Studies on the Induced Synthesis of Maleate cistrans Isomerase by Malonate

Transcription

1 Agricultural and Biological Chemistry ISSN: (Print) (Online) Journal homepage: Studies on the Induced Synthesis of Maleate cistrans Isomerase by Malonate Yoshichika Takamura, Tomiko Takamura, Masami Soejima & Teijiro Uemura To cite this article: Yoshichika Takamura, Tomiko Takamura, Masami Soejima & Teijiro Uemura (1969) Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate, Agricultural and Biological Chemistry, 33:5, , DOI: 1.18/ To link to this article: Published online: 9 Sep 214. Submit your article to this journal Article views: 98 Citing articles: 1 View citing articles Full Terms & Conditions of access and use can be found at

2 [Agr. Bioi. Chern., Vol. 33, No. 5, p , 1969] Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate Part III. Purification and Properties of Maleate cis-trans Isomerase Induced by Malonate By Yoshichika T AKAMURA, Tomiko T AKAMURA, Masami SoEJIMA and Teijiro UEMURA * Department of Agricultural Chemistry, Faculty of Agriculture, lbaraki University, lbaraki *Institute of Applied Microbiology, The University of Tokyo, Tokyo Received August 26, 1968 Maleate cis-trans isomerase in Alcaligenes faecalis IB-14 was induced by malonate and purified about 1-fold over the crude cell-free extract by treatments of ammonium sulfate fractionation, Sephadex G-1 gel filtration, DEAE-cellulose and DEAE-Sephadex A-5 column chromatography. The preparation was shown to be monodisperse on ultracentrifugal analysis and Svedberg value was found to be 3.84 S. The enzyme was most active at ph value around 8.3 and was stable over the range of ph 5. to 7. in the presence of dithiothreitol (DTT) for a few weeks, but in the absence of it, the enzyme activity was markedly decreased, especially in the alkaline region. The enzyme activity was inhibited by various sulfhydryl reagents and oxidizing agents, whereas it was not affected by metal chelating agents. The inhibition by Hg2+ and PCMB was overcome by the addition of sulfhydryl compounds such as DTT, 2-mercaptoethanol, L-cysteine and glutathione. It was observed that the enzyme did not require co-factor for its function. Kinetic studies showed that Michaelis constant for maleate was 2.8 x 1-a M and the enzyme did not catalyze the reverse reaction. l'vialeate and fumarate are isomeric, configuration of the former being cis while that of the latter trans, based upon this, early studies on the difference in their actions on biological systems were carried out to demonstrate the influence of chemical structure on activity. Many works have shown that fumarate is well utilized, while maleate is little. Indeed, in animal tissues maleate is a potent inhibitor and depression of respiration by it is the rule. 11 Fumarate, on the other hand, accelerates the endogenous respiration of animal tissues. Maleate also inhibits the fermentation of glucose by yeast, 21 but in plant tissues respiration is often stimulated by maleate of which metabolism has been thus demonstrated. Enzymes involved in metabolism of maleate in biological system have been reported for only two reactions, i.e., isomerization to fumarate, 3 " 41 and hydration to malate. 5 ' 61 I) J. L. Webb, "Enzyme and Metabolic Inhibitors," Vol. 3, Academic Press, New York and London, p. 285 (1966). 2) A. Jung and H. Muller, Helv. Chim. Acta, 5, 239 (1922). 3) E.J. Behrman and R. Y. Stanier,]. Bioi. Chern., 228, 923 (1957). 4) K. Otsuka, Agr. Bioi. Chern., 25, 726 (1961). 5) W. Sacks and.. Jensen,]. Bioi. Chern., 192, 231 (1951). 6) K. Otsuka and M. Hara, Presented at the Meeting of the Kanto Division of the Agricultural Chemical Society of Japan, Feb. (1964).

