Proc. Nat. Acad. Sci. USA Vol. 68, No. ;i, pp. 987-991, Mlay 1971 Demonstration of Imino Acids as Products of the Reactions Catalyzed by D- and L-Amino Acid Oxidases EDMUND W. HAFNER AND DANIEL WELLNER Department of Biochemistry, Cornell University Medical College, New York, N.Y. 10021 Communicated by Alton Meister, February 23, 1971 ABSTRACT It had long been thought, but never dem- from the enzyme as the primary oxidation product. Other in- onstrated, that imino acids are formed in the reactions terpretations, however, are not (see catalyzed by D- and L-amino acid oxidases (EC 1.4.3.3 and excluded Discussion be- 1.4.3.2). The formation of imino acids is now shown low). directly by allowing the amino acid oxidase reaction to Coffey, Neims, and Hellerman (9) recently observed that proceed in the presence of NaBH4, when the imino acid is when a mixture of D-amino acid oxidase and ['4C]D-alanine reduced to the corresponding racemic amino acid. Thus, was treated with sodium borohydride, the alanine moiety when NaBH4 is added to a mixture of D-amino acid oxidase and D-alanine, a significant amount of L-alanine is formed. became covalently attached to the enzyme; they subsequently Analogous results are obtained using L-amino acid oxidase found that the labeled protein gave e-N-(1-carboxyethyl)-L- and L-leucine. Since D-amino acid oxidase is active in the lysine on hydrolysis (10). Studies in this laboratory have con- presence of NaBH4, L-alanine continues to be formed un- firmed this finding and have also shown that i- and D-amino til most of the D-isomer is oxidized by the enzyme. This reaction provides a new method for inverting the con- acid oxidases labeled in this manner are still enzymatically figuration ofan amino acid. active (see Discussion). We thought that these results might be When NaBH4 is added to a system containing D-amino explained in terms of the formation of an imino acid. If a free acid oxidase plus D-alanine and L-lysine, free c-N-(1-carb- imino acid were released as the product of the amino acid oxi- oxyethyl)-L-lysine is formed. When bovine serum albumin dase reaction, it would be expected that its reduction by boro- is substituted for L-lysine, the same compound results upon acid hydrolysis. It is concluded that the amino acid hydride would yield a racemic amino acid. Thus, a significant oxidase reaction produces a free imino acid, which may amount of ialanine should be found when sodium borohy- be reduced by NaBH4 to a racemic amino acid or may dride is added to a system containing D-amino acid oxidase and form Schiff's bases by reaction with the -amino groups D-alanine. Experiments designed to demonstrate this reaction of proteins and of free lysine. and to elucidate the interaction between this postulated prod- The formation of a-imino acids as transient intermediates uct and the amino groups of proteins are described in this in the oxidative deamination of a-amino acids was postulated report. by Knoop (1) before Krebs' discovery of amino acid oxidases MATERIALS (amino acid: oxygen oxidoreductases, deaminating, EC 1.4.3.2 and 1.4.3.3) in animal tissues (2). Although such intermediates Crystalline D-amino acid oxidase was prepared from hog kid- are extremely unstable in aqueous solution and have never ney (11) and crystalline iamino acid oxidase from Crotalus been isolated from enzymatic reaction mixtures, their forma- adamanteus venom (12). Bovine serum albumin was from tion by amino acid oxidases is supported by some indirect Armour; catalase (EC 1.11.1.6) and FAD from Sigma; evidence. For example, evidence exists against an alternative D-alanine and D-methionine from Calbiochem; [1-14C]D-alanine mechanism of oxidation suggested by Dakin (3) involving the and [3H]NaBH4, from New England Nuclear. H-10 Antifoam formation of an a,jB-unsaturated intermediate (for a review was from Dow Chemical. All other reagents were analytical see ref. 4). More directly, it has been shown by Radhakrishnan grade. The D-alanine was treated with iamino acid oxidase and Meister (5) that in the reversal of the D-amino acid oxi- and reisolated on a Dowex-50 column. Subsequent analysis dase reaction, the formation of D-proline from Al-pyrroline-2- by reaction with D-amino acid oxidase showed that the treated carboxylate, which may be regarded as a substituted a-imino preparation contained, at most, 0.004% ialanine. acid, is considerably faster than that of other amino acids from the corresponding a-keto acid and ammonia. Spectro- METHODS photometric evidence was obtained by Taborsky (6) and by Determination of L-alanine Pitt (7) for the formation of an intermediate absorbing at 300 After the addition of HCl to the reaction mixture (Table 1) to nm in the course of oxidation of ityrosine by snake venom destroy NaBH4, the pH was adjusted to 8.1-8.3 with 3 N i-amino acid oxidase. The data presented, however, do not NaOH and the mixture was recharged with 1 mg of D-amino permit an unequivocal conclusion concerning the nature of acid oxidase, 0.12 gmol of FAD, and 2 mg of bovine serum this intermediate. Recently, Yagi et al. (8) reported evidence albumin. It was then incubated for at least 17 hr at 370C with for the formation of a product, less basic than either the start- vigorous shaking in dark test tubes. The mixture was depro- ing amino acid or ammonia, in the course of oxidation of D- teinized by the addition of 0.8 ml of 25% trichloroacetic acid alanine and other amino acids by D-amino acid oxidase. They followed by centrifugation, adjusted to pH 2.2 with 3 N interpreted their data to mean that an imino acid is released NaOH, and analyzed on a Beckman Model 120C amino acid 987 Downloaded by guest on September 26, 2021 988 Biochemistry: Hafner and Weilner Proc. Nat. Acad. Sci. USA 68 (1971) analyzer. In a number of experiments, the L configuration TABLE 1. Formation of Lalanine from D-alanine in the of the alanine was verified by demonstrating its disappearance presence of D-amino acid oxidase and sodium borohydride after treatment with -amino acid oxidase. In reaction mixtures containing benzoate, acidification L-Alanine with 0.5 ml of 3 N HC1 resulted in precipitation of benzoic formed Yield acid. This was filtered through Whatman no. 3 paper and Expt. Component omitted (umol) (%) the filtrate was extracted three times with 5 ml of ether. 1 None (NaBH4 added at 10 0.75 5.0 After removal of the residual ether with a stream of nitrogen, see)* the solution was treated with D-amino acid oxidase and 2 None (NaBH4 added at 0 0.71 4.7 analyzed as described above. time)t 3 D-Amino acid oxidase 0.005 ... Determination of e-N-(1-carboxyethyl)-L-lysine 4 NaBH4 <0.003 ... The protein solutions were mixed with an equal volume of 5 D-Alanine (replaced by so- <0.003 ... concentrated HCl, sealed under vacuum, and hydrolyzed at dium pyruvate + NH4Cl)t 110'C for 24 hr (Expts. 1 and 2, Table 6) or for 53 hr (Expt. 3). Analysis was carried out according to Spackman, Stein, * The complete system contained D-amino acid oxidase (1 mg), and Moore (13) with a Beckman 120C amino acid analyzer, FAD (0.12 iimol), bovine serum albumin (2 mg), sodium pyro- using PA28 resin, 0.2 N sodium citrate buffer, pH 3.20, a phosphate buffer, pH 8.3 (50,mol), and D-alanine (15 jsmol, added rate of 70 The buffer last) in a final volume of 1.5 ml. After 10 sec, 53 pmol of NaBH4 temperature of 550C, and a flow ml/hr. (freshly dissolved) in 0.2 ml of water was added. The reaction, change was delayed until methionine was eluted. The new carried out with stirring in a water-jacketed cup maintained at component [identified by Hellerman and Coffey (10) as c-N- 370C and with air blowing over the surface, was stopped after (1-carboxyethyl)-ilysine] appeared 12 min after methionine. 5 min by the addition of 0.2 ml of 3 N HCl. The L-alanine formed Correction was made for small amounts of alloisoleucine eluted was determined as described under Methods. in this position when control samples of protein were analyzed t Borohydride was added together with D-alanine. In other under identifical conditions. The same peak was observed experiments it was added 10 see later. without hydrolysis when the amino acid oxidase reaction was t 10 umol of each. carried out in the presence of free ilysine. Calculations were made using a ninhydrin color value of 1.07 leucine equivalents (10). TABLE 2. Test for imino acid accumulation in the absence of borohydride Radioactivity measurements '4C-labeled compounds were plated on aluminum planchets Time of Time of and counted in a Nuclear Chicago model D47 gas-flow NaBH4 Na benzoate L-Alanine counter fitted with a micromil window, or were dissolved in addition addition formed Yield scintillation fluid (14) and counted in A Nuclear Chicago Expt. (see) (see) (umol) (%) Unilux II scintillation counter. H-labeled compounds were 1 30 30 0.023 0.15 measured by scintillation counting. 2 30 31 0.027 0.18 3 31 30 0.022 0.15 RESULTS 4 30 60 0.387 2.6 Experiments showing ialanine production by reaction of 5 0 0 0.035 0.23 D-amino acid oxidase, D-alanine, and NaBH4 are presented in Table 1.