ON THE MECHANISM OF AN ANAEROBIC EXCHANGE REACTION CATALYZED BY SUCCINIC DEHYDROGENASE PREPARATIONS* BY SASHA ENGLARDi AND SIDNEY P. COLOWICK (From the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland) (Received for publication, January 5, 1956) Attempts in this laboratory to study with deuterium the possible mech- anism of an anaerobic hydrogen transfer reaction from reduced diphospho- pyridine nucleotide to fumarate, as catalyzed by heart particle preparations (l), necessitated an investigation of the possibility that these prepara- tions, which contain succinic dehydrogenase, also catalyzed an anaerobic exchange reaction described by Weinmann et al. (2). These workers ob- served that, when dideuteriosuccinate was incubated anaerobically with a rabbit kidney succinic dehydrogenase preparation, there resulted a signif- icant exchange of the hydrogen atoms of the medium with the deuterium atoms of the dideuteriosuccinate. They also noted that this rate of ex- change, although not influenced by the presence of methylene blue, was roughly equivalent to the rate of succinate oxidation in the presence of a suitable acceptor such as methylene blue (MB). This correspondence be- tween the rates of anaerobic exchange and oxidation was interpreted as lending support to the hypothesis that the same enzyme, namely succinic dehydrogenase, catalyzed both of the following reactions: (1) oxidation of succinate and (2) hydrogen exchange. (1) HOOC-CHT-CHz-COOH + MB + HOOC-CH=CH-COOH + MBHz HOOC-CH-CHCOOH + 2H+ --+ HOOC-CH-CH-COOH + 2D+ (2) I I I I D D H H The results presented in this paper show that, when normal succinate was incubated anaerobically with a heart particle preparation in a medium containing DzO, the exchange reaction was exceedingly slow compared with the rate of aerobic oxidation. The addition of fumarate greatly enhanced * Contribution No. 145 of the McCollum-Pratt Institute. Aided by a grant from the American Cancer Society, as recommended by the Committee on Growth of the National Research Council. t Postdoctorate Research Fellow of the American Heart Association, Inc. Pres- ent address, Department of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, New York 61, New York. 1019 by guest on April 27, 2019 http://www.jbc.org/ Downloaded from
Transcript
ON THE MECHANISM OF AN ANAEROBIC EXCHANGE REACTION CATALYZED BY SUCCINIC
DEHYDROGENASE PREPARATIONS*
BY SASHA ENGLARDi AND SIDNEY P. COLOWICK
(From the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland)
(Received for publication, January 5, 1956)
Attempts in this laboratory to study with deuterium the possible mech- anism of an anaerobic hydrogen transfer reaction from reduced diphospho- pyridine nucleotide to fumarate, as catalyzed by heart particle preparations (l), necessitated an investigation of the possibility that these prepara- tions, which contain succinic dehydrogenase, also catalyzed an anaerobic exchange reaction described by Weinmann et al. (2). These workers ob- served that, when dideuteriosuccinate was incubated anaerobically with a rabbit kidney succinic dehydrogenase preparation, there resulted a signif- icant exchange of the hydrogen atoms of the medium with the deuterium atoms of the dideuteriosuccinate. They also noted that this rate of ex- change, although not influenced by the presence of methylene blue, was roughly equivalent to the rate of succinate oxidation in the presence of a suitable acceptor such as methylene blue (MB). This correspondence be- tween the rates of anaerobic exchange and oxidation was interpreted as lending support to the hypothesis that the same enzyme, namely succinic dehydrogenase, catalyzed both of the following reactions: (1) oxidation of succinate and (2) hydrogen exchange.
(1) HOOC-CHT-CHz-COOH + MB +
HOOC-CH=CH-COOH + MBHz
HOOC-CH-CHCOOH + 2H+ --+ HOOC-CH-CH-COOH + 2D+ (2) I I I I
D D H H
The results presented in this paper show that, when normal succinate was incubated anaerobically with a heart particle preparation in a medium containing DzO, the exchange reaction was exceedingly slow compared with the rate of aerobic oxidation. The addition of fumarate greatly enhanced
* Contribution No. 145 of the McCollum-Pratt Institute. Aided by a grant from the American Cancer Society, as recommended by the Committee on Growth of the National Research Council.