3 Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate. Part III 719 Berhaman and Stanier (1957) 31 have observed that cis-trans isomerization of maleate was catalyzed by soluble extract of Pseudomonas sp. in the course of nicotinic acid oxidation. Otsuka (1961) 41 first isolated this enzyme from Pseudomonas sp. cells adapted to maleate and suggested that the partially purified enzyme requires glutathione or L-cysteine as co-factor for its function, but any of other properties of the enzyme was not reported. During the course of a study on the enzymatic production of L-aspartic acid from maleate and ammonia by Alcaligenes faecalis IB-14, it was found by one of the authors'- 91 that maleate cis-trans isomerase played an indispensable role in this reaction and that the enzyme was more effectively induced by malonate than by maleate although the reverse was the case in utility. Cells with much increased level of the enzyme thus obtained by the malonate induction led to purify the enzyme and to clarify in details its nature which is yet obscure. The present paper deals with the purification and properties of about 1-fold purified enzyme which was monodisperse on ultracentrifugal analysis. MATERIALS AND METHODS Organism and medium. Alcaligenes faecalis IB-14 was used throughout this work. The basal medium for culture consisted of I of peptone, I of meat extract,.196 of KHzPO.,.59 of MgS 4 7H 2. To the above medium I- 1 M of malonate was added as inducer and 5 X 1-2M of maleate as carbon source to promote the cell growth.joi Cultivation. Cells grown on the basal medium for one night at 3 C was used as seed. A 1 ml por- 7) Y. Takamura, I. Kitamura, M. Iikura, K. Kono and A. Ozaki, Agr. Bioi. Chern., 3, 338 (1966). 8) Y. Takamura, I. Kitamura, M. Iikura, K. Kono and A. Ozaki, ibid., 3, 346 (1966). 9) Y. Takamura, M. Soejima and T. Aoyama, ibid., 31, 27 (1967). 1) Y. Takamura, T. Nakatani, M. Soejima and T. Aoyama, ibid., 32, 88 (1968). tion of seed culture was inoculated on 1.2 liters of induction medium in a 5 liter flask which was incubated on rotary shaker at 3 C for 24 hr. Maleate cis-trans isomerase assay. lvialeate cis-trans isomerase was assayed photometrically by the following two methods. 1) Method of Otsuka. 4 1 Routine determination of the enzyme activity was carried out by this method. Enzyme activity was assayed by measuring the reduction of optical density of maleic acid at 24 mp in the presence of pig fumarase. Assay mixture contained 1.5 ml of phosphate buffer (1/15 M, ph 7.3),.5 ml of pig fumarase,.5 ml of enzyme solution. The reaction was initiated by addition of.5 ml of maleate (1/5 M, ph 7.3) at 28± l C. The unit of the enzyme activity when determined by this method is tentatively defined as follows. One unit of maleate cis-trans isomerase activity corresponds to the amount of enzyme which reduces.1 of optical density (-log T) at 24 mp in one minute. In steps of purification of the enzyme, the extent of purification was shown by the units per 1. of optical density at 28 mp of enzyme solution. This method was excellent in the sensitivity, but was not adequate to examine properties of the enzyme, since fumarase concomitantly added to the reaction system often disturbs the investigation of the isomerase reaction, and thus this method was applied specifically to the purification of the enzyme. 2) Method of Behrman and Stanier.31 The enzyme activity was further determined by this method especially at the investigation of the enzyme properties. Enzyme activity was assayed by measuring the increment of optical density at 295 mp based on the high absorption co-efficient of fumarate formed. Assay mixture contained 3. ml of M/2 borate buffer containing M/1 of maleate (ph 8.3) and.25 ml of purified enzyme (22 pg as protein). The unit of the enzyme activity when determined by this method is defined as follows. One unit of maleate cis-trans isomerase corresponds to the amount of enzyme which increases.1 of the optical density at 295 mp in 1 min at 28± 1 C. Determination of protein concentration. Protein concentration was determined by the method of Itszaki ) R. F. Itszaki, Anal. Biochem., 9, 41 (1964).

