J. Biochem. 86, 97-104 (1979) Substrate Specificity and Reaction Mechanism of Putrescine Oxidase Masato OKADA, Seiichi KAWASHIMA, and Kazutomo IMAHORI Department of Biochemistry, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113 Received for publication, January 9, 1979 Putrescine oxidase [EC 1.4.3.4] of Micrococcus rubens oxidizes many kinds of synthetic poly amines: triamines (spermidine types), tetramines (spermine types), and N-substituted putres cines. Polyamines possessing terminal 4-aminobutylimino groups in their structures were more active as substrates. Putreanine was oxidized at a rate comparable to that of putrescine, and was converted to 1-pyrroline and 8-alanine. Activities and Km values for polyamines were affected by the substituent attached to the 4-aminobutylimino group of the polyamine, and especially by its methylene chain length. It was also found that two types of oxidation occurred in the oxidation of polyamines by putrescine oxidase. When the moieties attached to the 4-aminobutylimino groups in polyamines were less hydrophobic, these polyamines were oxidized at the secondary amino groups to form 1-pyrroline. Polyamines which contained a hydrophobic substituent attached to the 4-aminobutylimino group were oxidized at the ter minal primary amino group of the 4-aminobutylimino moiety to form ammonia. N,N•Œ- Bis(4-aminobutyl)-1,3-diaminopropane ([‡U,4-3-4]) and N-(4-aminobutyl)-N•Œ-(3-aminopropyl) 1,3-diaminopropane ([‡U,4-3-3]) were oxidized to form 1-pyrrolinium salt derivatives as a result of oxidation of the terminal primary amino groups. It was concluded that the essential struc ture for substrates of putrescine oxidase is a 4-aminobutylimino group (NH2(CH2),NH-). Putrescine oxidase [EC 1.4.3.4], putrescine: oxygen which catalyze the degradation of spermidine oxidoreductase (deaminating) (flavin containing), also oxidize spermine (4-10). from Micrococcus rubens has a high substrate Swain and DeSa proposed a model for the specificity for amines (1). This enzyme oxidized active site of this enzyme based on studies of the putrescine, cadaverine, spermidine, and 1,6 interactions between the enzyme and competitive diaminohexane (1-3), but is exceptional in that it inhibitors: the enzyme has an anionic point and a does not oxidize spermine (1), since many enzymes hydrophobic region in the active site (3). How ever, this model failed to explain why putrescine oxidase oxidizes spermidine, but not spermine. Abbreviations: Me, methyl; Et, ethyl; n-Pr, n-propyl; The substrate specificity for polyamines other n-Bu, n-butyl; n-Am, n-amyl; [‡T, x-y], NH2(CH2) x- NH(CH2)yNH2; [‡V, x-y-z], NH2(CH2)xNH(CH2) than diamines, spermidine and spermine has not y- NH(CH2)zNH2; [‡V, R], NH2(CH2)4NH-R; [‡W, X], examined. In order to characterize the properties NH2(CH2)4NH(CH2)2-X. of the active site in more detail, it is necessary to Vol. 86, No. 1, 1979 97 98 M. OKADA, S. KAWASHIMA, and K. IMAHORI examine the substrate specificity for many poly 12 N HCl : H2O (60: 20: 20, v/v) (15). The amines. amines were detected by spraying 0.1 % ninhydrin In this paper, we report the activities of in acetone. IR spectra were taken with a Hitachi putrescine oxidase towards many kinds of synthetic EPI-G2 spectrophotometer, and NMR spectra polyamines (triamines, tetramines, and N-sub with a Hitachi Perkin-Elmer R-20A spectrometer stituted putrescine) and the structures of their at 90 MHz using tetramethylsilane as an external oxidation products. Based on these results, the standard. Liquid chromatographic analyses of essential structures of polyamines for activity as polyamines and their oxidation products were substrates, as well as the binding properties of the carried out using a Hitachi 835 amino acid ana enzyme, are discussed. lyzer in the buffer system with suitable modification (16). Activity of Putrescine Oxidase towards Poly MATERIALS AND METHODS amines-The maximal velocity (Vmax) and pH Materials-Putrescine oxidase of Micrococcus optimum of putrescine oxidase for polyamines rubens was purified to homogeneity by an affinity were determined from the amount of hydrogen chromatographic procedure (11). The specific peroxide evolved using the coupling assay method activity of the enzyme used in this study was with peroxidase and o-dianisidine (17) in 0.1 M 34.7 ƒÊmol/min/mg protein. Peroxidase and cata borate buffer at pH 7.5-10.5. lase were purchased from Boehringer, Mannheim, Km Values for Polyamines-Km values for Germany. Putrescine (free base and hydro polyamines were estimated from the competitive chloride salt), spermidine trihydrochloride, and inhibition by 1,8-diaminooctane of the oxidation spermine tetrahydrochloride were the products of of the polyamines. First, Km for putrescine was Nakarai Chemicals Ltd., Japan. Other diamines, determined from Lineweaver-Burk plots based on dimethylenetriamine, trimethylenetetramine, and the above assay method. Next, the Ki value for N-(3-aminopropyl)-1,3-diaminopropane were pur 1,8-diaminooctane, which is a competitive inhibitor chased from Tokyo Chemical Industry Co., Japan. of putrescine oxidase (3), was determined (Ki= Other polyamines were synthesized in our labo 9.2•~10-6M). The oxidation velocities for poly ratory (12). Other chemicals were of the highest amines in the presence (vi) and absence (v) of grade available. 