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 27, 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.