4 72 Y. T AKAMURA, T. T AKAMURA, M. SOEJIMA and T. UEMURA Ultracentrifugal sedimentation. The sedimentation analysis was carried out in a Hitachi model UCA-1 ultracentrifuge. Determination of equilibrium constant. The equilibrium constant was determined by radio-isotopic method as follows. The enzyme was incubated with 1,4-14C-maleate (Daiichi Kagaku Yakuhin Co.) in borate buffer (ph 8.3) at 3 C for 15 min, where the reaction was completely equilibrated. The reaction was terminated by the addition of concentrated hydrochloride. One tenth ml of ali quote of reaction mixture was spotted on filter paper Toyo Roshi No. 51 together with each of fumarate, maleate and malate as carriers. Acidic spots of fumarate, maleate on the paper were detected by spraying bromphenol blue and trimed off, then eluted with 2 ml of 796 ethanol in test tubes. Radioactivity of those acids was estimated with gas flow Geiger-Muller counter. Purification procedures of maleate cis-trans isomerase Step 1. Enzyme extraction. Alcaligenes faecal is IB-14 was grown in 12 liters of the basal medium supplemented with I-1 M of malonate as inducer and 5 x 1-2 M of maleate as carbon source at 3 C with rotary shaking. The cells were harvested by continuous centrifugation at 1, x g, and was washed with 1/5 M phosphate buffer (ph 6.85) containing.5 mm dithiothreitol (DTT) as reducing agent. The paste of the cells (12 gin wet weight) was suspended in an appropriate volume of the same buffer and was subjected to sonic disintegration at 1 kc, for 7 min. The cell debris and undisintegrated cells were removed by centrifugation at 15, X g, for 2 min. The cell-free extract thus obtained was further centrifuged at 15, X g for 12 min. Step 2. Fractionation with ammonium sulfate. To 7 ml of the supernatant solution by ultracentrifugation of the cell-free extract solid ammonium sulfate (22 g) was added to make.5 saturation at ph 6.85, at 2 C. After standing for 2 hr at 2 C the precipitate formed was removed by centrifugation at 15, x g for 2 min. The ammonium sulfate concentration of the supernatant was then increased at.7 saturation by the addition of solid ammonium sulfate (88.5 g) with stirring. After standing for 2 hr at 2 C, the precipitate was collected by centrifugation at 15, x g for 3 min and dissolved in 1/5 M phosphate buffer containing DTT (3 ml, total OD 28 omp= 539, total activity 1.97 x 16 units). Step 3. Gel filtration with Sephadex G-1. Twenty five ml of the above mentioned enzyme solution was subjected to Sephadex G-1 gel filtration. The gel was packed into ii column (3.5 X 115 em) and equilibrated with 1/5 M phosphate buffer containing.5 mm DTT, ph The enzyme solution (total OD 28omp =527, total activity 1.83 X 16 units) was passed through the column and washed with the same buffer. The buffer was allowed to flow at a rate of 3 ml per hour and each 1 ml fraction was collected. Step 1:. DEAE-cellulose column chromatography. The partially purified enzyme solution (6 ml, total OD 2 somp =6, total activity 6.6 X 15 units) obtained at the step 3 was then subjected to DEAE-cellulose column chromatography. The adsorbent was packed into a column (1.6 x 25 em) and equilibrated with 1/5 M phosphate buffer containing.5 mm DTT, ph The enzyme solution was passed through the column and, subsequently the column was washed with 5ml of the buffer of the same constituent. The maleate cis-trans isomerase adsorbed was eluated with a linear gradient increase in concentration of sodium chloride at a flow rate of 15 ml per hour. The reservoir contained 1 ml of the phosphate buffer containing.2 M NaCl, ph 6.85, and the mixing chamber contained 1 ml of 1/5 M phosphate buffer with the same ph. Step 5. DEAE-Sephadex A-5 column chromatography. The dialyzed enzyme solution obtained at the step 4 was subjected to DEAE-Sephadex A-5 column chromatography. The adsorbent was packed into a column (1.6 x 25 em) and equilibrated with 1/5 M phosphate buffer containing.5 mm DTT, ph The enzyme solution was passed through the column and washed with the same buffer. Maleate cis-trans isomerase was eluted with a linear gradient increase in sodium chloride concentration at a flow rate of 2 ml per hour. The reservoir contained 2 ml of 1/5 M phosphate buffer containing.3 M sodium chloride, ph 6.85, and the mixing chamber consisted of 2 ml of the same buffer without sodium chloride. RESULTS A. Purification of maleate cis-trans isomerase 1. Extraction and fractionation with ammonium sulfate More than 95 per cent of the maleate cis-