t Postdoctorate Research Fellow of the American Heart Association, Inc. Pres- ent address, Department of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, New York 61, New York.
the rate of deuterium incorporation into succinate under anaerobic condi- tions. Thus the exchange reaction studied here appears to be not a result of an increased rate of dissociation of the C-H bonds of succinate as sug- gested in Equation 2, but rather a reflection of the oxidation of succinate
TABLE I
Anaerobic Deuterium Incorporation into Succinate, Fumarate, and Malate -
*Each series represents one enzyme preparation with which all the experiments were carried out simultaneously.
t See the text, p. 1022. $ Malates in Series I and II isolated as cinchonine salt and in Series III as diphen-
acyl ester. 8 Experimental values. jj Values corrected for dilution and calculated on the basis that 1 atom of deuter-
ium per molecule of succinic acid, fumaric acid, cinchonine malate, and diphenacyl malate would correspond to a value of 16.7,25.0,3.57, and 5.55 atoms per cent excess, respectively.
7 Added as carrier at the end of the experiment. 1 mmole each for succinate and fumarate and 0.5 mmole for malate.
** Boiled enzyme.
(Equation 1) by a natural carrier in such a manner that the hydrogens of succinate are directly or indirectly donated to the medium (see “Discus- sion,” Equations 5 and 6). This possibility was also considered by Wein- mann et al. (2) and earlier by Erlenmeyer et al. (3). According to this view, the addition of unlabeled fumarate promotes the back-reaction by which the deuterium from the heavy water medium becomes incorporated into the succinate. This effect of fumarate might conceivably have in-
volved a prior incorporation of deuterium into fumarate if the fumarase present in our preparations had been non-stereospecific with respect to the methylene hydrogens of I-malate. However, experiments are reported herein which demonstrate the stereospecific behavior of fumarase, in agree- ment with similar results obtained by Fisher et al. (4).
On the assumption that the anaerobic exchange reaction is catalyzed by a single enzyme and that this enzyme is involved in the aerobic oxidation of succinate to fumarate, the data further suggest that all four methylene hydrogens of succinate were labeled as a result of the anaerobic exchange reaction. This result will be considered in terms of the stereospecificity of succinic dehydrogenase. A preliminary account of this work has appeared (5).
Methods
Enzyme Preparations, Assays, and Anaerobic Incubations-The heart muscle preparations were isolated from horse heart according to the method of Iieilin and Hartree, as described by Slater (6). The anaerobic reac- tion mixtures contained 2.44 mmoles of potassium phosphate buffer, pH 7.29, 11.0 mg. of cytochrome c, 1.0 ml. of enzyme preparation, and 1 mmole of sodium succinate or potassium fumarate or 1 mmole each of succinate and fumarate, in a total volume of 16.5 ml. at the indicated per cent D,O concentration by vo1ume.l The succinate was tipped from the side arm of a large Thunberg tube after flushing the remainder of the reaction mixture in the main compartment with nitrogen gas and evacuating the tubes for 5 to 8 minutes with an oil pump. Incubations were carried out in a water bath at 37”. The corresponding rates of succinate oxidation were deter- mined by following manometrically the oxygen uptake under aerobic con- ditions identical otherwise with those prevailing in the experimental an- aerobic incubations. These rates are expressed (Table I) as per cent of the total succinate added which could be oxidized in the experimental period. Fumarase activity was determined under aerobic conditions by measuring the rate of loss in absorption at 240 rnp in a Beckman DU quartz spectrophotometer (7), and was checked by measurements of total malate isolated at the end of the anaerobic reactions.
Separation, Isolation, Dilution, and Purijkation of Acids-Reactions were stopped at the indicated time by acidification with concentrated HzS04 to give a final concentration of approximately 2 N HzSOb. After filtration and washing the precipitate with 2.5 ml. of HzO, the combined superna- tant fluids were generally mixed with two weights of ether-washed Johns- Manville Celite No. 535. The acids were subsequently recovered by con- tinuous ether extraction for 16 to 20 hours. After removal of the ether
