distinct. Alkylamine and acetylglycine It should be noted that combinations of a-amino acids and ethyl ester were presumed to be accommodated at the respec- alkylamines with a total number of carbon atoms nearly iden- tive sites in place of the normal substrate. Although it is un- tical to that of lysine (C6) were most favorable for the oxida- certain whether such a mechanism works in the case of lysine tion of each 4-a-amino acid. In every case, oxygen evolution monooxygenase, activation of the fragmented substrates that was observed when catalase was added, indicating that an are susceptible to catalysis by the enzyme only when they are oxidative-rather than an oxygenative-reaction occurred combined is a common feature of both enzymes. If a mecha- under these conditions. nism such as the one proposed by Inagami and Murachi holds The alkylamine-dependent oxidation of -a-amino acids true for lysine monooxygenase, a-amino acids should be oxy- appeared to be associated with the lysine oxygenase activity of genated in the presence of alkylamines. However, the reaction the enzyme. First, the ratios of lysine oxygenation and alanine actually observed was not oxygenation, but oxidation of the oxidation were essentially constant at all steps of the enzyme amino acid, an abnormal reaction that appears to be due to a purification. Second, anaerobic reduction of the enzyme- misfit of both or either of the two fragments of substrate to bound FAD, an essential prosthetic group for lysine oxygena- the active site of the enzyme. A similar interpretation was tion (12), was observed only in the presence of both L-alanine also made in the oxygenase-catalyzed oxidation of ornithine and propylamine. Third, oxygenation of lysine was competi- (3). These oxidase reactions, demonstrated either with ana- tively inhibited by the presence of an alkylamine. logues of lysine or with fragments of lysine, may be compara- The competitive inhibition of lysine oxygenation by alkyl- ble in concept to the uncoupling of NADH oxidation from amines suggests a significant role for the e-amino group of oxygenation first proposed by Okamoto et al. (15) and further lysine in binding to the enzyme. A similar competitive inhibi- substantiated by White-Stevens and Kamin (16). It would be Norvolne Alonine C5/oc jNH2 C0 292 C3 HOOC¢y NHS C0 I0 C5 HOOC\\/\/ 355 C3 NOC, ,,HC1 23 SIN C2 C5 "Oc\/\V 92 C3~OCv\X / N C2 84 C5 NH C3 32 HNOOCz ,\/",IC3 100 C5 O\VV .NH C4 18 C3 HOOCv 4 C4 54 C5 OOC\ INHC5 8 C3C3 HOOC\,, \ N C/5 2 1 dC-Aminobutyrate Glyciine HOOC\ C4 mCo 167 C2 NHS CO 0 HOOC\ ,NH C C4OOC\ ,AftCI 186 C2 I 0 HOOC\ ,,NHg C2 C4 "~\/\ %/^J C2 164 C2 0 HOOC C4 ,43C3wl 88 \ . 9 C3 I C2 %4*%w HOOC 163r C4 HOOCC 84 C2 0 H HOOC C C4 °°C\A ,#C5 74 C2 \ ^ V,0-NH 5 0

FIG. 4. Oxidase activities of lysine monooxygenase with various combinations of i4a-monoamino acids and alkylamines. Oxygen con- sumption was followed by an oxygen electrode in a reaction mixture (2.2 ml) containing 1-a-monoamino acid (3 mmol), alkylamine (0.4 mmol), and enzyme (94 ,zg) at 24°. The activity with L-alanine and propylamine was expressed as 100%. Because of limited solubility, only 1.4 mmol of i-norvaline was present, and the activity with L-norvaline was compared with that observed with L-alanine at the same concentration. The carbon chain of an i4a-amino acid is indicated by a solid line, and that of an alkylamine by a broken line. Alkylamines tested were as follows: n-amylamine (C5), n-butylamine (C4), n-propylamine (C3), ethylamine (C2), methylamine (C1), and ammonia (C0). Downloaded by guest on September 28, 2021 3726 Biochemistry: Yamamoto et al. Proc. Nat. Acad. Sci. USA 69 (1972)