5 Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate. Part III 721 trans isomerase activity shown in sonicated cell-free extract was recovered from the supernatant obtained by centrifugation at 15, x g for 12 min and thirty per cent of inactive proteins involved in the cell-free extract was removed by this method. This result indicated that maleate cis-trans isomerase was a soluble enzyme of the cell (Step 1). The maleate cis-trans isomerase in the supernatant was considerably purified by salting out with ammonium sulfate (Step 2). The enzyme was fractionally precipitated by the salt of concentrations between.5 and.7 saturation and purified approximately 2.6-fold over the activity of the initial cell-free extract. About fifty per cent of the activity was recovered. 2. Gel filtration with Sephadex G-1 The elution pattern of the enzyme on Sephadex G-1 is shown in Fig. 1. Early major fraction was an inactive protein and almost all of the enzyme activity was found in later minor peak in shoulder. Thus gel filtration was quite effective for purification of maleate cis-trans isomerase of this bacterium and thus the enzyme was purified 13-fold over the activity of the initial cell-free extract (Step 3). Active fractions, tube number 46 to 54, were combined together and dialyzed against 1/5 M phosphate buffer containing.5 mm DTT and subjected to next step. 3. Chromatographic purifications on DEAE-cellulose and DEAE-Sephadex A-5 columns Figure 2 illustrates the elution pattern of the partially purified enzyme on DEAE-cellulose column. The enzyme activity was 25,< e '. '3 'i: 15 c 2 1 1'-- u u "' '" rjl '" 8-4;- 3L I 4 8 Effluent volume (ml) FIG. l. Gel-filtration of Maleate cis-trans Isomerase by Sephadex G-1. On a column of Sephadex G-1, 3.5 x 115 em, equilibrated with a M/5 phosphate buffer solution containing.5 mm DTT, ph 6.85, was charged 25 ml enzyme protein solution and developed 8 ml of the buffer solution at a flow rate of 3 ml per hour. Fractions within the horizontal arrow were combined. e-e Maleate cis-trans isomerase activity - Protein concentration, Emt' Fraction No. FIG. 2. Chromatography of Maleate cis-trans Isomerase on a DEAE-cellulose Column. On a column of DEAE-cellulose, 1.6 X 25 em, equilibrated with M/5 phosphate buffer containing.5 mm DTT, ph 6.85, was charged 6 ml enzyme protein solution. The column was then washed with 5 ml of the buffer solution. Elution of the enzyme was performed by a linear gradient increase in concentration of sodium chloride. The eluate was fractioned in 5 ml. Fractions within the horizontal arrow were combined. Symbols are the same as in Fig. I.

6 722 Y. TAKAMURA, T. TAKAMURA, M. SOEJIMA and T. UEMURA XIO' 13 '" IOJ..:: <3 T' '".3 ;;;: '". UJ U.2.2 <) " U.1 Z (I '" / /, <) i k1 U " <) -3 " - / / / Fraction No. FIG. 3. Chromatography of Maleate cis-trans Isomerase on a DEAE-Sephadex A-5 Column. The enzyme solution, 7 ml, of fractions from No. 15 to 2 in Fig. 2, was charged on a column of DEAE-Sephadex A-5, 1.6 X 25 cm, equilibrated with M/5 phosphate buffer containing.5 mm DTT. The column was then washed with 5ml of the buffer solution. Elution of the enzyme was performed by a linear gradient increase in concentration of sodium chloride. The eluate was fractionated in 5 mt. Fractions within the horizontal arrow were combined. Symbols are the same as in Fig. 1. found in the shoulder of the second peak and the enzyme was purified about 52-fold over the activity of the initial cell-free extract, corresponding to 4-fold that of the sample charged on this column. Recovery of the enzyme activity was 15 per cent, whereas that of protein was 37 per cent. That indicates that FIG. 4. Ultracentrifugal Pattern of Purified Maleate cis-trans Isomerase. The sedimentation run was operated at 6, rpm at 15 C. The enzyme concentration was 9.9 mg per ml of M/5 phosphate buffer containing.5 mm DTT, ph The photograph was taken at 9 min after reaching the full speed at angle 7 more than 6 per cent of protein was not eluted at the concentration of.2m NaCl. Active fractions, number 15 to 2, were combined and dialyzed. The dialyzed solution was concentrated by collodion bag at O C, and then subjected to rechromatography of DEAE-cellulose column under the same conditions. More than fifty per cent of protein of the sample was removed as inactive protein by rechromatography on DEAE-cellulose column and the enzyme was purified 95-fold over the activity of the initial cell-free extract (Step 4). Figure 3 illustrates the elution pattern of the enzyme on DEAE-Sephadex A-5 column TABLE 1. SUMMARY OF PURIFICATION PROCEDURE Fractions Specific Total Recovery Protein Purificaactivit. unit activo unit Recovery OD28omp tion Original enzyme solution x 16 1:'6 29,4 1% I 15,xg Supernatant x , (NH,hSO, 57"" 347 I.97 x , Sephadex G-1 gel-filtration 1, x DEAE-cellulose column chromatography (First) 7, 5.65 X (Second) 12,7 2.32x DEAE-Sephadex A-5 column chromatography 12,9 1.74x