1 The heavy water used in these experiments was obtained on allocation from the United States Atomic Energy Commission.
under a vacuum in the presence of 5 ml. of water, the aqueous solution was adjusted to pH 8.0 to 8.5 with KOH and passed through Dowex 1-formate columns, and the acids were separated by a slight modification of the method described by Busch et al. (8): Subsequent to desiccation, the fractions representing individual peaks were combined by dissolving in HzO. The identity of each peak was determined by its position of emer- gence from the Dowex 1-formate columns (titration with NaOH) and by ascending paper chromatography in the butanol-propionic acid-Hz0 sol- vent described by Benson et al. (9), and then spraying the dried paper with bromophenol blue (10). Prior to isotope dilution by addition of carrier, each acid was quantitatively determined as presently described. Succinate was determined manometrically by measuring the total oxygen uptake upon incubation with the succinic oxidase preparation from heart. Fu- marate was measured by determining the optical density at 240 mp in the Beckman spectrophotometer (7). Malate was determined fluorometri- tally by the method of Speck as described by Loewus et al. (11). After appropriate isotope dilutions with the respective acids, succinate and fu- marate were isolated as the free acids and further purified by vacuum sub- limation. Malate in some experiments was isolated as the cinchonine salt (12, 13), which was recrystallized once from methanol-acetone and finally from water-acetone. In other experiments malate was isolated as the diphenacyl ester (11) and recrystallized from benzene-petroleum ether.
Deuterium Analyses-These were carried out by combustion of the sam- ples to water, and reduction of the water over zinc dust to Hz plus HD. The deuterium content of the gas was then determined by mass analysis.
Results
Influence of Fumarate on Anaerobic Deuterium Incorporation into Suc- cinate-In contrast to the previous findings of Weinmann et al. (2), the present preparations, when incubated anaerobically with succinate in a medium containing DzO, exhibited an extremely low rate of incorporation of deuterium into succinate compared to the corresponding rate of aerobic oxidation (Table I, Experiment 1 of Series I, II, and III). As indicated in Table I (Series I, Experiments 1 and 3, Series II, Experiments 1 and 4, and Series III, Experiments 1 and 4), the addition of fumarate, although
2 This modification involved the initial elution of succinate and malate in one fraction with 90 to 100 ml. of 2.5 N formic acid. Changing to 5 N formic acid resulted in the almost, immediate elution of fumarate as a distinct peak, followed by the emergence of inorganic phosphate. The 2.5 N formic acid eluates containing both succinate and malate were lyophilized, dissolved in water, neutralized to pH 8.0 to 8.5 with KOH, and again passed through Dowex l-formate columns. Succinate and malate were then separated by elution with continually increasing formic acid con- centrations as described by Busch et al. (8).
occasionally inhibiting aerobic succinate oxidation, invariably greatly stimulated the incorporation of deuterium into succinate under anaerobic conditions.
The effective stimulation of deuterium incorporation into succinate by fumarate, if interpreted in terms of a simple reversal of the classical suc- cinic dehydrogenase reaction, could mean that the deuterium appearing in the succinate either arose directly from the medium in the course of reduc- tion of unlabeled fumarate or arose from a reduction of previously labeled fumarate. The latter possibility was ruled out by the observations de- scribed in the following section.
Xtereospecijic Behavior of Fumarase-The succinic dehydrogenase prep- arations used throughout this study contained sufficient fumarase to estab- lish rapidly the fumarate-malate equilibrium. Such an equilibration, if catalyzed in the non-stereospecific manner described by Equation 3, would yield labeled fumarate. Hence, the finding of deuterium in succinate and the stimulation of this deuterium incorporation by fumarate might have at least partially reflected a prior incorporation of deuterium into fumarate by a non-stereospecific behavior of fumarase.
COOH COOH COOH
I I I (3) “i
+DzO HC-OD
-HOD , I - iH
CH DCH DC
I COOH
I tiOOH
I COOH
That such was not the case can be noted from an experiment in which fu- marate alone was incubated with the enzyme preparation under conditions identical to those in which the exchange reaction was determined (Table I, Series II, Experiment 5). Thus, while the malate exhibited an extremely high deuterium content, as anticipated, the fumarate was exceedingly low in its content of deuterium. The reaction proceeded long after the fu- marase equilibrium had been reached,3 so that there was ample opportunity for the fumarate to become more highly labeled if the fumarase behaved in a non-stereospecific manner, according to Equation 3. Similarly, in the experiments designed to test the influence of fumarate on the extent of deuterium incorporation into succinate (Table I, Series I, Experiments 2 and 3, Series II, Experiments 2, 3, and 4), while the succinate exhibited in- creased deuterium incorporation with time, the deuterium content of fu- marate, if significant at all, was extremely low, and the deuterium content of malate remained high and essentially constant, at close to the theoretical
8 The half time for reaching fumarase equilibrium was approximately 2 hours with the enzyme preparation used in Series II and 44 hours with the enzyme preparation used in Series III.
