Inhibition of the Succinic Oxidase System by S o m e Structural Analogs of Succinic and Malonic Acids Frank Tietze 1 and Irving M. Klotz From the Deparlment of Chemistry, Northwestern University, Evanston, Illinois Received July 31, 1951 INTRODUCTION
0nly fragmentary information exists in the literature relative to the possible inhibitory action of succinate analogs formed by replacement of one or more carboxyl groups of malonic or succinic acids by other acidic groups. It has been observed by Everett and Krantz (1) that the salt of a sulfon-succinic acid 2 inhibits the endogenous uptake of oxygen by frog skin or muscle. Previous work in this laboratory (2) has indicated further that 1,2-ethanedisulfonic acid, as well as ~-sulfopropionic acid, is capable of producing a marked inhibition of the succinic oxidase system of mouse liver. Similar results have been reported by Ajl and Werkman (3) for the effect of the disulfonate on extracts from Escherichia coli. An extension of these studies has seemed desirable and hence a number of new analogs of succinic and malonic acids have been examined. EXPERIMENTAL
Measurement of Enzyme Activity The uptake of oxygen following addition of sodium succinate to a buffered mouse liver homogenate (4) was determined in the conventional Warburg manometric apparatus maintained at a temperature of 36.8 4- 0.04~ In order to localize any inhibition, a procedure was employed whereby participation of the cytoehrome system was eliminated by the addition of potassium cyanide to the system. Simultaneous 1Junior Fellow of the National Institutes of Health, 1947-49. Present address: Department of Biochemistry, School of Medicine, University of Washington, Seattle 5, Washington. Formula probably HOOC--CH--CH~--COOH, but is not given explicitly by the
FRANK TIETZE AND IRVING M. KLOTZ
addition of methylene blue provided an alternative pathway of electron transfer; preliminary runs determined the amount of methylene blue necessary for maximum rate of oxidation. Inasmuch as carbon dioxide production was found to be negligible compared to oxygen consumption, no alkali was added to the center well of the reaction flasks. The final concentrations of reagents were generally as follows: sodium suceinate, 0.01-0.02 M; potassium cyanide, 0.002 M; methylene blue, 0.004~0.009 M; inhibitor, 0.01-0.05 M. All experiments were carried out in 0.066 M phosphate buffer (5) at pH 7.4. Oxidation rates were obtained by plotting the uptake of oxygen against time and drawing the best straight line through the points. In general the oxygen consumption was a linear function of the time.
Reagents Sodium succinate hcxahydrate (Merck) of c.P. grade was purified by crystallization from doubly distilled water. The methylene blue was of U.S.P. grade and was used without further purification, trans-l,2-Cyclopentanedicarboxylic acid was obtained through the courtesy of Prof. R. C. Fuson of the University of Illinois. The authors are also indebted to Prof. Raymond P. Mariella of Northwestern University for a generous sample of 1,1-cyclobutanedicarboxylic acid. The pure sodium salts of 1,2-ethanedisulfonic and arsonoacetic acids were prepared by the methods of Kohler (6) and Palmer (7), respectively. Potassium methionate was prepared by the method of Lauer and Langkammerer (8). g-Phosphonopropionic acid was prepared from its triethyl ester by the pyrolytic method of Arbuzov, Konstantinova, and Anzyfrova (9) ; the procedure for the synthesis of the triethyl ester followed closely that employed by Nylen (10) for the preparation of triethyl phosphonoacetate. 3 The melting point of the final product after crystallization from aqueous acetone was 159-162~ Although this was considerably lower than that reported by Arbuzov et al., 170~ no further purification was attempted because of the small amount of material ohtained, cis-l,2-Cyclopentanedicarboxylicacid was prepared from the trans-isomer by the method of Wassermann (12). After two recrystallizations from water the product was found to melt at 134-138~ (uncorr.). Melting points previously recorded in the literature are those of Wassermann, 134-135~ (12); and Perkin, 141~ (13). Pure o-sulfobenzoic acid was prepared as follows: Technical (93%) o-sulfobenzoie acid anhydride was added to an excess of water and the mixture was boiled under reflux for several hours. The resulting solution was clarified with charcoal, evaporated to a small volume, and allowed to cool, whereupon solidification of o-sulfobenzoic acid trihydrate occurred. The product was purified by another crystallization from water. When necessary, the compounds described above were neutralized to pH 7.4 (Beckman pH meter, model G) with dilute aqueous sodium hydroxide prior to addition to the Warburg flasks. ]~E~ULTS AND DISCUSSION T h e results o b t a i n e d w i t h each c o m p o u n d are s u m m a r i z e d in T a b l e I. T h e i n h i b i t i o n of the succinic oxidase s y s t e m b y t h e v a r i o u s a n a l o g s 3The diethyl phosphite necessary for this synthesis was prepared by the second method of McCombie, Saunders, and Stacey (11).