7 Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate. Part III 723 chromatography. A symmetrical protein peak with constant specific activities around 13, units of maleate cis-trans isomerase appeared in eluate at.2 M concentration of sodium chloride and the enzyme activity went parallel to the protein concentration. The enzyme was purified about 97-fold over the activity of initial cell-free extract (Step 5). Ultracentrifugal analysis confirmed the enzyme preparation to be monodisperse (s 2 o,w= 3.84) (Fig. 4). In Table I is given the summary of purification procedure of the maleate cis-trans isomerase induced by malonate, together with specific activity and recovery yield of activity at each step of the procedure. B. Properties rif the purified maleate cis-trans zsomerase I. Effect rif ph on activity and stability rif the enzyme Figure 5 illustrates the effect of ph on the maleate cis-trans isomerase activity. Optimal activity was obtained at ph 8.3. To check ph-stability the enzyme was diluted with 1/5 M buffer solutions of various ph values ph FIG. 5. Effect of ph on Maleate cis-trans Isomerase Activity. The enzyme activity was assayed for M/1 substrate in M/2 buffers as follows; phosphate-borate buffer, from ph 6. to 8. 7; borate-carbonate buffer, from ph 9. to 9.6. i: :E 1 "' 5 tj:.s "' E e::: "' Preincubation ph FIG. 6. Stability-pH Curves of Maleate cis-trans Isomerase. Maleate cis-trans isomerase was preincubated in various buffer solutions as follows at 3 C; acetate buffer, from ph 4. to 6. (6-6); phosphate buffer, from ph 6. to 8. (e-e); borate buffer, from ph 8. to 1. (-Q). Preincubation for 1 min ---- Preincubation for 5 hr contammg 1 mm 2-mercaptoethanol, and incubated at 3oC for 1 min to 5 hr. As shown in Fig. 6, the enzyme was considerably unstable, since a significant decrease in the activity was found at ph values below 5 and above 7 for 1 min incubation. Especially, at alkaline region the decrease of activity was remarkable and more than 8 per cent of the activity was lost by incubating the enzyme at ph 1 for 5 hr. The enzyme was the most stable in acetate buffer at ph 6, while less stable in phosphate buffer at the same ph value. 2. Effect of temperature on the stability The purified enzyme was incubated in 1/5 M acetate buffer (ph 6.) at various temperature for 15 min and the remaining activity was measured. As shown in Fig. 7, the maleate cis-trans isomerase was stable below 3 C and almost lost its activity above 5 C for 15 min. 1

8 724 Y. TAKAMURA, T. TAKAMURA, M. SOEJIMA and T. UEMURA 1 ooe Temperature ("C) FIG. 7. Effect of Temperature on the Activity of Maleate cis-trans Isomerase. Maleate cis-trans isomerase was preincubated in M/5 phosphate buffer containing.5 mm of DTT, ph 6.2, at various temperature as indicated. The remaining activities were assayed by the method described in text. 3. Effect of various metal ions and inhibitors on the activity Effects of various metal ions and inhibitors on the enzyme activity were examined. Presence of w- M of Hg 2 + and Cu 27 inhibited the enzyme activity, while at Li+, Zn2+, Co 2 +, Fe 2 +, Ni 2 +, Mn 2 + and Mg 2 + were not inhibitory (Table II). TABLE II. EFFECTS OF VARIOUS METAL IONS ON THE ACTIVITY OF MALEATE cis-trans ISOMERASE Metal ions Concentra- Relative Inhibition added tion (M) activity (O;j) (;:?i)) None 1 Hg Cu 2 +!O Li Zn2+!-3 1 Co Mn2+!-3 13 FeZ Fe Ni Mg2+!-3 15 All reactions were carried out in borate buffer at ph 9.. TABLE III. EFFECT OF VARIOUS INHIBITORS ON THE ACTIVITY OF MALEATE cis-trans ISOMERASE Inhibitors Concentra- Relative Inhibition added tion (M) activity (5') (96) None 1 KCN EDTA IAA NBS Semicarbazide Nai PCMB Maleate cis-trans isomerase was preincubated with and without inhibitor in M/2 borate buffer, ph 8.3 at room temperature for 1 min and then remaining activities were assayed by the method as described in the text. Table III summarizes effects of metal chelating agent and sulfhydryl reagents on the enzyme act1v1ty. Sulfhydryl reagents such as p-chlor-mercuribenzoate (PCMB) and monoiodo acetate (IAA) at a concentration.5 2 1'<1.4 = "' Ul ( 3) "'... "' u c. " t' ( 4) u "' E "' " Reaction time (min) FIG. 8. Reversal of Inhibition of Hg2+ Ion by Sulfhydryl Compounds. Enzyme reaction was carried out in the presence (-) and absence (x-x) of 1-4M of Hg2+ ion, at ph 8.3. After 3 min incubation, 1-3M of sulfhydryl compounds were added to the reactions mixture as indicated by an arrow; (I) DTT, (2) 2-mercaptoethanol, (3) L-cysteine, (4) glutathione, (5) sodium thioglycolate.