values of 0.82 and 0.86 atom of deuterium per molecule, respectively (as- suming no isotope effect). These results thus tend to strengthen the belief that the deuterium incorporated anaerobically into succinate in the absence or presence of fumarate arises directly from the medium and not from labeled fumarate.
The finding that fumarase in its back-reaction behaves with strict speci- ficity in removing the identical molecule of Hz0 from E-malate as was incorporated into fumarate in its forward reaction, is in accordance with Equation 4, and in agreement with similar results obtained by Fisher et al. (4) with crystalline fumarase.
COOH COOH I I
HC-OD (4) “1
+DnO ) I
CH ’ -DzO D-CH I I
COOH COOH
VariabiEity of Enzyme Preparations in Extent of Deuterium Incorporation into Fumarate-The previous conclusions regarding the stereospecific be- havior of fumarase and the probability that the deuterium incorporated into succinate during the anaerobic exchange reaction arises directly from the medium are based on the low deuterium values recorded for fumarate (Table I, Series I and II). In other experiments, however, a higher deu- terium incorporation into fumarate was observed. Thus, when fumarate was permitted to undergo the fumarase-catalyzed equilibrium in a medium enriched with DzO (Table I, Series III, Experiment 5), the deuterium content of the reisolated remaining fumarate was significantly increased over that of a similar experiment recorded for a different enzyme prepara- tion (Table I, Series II, Experiment 5). Moreover, in contrast to the previously discussed experiments in Table I (Series I and II), this same preparation (Series III), as well as that used for the experiment recorded in Table I, Series IV, when incubated anaerobically with succinate in the presence of fumarate, led to even higher deuterium incorporation into fumarate. The increase in deuterium levels of succinate, with time, was accompanied by similar high increases of deuterium incorporation into fumarate and a concomitant increase of malate labeling (due to malate originating from labeled fumarate), eventually exceeding the theoretical value of 0.86 atom of deuterium per molecule for malate derived from un- labeled fumarate (Table I, Series III, Experiments 2, 3, and 4). Although the reason for the differences in behavior of the various preparations made in practically identical fashion is not clear at the present time, it should be noted that certain correlations, to be discussed later, exist between the
oxidizing activities of the various preparations and their activities with respect to deuterium incorporation into both succinate and fumarate.
Deuterium Distribution in Enzymatically Produced Deuteriosuccinate- The original postulate of Ogston (14) that citrate as an enzyme substrate complex may behave as an asymmetrical molecule was subsequently sup- ported by the experiments of Potter and Heidelberger (15) and Lorber et al. (16). Recent studies on reactions involving hydrogen transfers to and from pyridine nucleotides have shown that these reactions are sterically specific with respect to the pyridine nucleotide (11, 17-20) and with re- spect to ethanol as well, when studied in the case of alcohol dehydro- genase (21). On the assumption that the exchange observed in the present experiments is catalyzed by a single enzyme and that the same enzyme is concerned in the aerobic oxidation of succinate, the occurrence of a similar sterically specific behavior for succinic dehydrogenase could be tested. Thus, if the exchange reaction resulted in succinate labeled specifically in two of its four methylene hydrogen positions, its oxidation by the same enzyme should yield essentially unlabeled fumarate. Such an experiment, in which deuteriosuccinate obtained by enzymatic exchange of deuterium into normal succinate in the presence of fumarate was enzymatically oxi- dized to fumarate, is recorded in Table II, and clearly indicates that the resulting fumarate retained (per molecule) about half the deuterium orig- inally present in the succinate. These findings, therefore, lend themselves to the interpretation that during the course of the anaerobic exchange reaction all four methylene hydrogens of succinate became labeled. The apparent discrepancy between the deuterium content of succinate before and after 50 per cent oxidation is probably due to an isotope effect which causes preferential oxidation of the unlabeled succinate molecules (22) lead- ing to a higher apparent deuterium content of the residual succinate and a somewhat lower than theoretical content of the resulting fumarate. It may be pointed out that identical results were observed with the deuterio- succinate obtained from experiments in which the respective fumarate pools were unlabeled or labeled (Table II, Experiments 1 and 2, respec- tively) .