1,8-diaminooctane at various concentrations (i) 1-(3-Hydroxy-l-oxoinden-2-yl)-1-pyrrolinium were measured, then Km values for the polyamines Hydroxide, Inner Salt ([V])-This compound were calculated from v/vi vs. i plots based on the (13) was prepared by the condensation reaction of equation: v/vi=1+i/Ki[Km/(Ki+s)], where Ki is proline with ninhydrin according to the method the inhibitor constant of 1,8-diaminooctane and of Grassmann and Arnim (14). NMR (in CDCl3) s is the concentration of polyamine. S 9.76 (s, 1H, imine proton), 8.00 (2d, 4H J=2Hz, Determination of 1-Pyrroline-l-Pyrroline indene ring protons), 5.43 (t, 2H, J=8Hz, 5-H), formed by the oxidation of polyamines was deter 3.60 (m, 2H, 3-H), 2.80 (quin, 2H, J=8Hz, 4-H). mined according to the method described by Holmstedt et al. (18, 19), except that a value of 2.1 •~ 103 M-1 • cm-1 was used for the molar extinc tion coefficient of the adduct between 1-pyrroline and o-aminobenzaldehyde at 435 nm (18). The reaction mixture contained 0.5 ml of 0.1 M borate (pH 9.0), 10 pl of 50 mM polyamine (0.50 ƒÊmol), and 20 ƒÊl (ca. 1.0 unit) of putrescine oxidase. After incubation for 16 h, 0.1 ml of 0.6 M phosphate (pH 5.5) was added to the incubation mixture. After 1 h, the absorbance at 435 nm was mea Measurement-TLC was carried out on sured. Avicel SF cellulose (Funakoshi Pharmaceutical Determination of Ammonia-Ammonia was Co.) in the following solvent system : 1-propanol: determined with an amino acid analyzer using J. Biochem. SUBSTRATE SPECIFICITY OF PUTRESCINE OXIDASE 99 ammonium sulfate as a standard. Nessler's reagent TABLE I. Vmax, Km, and pH optimum values of (20) was not applicable in this case because a putrescine oxidase for polyamines, including diamines. The reaction mixtures (0.555-0.570 ml) contained 2.5 precipitate was formed in some cases. The ƒÊ mol of polyamines, and 0.06-1.10 units of purified enzymatic reactions were carried out by methods putrescine oxidase. Vmax and Km were determined by similar to that described for the determination of the methods described in " MATERIALS AND 1-pyrroline. When the reaction stopped, 5011 METHODS." Vmax values are relative ratios based aliquots were withdrawn from the incubation on the velocity of putrescine oxidation at pH 9.0. mixtures and diluted to 550 pl with water. These diluted mixtures were used as samples for the amino acid analyzer. RESULTS Activities of Putrescine Oxidase towards Poly amines-Vmax, pH optimum, and Km for various polyamines were determined, as summarized in Table I; most of the polyamines examined were active as substrates, and polyamines possessing 4. aminobutylimino groups were more active. This agrees with the model which Swain and DeSa proposed for the active site of putrescine oxidase (3): the length of the 4-aminobutylimino group corresponds to the distance from the active (cata lytic) point to the anionic point which has a binding role. It is interesting that the activity for [IV, COOH] (putreanine) (21) is comparable to that for putrescine, in spite of its higher Km value. So far, no enzymes other than putrescine oxidase have been reported that can degrade putreanine. On the other hand, Km values for polyamines tend to decrease with increasing methylene chain length in their structures. This tendency is clear when Km values for the members of the [‡T,4-y] or [‡V,R] group are compared. This is comparable to the known tendency that Ki values for com petitive inhibitors of putrescine oxidase decrease as the methylene chain length increases (3), sug gesting the existence of a hydrophobic interaction between the enzyme and the substituent on the 4-aminobutylimino group in polyamines. Formation of 1-Pyrroline and Ammonia on Polyamine Oxidation by Putrescine Oxidase Putrescine oxidase oxidizes putrescine to form 1-pyrroline, ammonia, and hydrogen peroxide, and converts spermidine to 1-pyrroline and 1,3 diaminopropane, generating hydrogen peroxide ND: Not determined because the formation of H2O2 (1, 2). However, it has not been clarified whether all the [I,4-y], [‡U,4-y-4], [‡V,R], and [IV,X] types was observed only for a short initial period. Other of polyamines are also oxidized to form 1-pyrroline polyamines, [I, 2-2], [‡U, 2-2-2], [‡U, 4-8-4], and [‡U, 6-6-6] were not active as substrates.