9 Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate. Part III 725 of 1-3 M inhibited the enzyme at preincubation the cell growth. Then, the effect on the for 1 min at room temperature. Oxidiz ing agent such as sodium periodate also inhibited enzyme activity of various organic acids and some other compounds which were closely the enzyme. On the other hand, related to the metabolism of maleate was w-a M of EDT A did not inhibit the enzyme examined. activity. Thus, it was mentioned that maleate Table IV illustrated that any of compounds cis-trans isomerase did not require metal ion tested was not effective on the enzyme activity and neither increase nor decrease in for its activity and that it had a sulfhydryl group(s) for the active site which was susceptible to oxidation. revealed that malonate was not an activator enzyme activity was observed. Thus, it was The inhibition of the enzyme by PCMB or of the enzyme but an inducer to provoke the Hg 2 + was completely recovered by the addition of DTT, 2-mercaptoethanol, L-cysteine formation of the enzyme. or glutathione. As shown in Fig. 8, when 5. Co-factor requirement of the enzyme the enzyme solution was incubated with 1-4 M Otsuka 4 of Hg 2 ) reported that partially purified enzyme isolated from Pseudomonas sp. was in +, cis-trans isomerization of maleate did not proceed at all. After three minutes, activated by dialysis against M phosphate M of each one of DTT, 2-mercaptoethanol, buffer (ph 7.3) for one hour at ooc and that glutathione and L-cysteine was added to the the dialyzed enzyme was reactivated by the reaction mixture, then immediately the isomerization commenced. Among those reduc This finding led him to a tentative conclusion addition of either L-cysteine or glutathione. ing agents, DTT was the most effective, while that the maleate cis-trans isomerase in Pseudomonas sp. requires either L-cysteine or gluta sodium thioglycolate showed no activation. thione as co-factor for its activity. Then the 4. Effect cif various organic acids on the activity co-factor requirement of this enzyme was As reported previously, the formation of further inquired with the purified preparation maleate cis-trans isomerase in A. faecalis was isolated from A. faecalis. induced by malonate, whereas it was repressed by various dicarboxylic acids such as mg as protein) was dialyzed against 1 ml Two ml of the purified enzyme solution (3 malate, oxalacetate, fumarate and succinate of lj5m acetate buffer (ph 6.) containing which were rapidly metabolized to serve for.2 M of 2-mercaptoethanol for 24 hr with stirring at SOC and the remaining activity TABLE IV. EFFECT OF V ARlO US CARBOXYLIC was measured. ACIDS ON THE ACTIVITY OF MALEATE As shown in Fig. 9, the rate of cis-trans cis-trans ISOMERASE isomerization of maleate shown by the dialyzed Relative Inhibition Compounds added activity coal enzyme was approximately the same as that None 1 by non-dialyzed preparation. However, when Fumarate 97 3 the enzyme was dialyzed against buffer without Malate 1 any addition of the reducing agents such Succinate 1 as 2-mercaptoethanol, DTT or L-cysteine, the Malonate 1 rate of isomerization of maleate was remarkably reduced. This result indicated that the Citrate 1 L-A sparta te 1 enzyme, because of its SH-nature, was susceptible to oxidation and that considerable The enzyme activity was assayed for M/1 substrate in M/2 borate buffer at ph 8.3. Carboxylic acids were added at a concentration of 1-3M. amount of the enzyme was inactivated during the process of dialysis. Thus, maleate cis-