Use of Enzymatically Formed Deuteriosuccinate As Substrate for Anaerobic Exchange Reaction-In order to duplicate more closely the experimental procedure followed by Weinmann et al. (2), deuteriosuccinate samples iso- lated from various experiments listed in Table I were incubated anaero- bically with the heart particle preparations in a normal water medium with or without added fumarate. The results are outlined in Table III.
The main point to note is the relatively small decrease in the deuterium content of the deuteriosuccinate, even when incubated with fumarate. While this finding might at first inspection suggest a discrepancy with the
experiments reported in Table I, on closer examination it becomes clear that the difference is due to a difference in sensitivity of the two methods employed for the detection of exchange. This can be illustrated by a con- sideration of the following figures. The enzyme preparations in Experi- ments 2 and 3, Table III, when used for similar time periods in experiments with normal succinate and heavy water (Table I), showed about 0.6 per cent of maximal theoretical exchange without fumarate and about 9 per
TABLE II
Enzymatic Oxidation of Enzymatically Labeled Deuteriosuccinate
I Deuterium content
Acid isolated and conditions I Experiment 1 Experiment 2
Deuteriosuecinate obtained from anaerobic exchange re- action.......................
Succinate after approximately 50% enzymatic oxidation of above deuteriosuccinate
Fumarate resulting from above oxidation of deuteriosucci- nate.........................
No dilu- tion
2.1
3.5
Atom Atom per Dilution y$;$ moleculet factors
-___-
Atom per cent excess*
0.502 0.0301 4.1 0.402
0.341 0.0430 4.4
0.098 0.0137 5.0
0.505
0.222
Atom per m&c&t
0.0989
0.133
0.0444
Experiment 1, n-succinate from Experiment 3, Series I, Table I, oxidized with the enzyme from Series IV; Experiment 2, n-succinate from Series IV, Table I, oxidized with the enzyme from Series III.
* Experimental values. t Values corrected for dilution and calculated on the basis that 1 atom of deuter-
ium per molecule of succinic acid and fumaric acid would correspond to a value of 16.7 and 25.0 atoms per cent excess, respectively.
cent with fumarate. If a similar degree of exchange had occurred, start- ing with deuteriosuccinate in Table III, the exchange would certainly have been undetectable in the experiments without fumarate and would have been just on the border of significance with fumarate. The actual results in Experiments 1 and 3, Table III, are in agreement with this expectation, while those in Experiment 2 show somewhat higher exchange values than would have been expected from the above considerations.
Our inability to observe rapid exchange of deuterium into or out of suc- cinate, when incubated alone anaerobically with the Keilin and Hartree heart particle preparations, is not in agreement with the rapid exchange of
hydrogen into dideuteriosuccinate originally reported by Weinmann et al. (2), who used a rabbit kidney preparation. The discrepancy between these findings and those of the earlier workers might perhaps be due to differences in the nature of the enzyme preparations used. Similar experi- ments, however, with dialyzed and undialyzed supernatant fractions and
TABLE III
Enzymatic Anaerobic Exchange of Enzymatically Labeled Deuteriosuccinate with Normal Water
Treatment of deuteriosuccinate obtained from anaerobic
exchange reaction
None.. ._........._.......
After anaerobic incubation with succinic dehydrog- enase....................
After anaerobic incubation with succinic dehydrog- enase in presence of equivalent concentration of fumarate.............
-
-
--
-
Deuterinm content of succinate
Experiment 1 Experiment 2 Experiment 3
Da&g *;F Atom
Per E E *;zrm Atom c E *;zrm Atom
cent ex- Ill&-
gs cent 22 ex-
,yi- $2 cent Per
cess* de n cess* cl&St a! ,z;*
mole-
n cu1et
~--_-~_-~
No dilu- 0.5020.03014.10.4020.09894.10.3560.0876 tion
2.3 0.2090.02885.00.2650.07952.90.5030.0876
5 40.2200.07132.90.4400.0765 I
Experiment 1, n-succinate from Experiment 3, Series I, Table I, incubated anaerobically for 16 hours with the enzyme preparation from Series IV; Experiment 2, n-succinate from Experiment 1, Series IV, Table I, incubated anaerobically for 10 hours with the enzyme preparation from Series III; Experiment 3, n-succinate from Experiments 2, 3, and 4, Series III (pooled), incubated anaerobically for 4 hours with the enzyme preparation from Series II.