TABLE I Effect of Vario~.s Inhibitors on the Oxidation
Inhibitor 1,2-Ethanedisulfonic acid HO~SCH2CH2SOsH
Methionic acid HOsSCH2SOaH Arsonoacetic acid HO~CCH,AsO3H~ None trans- 1,2-Cyclopen tanedicarboxylic acid CI-I:
of S~ccinate by M o , s e Liver Homogenate
Concentration of suceinate M 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
Concentration of irihibitor M 0.000 0.0144 0.020~ 0.00 0.01 b 0.05 b 0.00 0.01 0.05 0.00 0:01 0.05 0.00 0.01 0.05 0.00 0.029
O: consumption cu. ram./hr. 96.7 78.8 58.1 91.5 77.4 57.5 107 107 107 119 90 73 91 91 91 117 62
0.01 0.01 0.01
0.00 0.01 0.05
167 131 68
0.01 0.01 0.01
0.00 0.01 0.05
188 131 81.5
HOOC/ cis-l,2-Cyclopentanedicarboxylic acid 1,1-Cyclobutanedicarboxylic acid CH~ COOH
~'CH/ ~COOH Malonie acid HO~CCH2CO~H Phthalic acid
o-Sulfobenzoic acid -
Sample obtained through the courtesy of Professor S. M. McElvain, University of Wisconsin. b Crystalline sodium salt prepared by method of Kohler (6). 357
M. K L O T Z
reported in this paper are in general of a low order of magnitude in comparison with the more familiar analogs, e.g., malonic acid. A previous communication (2) has dealt with the inhibitory effects of the two sulfonic acid analogs of succinic acid on the succinic oxidase system of mouse liver. Since this study did not localize the site of action of either inhibitor, the effect of 1,2~ acid on the reduction of methylene blue was included in the present investigation. The inhibitions obtained here (Table I) are lower than those previously reported for the addition of this compound to the complete succinic oxidase system. Although this drop may indicate a partial attack by the analog on the cytochrome component or at some intermediate locus in the chain, the major inhibitory action appears to be at the dehydrogenase level. This conclusion is consistent with the observation that 1,2-ethanedisulfonate has no detectable effect on the endogenous metabolism of the homogenate. If one proceeds from the sulfonate to the phosphonate analog of succinic acid, the inhibitory effect drops to zero. A similar trend is observed in the malonate analogs where the order of activity is malohate > methionate > arsonoacetate. The absence of inhibition by the phosphonate and arsonate analogs may be due to the extra negative charge carried by these dibasic substitutents. If the enzyme is also negatively charged it would repel the trivalent ions more than the bivalent carboxyl or sulfonate compounds. In an interpretation of the effects of the cyclopentanedicarboxylic acids it is necessary to realize first that the bulk of the rings in the cyclic acids introduces a steric factor which may reduce substantially the ability of the molecule to combine with the active site on the enzyme. It is generally true that the alkyl malonie acids are less effective as inhibitors of the succinic oxidase system than the parent compound. Furthermore, as has been demonstrated in the present studies (Table I) 1,1-cyclobutanedicarboxylic acid, a malonate analog, is much less effective an inhibitor than is malonate itself, and in fact is about as active as the 1,2-cyclopentanedicarboxylic acids. Thus the presence of a ring in these compounds interferes with their ability to combine with the enzyme. With the consequent relatively small inhibitions by either form of the eyclopentanedicarboxylic acid, as well as by their coplanar counterparts, phthalic or sulfobenzoic acids, it is not possible to establish which configuration corresponds most closely to the substrate in the activated complex.
SUCCINIC OXIDASE INHIBITION
Methionic and 1,2-ethanedisulfonic acids, analogs of malonic and succinic acids, respectively, inhibit the oxidation of succinate. Arsonoacetic and B-phosphonopropionic acids, however, exert no inhibitory action. Other analogs containing rings which fix the spatial orientation of the carboxyl groups (cis- and trans-l,2-cyclopentanedicarboxylic acids, 1,1-cyclobutanedicarboxylic acid, phthalic acid, and o-sulfobenzoic acid) show inhibitory properties, but they cannot be sufficiently differentiated in their effects to establish the configuration of the enzyme-subs,rate complex. REFERENCES 1. EVERETT,G., AND KRANTZ,J. C., JR., Proc. Soc. Exptl. Biol. Med. 55, 220 (1944). 2. KLOTZ,I. M., AND TIETZE, F., J. Biol. Chem. 168, 399 (1947). 3. AJL, S. J., ANn WERKMAN, C. H., Proc. Natl. Acad. Sci. U. S. 34, 491 (1948). 4. POTTER, V. R., AND ELVSHJEM, C. A., J. Biol. Chem. 114, 495 (1936). 5. LATIMER,W. M., AND HILDEB1L~ND,J. ~-~., Reference Book of Inorganic Chemistry, p. 509. The Macmillan Company, New York, 1940. 6. KOHLER,E. P., Am. Chem. J. 19, 728 (1897). 7. PALMER, C. S., in Organic Syntheses, Coll. Vol. I, pp. 73-5. John Wiley and Sons, New York, 1941. 8. LAUER,W. M., AND L&NGKAMMERER,C. M., J. Am. Chem. Soc. 57, 2360 (1935). 9. ARBUZOV,A. E., KONST&NTINOVA,T., AND ANZYFROVA,T., Izvest. Akad. Nauk S.S.S.R., Otdel. Khim. Nauk 1946, 179; C. A. 42, 6315 (1948). 10. NYLON,P., Bet. 57, 1023 (1924). 11. McCoMmE, H., SAUNDERS,B. C., AND STAC~Y,G. J., J. Chem. Soc. 1945, 380. 12. WASSERMANN,A., Helv. Chim. Acta 13, 223 (1930). 13. PERKI~, W. J., JR., J. Chem. Soc. 65, 572 (1894).