10 726 Y. TAKAMURA, T. TAKAMURA, M. SOEJIMA and T. UEMURA E ' >. :>": E o ;;: '"1.3 -e =.2 "' '" '" Ei """ ;:;... '" " u Reaction time (min) FIG. 9. Effect of Dialysis on the Activity of Maleate cis-trans Isomerase. Two ml of the purified enzyme solution (3 mg as protein) was dialyzed against 1 ml of M/5 acetate buffer (ph 6.) containing M/5 of 2 mercapto ethanol for 24 hr with stirring at 5 C. e-e Dialysed enzyme. - Control. trans isomerase of A. jaecalis induced by malonate does not require any co-factor for its function, although reducing agents such as DTT, 2-mercaptoethanol, L-cysteine and glutathione etc., are effective to activate sulfhydryl group of the enzyme which is involved in an active site of this enzyme. 6. Substrate specificity As shown in Table V, significant change of optical density at 295 m,u was observed only with maleate as substrate. Methylmaleate TABLE V. SUBSTRATE SPECIFICITY OF MALEATE cis-trans ISOMERASE Substrates Maleate Fumarate Methylmaleate Methyl fumarate L-Malate D-Malate Changes in Eml' per minute, x 1-s 97 The enzyme activity was assayed for M/1 substrate in M/2 borate buffer, ph 8.3 at 295 mp based on a change of absorption co-efficient of product. (citraconate) and methylfumarate (mesaconate) were not isomerized at all. Since the reduction of optical density at 295 m,u was not observed when the purified enzyme preparation was incubated with maleate or fumarate, it is evident that both malease 51 and fumarase were not contaminated in this preparation. 7. Kinetics The effect of substrate concentration on the reaction rate is studied. The Michaelis constant Km, as calculated by the method of Lineweaver and Burk/ 21 was 2.8x w-a M, where velocity (v) was expressed as increment of optical density at 295 m,u per minute and concentration(s) of maleate as substrate as mole per liter (Fig. 1). Figure 11 shows a typical time course of the maleate cis-trans isomerase reaction which was carried out at 3 C, starting from maleate. The curve represents the time course of formation of fumarate expressed as increase of Emp based on high absorption co-efficient of fumarate. It is seen that the equilibrium 4 ' 3 X '"' :::. 1 Km 2.78 X1-3 M /S FIG. 1. Lineweaver-Burke's Plot of the Effect of Substrate Concentraion on Maleate cis-trans Isomerase. The enzyme activity was assayed by the method described in text. Velocity (v) was expressed as increment of Emp per minute at 3 C, ph 8.3, and concentration (S) of maleate as substrate as mole per liter. 12) Lineweaver and D. Burk, J. Am. Chern. Soc., 56, 658 (1934).

11 Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate. Part III 727 ''t I.$.4 "' "' H " "'.3 ".2 t Enzyme Maleate initially provided was almost converted to radioactive fumarate after 15 min incubation, where the residual radioactivity of maleate was within a limit of experimental erra. Thus, at the equilibria! point there may be less than 2% maleic acid and more than 98% fumaric acid. This result was consistent with K determined by Davis and Evans 13 ' from thermodinamic parameters Reaction time (min) FIG. 11. Time Course of Maleate cis-trans Isomerase Reaction and Equilibrium. The reaction mixture contained 3. ml of M/1 maleate in borate buffer, ph 8.3 and.25 ml of purified enzyme (22 pg as protein). After reaching plateau of the reaction,.1 ml of purified enzyme and.5 ml of substrate were further added to the reaction mixture as arrows indicated. TABLE VI. ISOMERIZATION OF 14 C-MALEATE TO FUMARATE BY PURIFIED ENZYME Reaction Incubation Radioactivity systems time (min) Maleate Fumarate Malate Complete 9167 cpm 222 cpm 61 cpm Complete enzyme The complete system contained 3.ml of M/1 14C-ma1eate together with carrier in M/2 borate buffer, ph 8.3 and.25 ml of purified enzyme (22 pg as protein). For experimental conditions see the text. is virtually reached at 15 min incubation, since isomerization did not proceed by further addition of fresh enzyme. The equilibrium concentration of maleate and fumarate was obtained from the concentration at this time. The concentration of fumarate formed was determined by both photometric and radioisotopic methods. It was revealed by photometric method that maleate was almost completely isomerized to fumarate and the reverse reaction of isomerization from fumarate to maleate was not observed. Radio-isotopic experiment also confirmed this conclusion. As shown m Table VI, radio-active maleate DISCUSSION Cis-trans isomerization is an intramolecular rearrangement reaction and thus no change in empirical formula occurs. The product is usually the thermodynamically more stable isomer, i.e., trans form. Such a type of reaction does not occur so frequently in biological systems. In the references so far concerned, four enzymes which catalyze cis-trans isomerization reaction have been known in biological systems. 