* Experimental values. t Values corrected for dilution and calculated on the basis that 1 atom of deuter-
ium per molecule of succinic acid corresponds to a value of 16.7 atoms per cent excess.
washed residues from rabbit kidney homogenates prepared according to Colowick et aZ. (23, 24), invariably yielded extremely low values of anaero- bic deuterium incorporation into succinate compared to the rate of aerobic succinate oxidation. It should be emphasized that, in the experiments of Weinmann et al. (2), extremely large quantities of enzyme were used and methylene blue reduction rather than oxygen uptake was used as the basis for comparison with the rate of anaerobic exchange. These differences in experimental conditions and procedure may account in part for the ap- parent discrepancies observed.
in which Equation 5 represents succinate as a reductant of a natural car- rier X and Equation 6 represents fumarate as reoxidizing the reduced car- rier X in a medium enriched with D20. Alternatively, the reduction of X would yield XH2, the hydrogens of which would be rapidly exchangeable with the medium DzO. The resulting XDZ would subsequently be re- oxidized by fumarate, yielding X and deuteriosuccinate. According to this scheme, the addition of fumarate would promote the incorporation of deu- terium into succinate by establishing a new equilibrium position in which the rates of both Equations 5 and 6 would be increased.4 The available data do not permit a definitive conclusion as to the involvement of only one enzyme in the catalysis of both Equations 5 and 6. Thus, while Equa- tion 6 in all probability is due to the generally described succinic dehydro- genase, the possibility must be entertained that Equation 6 may be cata- lyzed by an enzyme similar to the fumaric reductase isolated from yeast by Fischer et al. (25-27). However, in such a case, the carrier X would have to be capable of interacting with both enzyme systems. Massey et al. (28) have recently reported evidence supporting the view that a single enzyme is responsible for Equations 5 and 6. Using a highly purified flavo- protein from beef heart, they showed that the enzyme served as both a succinic dehydrogenase and a fumarate reductase, and that the reduced enzyme could be reoxidized by fumarate.
Signijicance of Fumarate Labeling-According to the scheme proposed, the deuterium appearing in the succinate should arise directly from the medium and not from a reduction of labeled fumarate. The results out- lined in Table I, Series I and II, which show that the fumarate remains essentially unlabeled as the deuterium incorporation into the correspond- ing succinate increases with time are, therefore, in agreement with the pro- posed mechanism. As already pointed out, the fumarase in these prepara- tions functions stereospecifically and does not lead to labeling of fumarate. The small amount of deuterium found in the fumarate in these experiments, if indeed significant at all, probably arises from deuteriosuccinate by the non-stereospecific reversal of Equation 6 as formulated by the Equation 7.
4 At the new equilibrium position, Equation 6 would be increased in rate simply because of the increased fumarate concentration, while Equation 5 would be in- creased in rate because of the increased concentration of oxidized carrier resulting from fumarate addition.
The low values for fumarate labeling probably reflect (a) the low concen- tration of labeled succinate undergoing dehydrogenation during the experi- mental period and (b) the dilution of the small amount of isotopic fuma- rate thus formed by the initially added normal fumarate. It should be added that the non-stereospecific reversal of Equation 6 would be expected to lead eventually to extensive labeling of fumarate. It can be shown from a theoretical consideration of this process that, whereas the rate of labeling of succinate is directly proportional to the concentration of unlabeled suc- cinate remaining at any time, the rate of labeling of fumarate is directly proportional to the concentration of labeled succinate (minus labeled fu- marate) at any time. The theoretical time-course for labeling of fumarate would, therefore, involve a lag period. Thus, one might expect that under conditions by which succinate labeling became extensive (e.g. with longer incubation or with more active enzyme preparations) the degree of fuma- rate labeling would become larger. The high degree of fumarate labeling shown in Table I, Series III and IV, is, therefore, not in itself inconsistent with the proposed hypothesis. However, the fact that the isotope levels in the corresponding succinate were no higher than those of the experi- ments in Series I and II and that the rate of fumarate labeling was constant with time and thus independent of the concentration of labeled succinate, makes it difficult to reconcile the results of Series III and IV with those of Series I and II. Nevertheless, even the results summarized in Table I, Series III, are in agreement with the proposed scheme in that the bulk of the deuterium in the succinate arises directly from the medium and not secondarily from labeled fumarate, and that the extent of fumarate label- ing depends on the presence of succinate. Thus, in all experiments the deuterium content per molecule of fumarate was always less than that of succinate and, by comparison of Experiments 4 and 5 (Table I, Series III), one notes that the presence of succinate increased the extent of fumarate labeling by a factor of approximately 3-fold. The appearance of deuterium in fumarate during the anaerobic incubation of normal fumarate alone with the enzyme preparation in heavy water (Table I, Series III, Experi- ment 5) may possibly reflect a prior net synthesis of small amounts of deu- teriosuccinate attributable to traces of reducing substances in this enzyme preparation.