of interest to look for conditions under which the "fragmented 6. Fujisawa, H. & Hayaishi, 0. (1968) "Protocatechuate 3,4- I. J. substrates" are oxygenated rather than being oxidized. Should dioxygenase. Crystallization and characterization," Biol. Chem. 243, 2673-2681. such an attempt materialize, it would provide a clue to a better 7. Buttery, P. J. & Rowsell, E. V. (1971) "Enzymic assays for understanding of the reaction mechanism of oxygenases in ammonia and L-glutamine in tissue extracts," Anal. Bio- general. chem. 39, 297-310. 8. Black, S. (1955) in Methods in Enzymology, eds. Colowick, We thank Prof. H. Katsuki and Dr. M. Tokushige, Kyoto S. P. & Kaplan, N. 0. (Academic Press, New York), Vol. 1, University Faculty of Science, for helpful advice in the prepara- pp. 508-511. tion and identification of hydrazones of keto-acids and aldehydes, 9. Katsuki, H., Tanegashima, C., Tokushige, M. & Tanaka, S. and Dr. H. Yamamoto, Kyoto University Faculty of Engineer- (1972) "Studies on the isomerization of 2,4-dinitrophenylhy- ing, for recording the infrared absorption spectra. Thanks are drazones of some aliphatic a-keto acids and the preparation also due to Miss M. Ohara for her kind help in the preparation of their geometrical isomers," Bull. Chem. Soc. Jap. 45, of this manuscript. This work has been supported in part by the 813-817. Scientific Research Fund of the Ministry of Education of Japan, 10. Ariga, N. (1972) "Thin-layer chromatography of keto acid and by grants from the Waksman Foundation of Japan, the 2,4-dinitrophenylhydrazones," Anal. Biochem., in press. Naito Foundation, the Tanabe Amino-Acid Research Founda- 11. Ariga, N. (1971) "Methods for determination of carbonyl tion, and the Squibb Institute for Medical Research. T. Y. was compounds by 2,4-dinitrophenylhydrazine and their applica- the recipient of a Postdoctoral Fellowship of the Naito Founda- tion to the assay of aldehyde dehydrogenase," Anal. Bio- tion. chem. 43, 446-453. 1. Takeda, H. & Hayaishi, 0. (1966) "Crystalline L-lysine oxy- 12. Yamamoto, S., Hirata, F., Yamauchi, T., Nozaki, M., Hiro- genase, "J. Biol. Chem. 241, 2733-2735. mi, K. & Hayaishi, 0. (1971) "New spectral species of L-ly- 2. Takeda, H., Yamamoto, S., Kojima, Y. & Hayaishi, 0. sine monooxygenase, a flavoprotein," J. Biol. Chem. 246, (1969) "Studies on monooxygenases. I. General properties of 5540-5542. crystalline L-lysine monooxygenase," J. Biol. Chem. 244, 13. Inagami, T. & Murachi, T. (1963) "Induced activation of the 2935-2941. catalytic site of trypsin," J. Biol. Chem. 238, PC1905-1907. 3. Nakazawa, T., Hori, K. & Hayaishi, 0. (1972) "Studies on 14. Inagami, T. & Murachi, T. (1964) "The mechanism of the monooxygenases. V. Manifestation of amino-acid oxidase specificity of trypsin catalysis. III. Activation of the catalyt- activity by L-lysine monooxygenase," J. Biol. Chem. 247, ic site of trypsin by alkylammonium ions in the hydrolysis of 3439-3444. acetylglycine ethyl ester," J. Biol. Chem. 239, 1395-1401. 4. Kornberg, A. (1955) in Methods in Enzymology, eds. Colow- 15. Okamoto, H., Nozaki, M. & Hayaishi, 0. (1968) "A role of ick, S. P. & Kaplan, N. 0. (Academic Press, New York), sulfhydryl groups in imidazoleacetate monooxygenase," Bio- Vol. 1, pp. 441-443. chem. Biophys. Res. Commun. 32, 30-36. 5. Katsuki, H., Yoshida, T., Tanegashima, C. & Tanaka, S. 16. White-Stevens, R. H. & Kamin, H. (1970) "Uncoupling of (1971) "Improved direct method for determination of keto oxygen activation from hydroxylation in a bacterial salicy- acids by 2,4-dinitrophenylhydrazine," Anal. Biochem. 43, late hydroxylase," Biochem. Biophys. Res. Commun. 38, 349-356. 882-889. Downloaded by guest on September 28, 2021