4 ' Of these enzymes, maleate cis-trans isomerase is the typical one in view of the fact that maleate and fumarate are the simplest geometric isomers of carboncarbon double bond. Previously, the inducibility of maleate cistrans isomerase in A. faecalis was studied and the formation of this enzyme was found to be remarkably promoted by malonate. Therefore, the cultivation of cells in the medium containing malonate as inducer was essentially adequate for the purification of the enzyme because of the pronounced elevation of the cell enzyme level. In this case, 1-1 M malonate was added to the medium together with 5 X 1-2 M maleate, since the latter promoted the cell growth without prevention of the enzyme formation ) M. Davies and F. P. Evans, Trans. Faraday Soc., 51, 156 (1956). 14) S. W. Edwards and W. E. Knox,]. Biol. Chern., 22, 79 (1956). 15) R. Hubbard, J. Gen. Physiol., 39, 935 (1956). 16) S. Sugiyama, K. Yano, H. Tanabe, K. Komagata and K. Arima, ]. Gen. Appl. Microbial., 4, 223 (1958).

12 728 Y. TAKAMURA, T. TAKAMURA, M. SOEJIMA and T. UEMURA Otsuka 41 first isolated the enzyme from Pseudomonas sp. grown on the medium containing.1% maleic acid as carbon source, but the enzyme level of the cell was very low. Although maleate is the intrinsic inducer of this enzyme, its inducing activity is less than that of malonate. 91 Indeed, in A. faecalis,.1% (i.e. ca. l.2xl- 2 M) of maleate was a concentration to induce poor formation of maleate cis-trans isomerase. At the concentration of 5 X 1-2 M, less than one twentieth of the enzyme activity of cells induced with malonate was observed in cells induced with maleate. Thus, it is well mentioned that the induction of the enzyme by malonate in place of maleate effectively favoured to obtain its purified preparation. Consistently with the result obtained by Otsuka, 41 maleate cis-trans isomerase is very unstable since it possesses sulfhydryl group(s) which is susceptible to oxidation. Reducing agents such as DTT, 2-mercaptoethanol, L cysteine and glutathione were thus indispensable for both activation and protection of the enzyme. Of these reducing agents, DTT was the most effective, and accordingly phosphate buffer containing.5 mm of DTT was used throughout this experiment. In the absence of DTT or 2-mercaptoethanol the enzyme activity was remarkably reduced by sonic oscillation and dialysis etc. In view of the fact that inactivation of the enzyme by Hg 2 + or PCMB was reversed by the addition of higher concentration of DTT, 2-mercaptoethanol and that the purified enzyme dialyzed against phosphate buffer containing DTT or 2-mercaptoethanol showed approximately the same activity as that of non-dialyzed enzyme, it was mentioned that L-cysteine and glutathione which were assumed by Otsuka as an essential co-factor are a simple activator for the enzyme, playing the similar role to that of such sulfhydryl compounds as DTT and 2-mercaptoethanol. However, the aged preparation of this enzyme seemed to receive a irreversible damage, so that it could not be restored by incubation with sulfhydryl compounds. From the elution pattern on Sephadex gel filtrations and Svedberg value (3.84 S) obtained by sedimentation analysis, the molecular weight of the maleate cis-trans isomerase might be assumed to be less than one hundred thousand. Acknowledgements. The authors wish to express their thanks to Assistant Prof. Dr. K. Aida of Institute of Applied Microbiology, the University of Tokyo, for his kind guidance and encouragement during the course of this investigation. They are also grateful to Mr. T. Sai for his helpful suggestions. Thanks are also due to Mr. T. Ando for technical assistance. This work was supported by a research grant of the Ministry of Education, Japan.