Although, as indicated before, both sets of data obtained in this study (i.e. low and high fumarate labeling accompanying succinate labeling) are compatible with the proposed scheme, the reasons for the differences in behavior of different preparations are elusive and not clear at the present time. Nonetheless, there were certain differences in the history and prop-
erties of these preparations which appear to be worth noting. Thus, the preparations made from relatively fresh tissue showed high succinoxidase activity in the presence of fumarate and were only slightly inhibited by addition of the latter. These preparations (Table I, Series I and II) ex- hibited high rates of deuterium incorporation into succinate with corre- spondingly little labeling in the fumarate. On the other hand, prepara- tions from tissues stored in the frozen state for periods ranging from 2 to 34 months were relatively less active with respect to succinate oxidation in the presence of fumarate and were inhibited as much as 50 per cent by the latter. These same preparations (Table I, Series III and IV) gave equal or lower rates of deuterium incorporation into succinate but much higher levels of fumarate labeling. One could possibly invoke the presence in these latter preparations of an enzyme stimulated by succinate, which in some unknown manner introduces deuterium into fumarate, in order to explain the two different sets of data obtained in this study. It should be noted, however, that such an explanation is presented only as a possible means of reconciliation between two sets of data and is completely devoid of experimental basis.
Stereochemistry of Dehydrogenation of Xuccinate-As pointed out in a pre- vious section, on the assumption that the exchange reaction is catalyzed by a single enzyme and this same enzyme is concerned in the oxidation of succinate to fumarate, the data presented in Table II would indicate that during the anaerobic exchange reaction all four hydrogen positions of suc- cinate became labeled. This random labeling of succinate has been re-
DIAQRAM 1. Succinic acid model
ferred to above as resulting from the “non-stereospecific” nature of Equa- tions 4 to 7. In order to aid in the discussion of the stereochemistry of this process, Diagram 1 accompanying has been reproduced. In this dia- gram, the hydrogen atoms marked HI and HI’ are stereochemically indis-
tinguishable. The atoms marked H2 and Hz’ are also stereochemically indistinguishable. HI or HI’ is stereochemically readily distinguishable from Hz or HZ’, e.g. by the following test: When examined from a point outside the molecule on the axis joining the methylene carbon atoms, the HI, Hz, and COOH groups attached to the proximal methylene carbon will always appear in that order, reading clockwise. Such a test for the asym- metry of a carbon atom bearing two like groups has been suggested pre- viously by Schwartz and Carter (29). The Ogston (14) test of 3 point attachment may also be applied, but is in no way a unique criterion for stereochemical asymmetry.
Whenever dehydrogenation occurs by removal of a pair HI, Hz’, this is stereochemically indistinguishable from removal of the pair HI’, Hz, and there is no enzyme or other device which could make that distinction. On the other hand, when dehydrogenation occurs by removal of the pair HI, H,‘, this is stereochemically readily distinguishable from removal of the pair Hz, Hz’, and an enzyme could readily make that distinction. The fact that the enzyme studied here does not distinguish between the 4 hy- drogen atoms of succinic acid must mean either that (a) the enzyme is catalyzing the removal of a pair HI, Hz’ (= HI’, Hz), in which case there is no chance for selective action on one pair of hydrogens, or that (b) the enzyme is catalyzing indiscriminately the removal of either an HIHI’ or an HzH2’ pair, in spite of the fact that there is a theoretical possibility for selective action. In view of the fact that other enzymes exert stereospeci- ficity with respect to methylene hydrogens in all known cases so far studied, it would appear more likely that (a) rather than (6) is operative here.
Thus far, no statement has been made regarding the configuration of the carboxyl groups of the succinate or the steric mechanism of the reac- tion, since the argument is independent of these considerations. If the staggered configuration is assumed, (a) corresponds to a trans elimination of 2 hydrogen atoms, HIHZ’ (= H1’H2), whereas (b) would correspond to the indiscriminate cis elimination of pairs H1H1’ and HzH2’.6 Whether (a) or (b) applies, the random labeling in the succinate would appear to justify the application of the term “non-stereospecific” to the action of the enzyme.
Quantitative Aspects of Rate of Exchange Reaction-Whereas Weinmann et al. (2) were able to demonstrate high rates of anaerobic exchange of succinate hydrogens with the medium in kidney preparations, and regarded the exchange rate as reflecting closely the oxidation rate, other reports, including the present one, suggest that the exchange rate is relatively small compared with the possible rate of succinate oxidation. Erlenmeyer et al.
6 The authors are grateful to Dr. Harvey F. Fisher for bringing these considera- tions to our attention.
(3) incubated deuteriosuccinate anaerobically with a concentrated beef muscle paste, and under such conditions oxidized the suceinate by the drop- wise addition of methylene blue over a period of 6 hours. The fumarate formed as well as the unchanged succinate showed an approximately 20 per cent higher hydrogen to deuterium ratio than the initial succinate. The extent of exchange obtained by them, in the presence of continually formed fumarate, closely corresponds to the values obtained in the present study when succinate and fumarate were incubated together. Even with the 14- to 15-fold stimulatory effect of fumarate, the extent of deuterium incorporation into succinate does not approach the values obtained by Weinmann et al. (2). Nevertheless, the observed exchange rates are highly significant and readily measurable by the techniques described herein.
However, difficulties in demonstrating with certainty deuterium ex- change into succinate as catalyzed by succinic dehydrogenase preparations have been experienced by Geib and Bonhoeffer (30). Although these au- thors mention their repeated attempts under varying conditions to demon- strate such an exchange reaction, no data are presented nor any descrip- tion of the various experimental conditions employed. More recently, Swim and Krampitz (31), in attempting to ascertain the, quantitative significance of the tricarboxylic acid cycle in Escherichia coli, studied the anaerobic oxidation of acetate in the presence of fumarate added as an oxidant. Under these conditions, experiments with acetate-l-C4 showed that most of the isotope was located in the succinate and that the residual fumarate as well as respiratory carbon dioxide were essentially unlabeled. Although these results may seem to contradict the present interpretation of the anaerobic exchange reaction, as based on the stimulatory effects of added fumarate, one should take into consideration the increased com- plexity of the electron transfer reactions which must occur during the course of acetate utilization in the E. coli system. It is clear that a pref- erential oxidation of electron donors other than succinate could explain the observed results.
The authors are indebted to Dr. Theodore Enns of The Johns Hopkins University School of Medicine, working under Veterans Administration contract No. VlOOl-M527, for his kind cooperation in carrying out the deuterium analyses.
SUMMARY
1. Heart particle preparations catalyze a slow anaerobic exchange of deuterium from water into succinate. The rate of this exchange reaction is extremely slow compared to the rate of theoretical aerobic oxidation and is stimulated about 15-fold by the addition of fumarate.
2. The deuterium appearing in the succinate arises directly from the medium and not secondarily from labeled fumarate.
3. Equilibration of fumarate with malate in DzO by fumarase does not lead to appreciable fumarate labeling, thus indicating a stereospecific be- havior of fumarase, in agreement with the conclusions of Fisher et al.
4. Deuteriosuccinate obtained by the anaerobic enzymatic exchange re- action, when oxidized aerobically by the same enzyme preparation, yields fumarate which retains half the isotope content (as atoms D per molecule) found in the original succinate. These results are discussed in terms of the stereochemistry of the succinic dehydrogenase reaction, and it is sug- gested that the term “non-stereospecific” be applied to the action of the enzyme in this case.
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