69. On the Mechanism o f Thiamine Action, II.

Transcription

1 302 [Vol. 27, 69. On the Mechanism o f Thiamine Action, II. Department By Shunzi MIZUHARA, Ryohei TAMURA, and Hidetaka ARATA. of Biological Chemistry, Okayama University Medical School. {Comm. by T. SHIMIZU, M.J.A., June 12, 1951.} In 1946, a remarkable theory on the mechanism of thiamine action was suggested by Karrer,~'~ in which he considered that the decarboxylation of pyruvic acid was performed by the sulfhydryl group of thiamine. In 1942, Green et reported the carboxylase obtained from the pig heart capable of converting pyruvic acid into acetoin and CO2. In 1943, Ugai et showed that the thiamine and the other quaternary thiazol derivatives catalysed f uroin condensation. Mizuhararepeated Ugai's experiments and in a previous paper reported that in 50 alcoholic solution about the same quantities of f uroin were produced from f urf urol anaerobically in the presence of thiamine in both cases when two equivalents of alkali added to one molecule of thiamine and when three equivalents were added. Thiaminedisulfide cou'd not catalyse the furoin condensation. Then the authors found that, in aqueous solution, acetoin and CO were formed from pyruvic acid and acetaldehyde in the presence of thiamine when two equivalents of alkali were added to one molecule of thiamine, but not formed when one or three equivalents of alkali were added. Under the same conditions, acetoin was not produced in the presence of thiaminedisulfide. Recently in this laboratory, Aratastudied the oxygen consumption and methylene blue reduction by thiamine, and found that in 50 ~% alcoholic solution the greater part of thiamine was in the form of pseudobase when two or three equivalents of alkali were added to one molecule of thiamine. In aqueous solution, the same relationship was observed when two equivalents of alkali were added, but it was found that the most part of thiamine was in the form of a sulfhydryl compound when three equivalents of alkali were added. So it is considered that acyloin condensation and decarboxylation of pyruvic acid are performed by the pseudobase form of thiamine. Moreover, in the chemical equilibrium between ammoniumbase and pseudobase, the most remarkable changes in the thiamine molecule is the quaternary thiazol nitrogen change into tertiary, and its tertiary nitrogen gain of a to le pair. So we are led to the

2 ' lv. 6.1 On the Mechanism of Thiamine Action. II. 303 conclusion that decarboxylation and acyloin condensation are not performed by the sulfhydryl group of thiamine, but by the lone pair of thiazol nitrogen atom of the pseudobase form of thiamine. We can show the following reaction scheme for the acyloin condensation and decarboxylation of pyruvic acid by thiamine : Pyruvic acid 0 CH -Ic-COOH -N - i i HO BiPseudobase -~ IOH CH3-C-COOH N+ 3 OH CH3-C N+ Il R 3 OH CH3--C ~ N+ ~ ~" Acetaldehyde 3 l R3 By the coordination of carbocation of a-carbonyl gr group of pyruvate is drawn of a-carbonyl group. The structure of 0^C carbon dioxide molecule th at it has a tendency to change into carbon dioxide molecule by e mitting electron. Furthermore, by the influence of --I effect of coo rdinated thiamine molecule (--N=R~;), which has linked with a-car y carbon,, the electron of --C--C 1 bon bound between carbonyl and carboxyl group is drawn to the a-carbonyl carbon. Owing to these reasons, a carbon dioxide is liberated and a-carbonyl carbon turns into carboanion. Then a carbocation off acetaldehyde links with the carboanion above indicated, and the acyloin condensation is carried out. Experimental OH (0 j Evidence. Methods : -- For the determination of acetoin, a colorimetric method described by Westerfeldtt~~ was used. The distinction of color was read in a Pulflich photometer using a 1 cm. cell and a light filter No. S-53, and the amounts of acetoin (ry) of the solution (5 ml.) were calculated by multiplying the factor 35 by the distinction. For the determination of COQ, the Warburg manometers were OH CH3-- C---C--CH p CH3 C0 CH--CH H N Acetoin --N H/\/ HO a lone pair of thiazoi nitrogen with a oup of pyruvate, a proton of carboxyl to the negative charged oxygen atom --0~ group appeared is so similar to I L- 3

3 304 S. MIZUHARA, R. TAMURA, and H. ARATA. [Vol. 27, used. Pyruvic acid determination was by using 2, 4-dinitrophenylhydrazine. performed colorimetrically (I) Acetoin Formation by Thiamine and Cocarboxylase : The mixture ml. M/10 pyruvic acid, 0.3 ml. M-acetaldehyde and 0.5 ml. M/10 thiamine or cocarboxylase -- was placed in a Thunberg tube, pyruvic acid neutralized with n-naoh, and two or three equivalents of alkali (n-naoh) were added to thiamine or cocarboxylase. Then the liquid amount of each tube is amended to 2 ml. with distilled water and evacuated. After being kept at 37 C for ten hours it was diluted ten-fold with distilled water. 5 ml. of the solution were used for the determination of acetoin and 0.5 ml. for the pyruvic acid. The results obtained are shown in Table I. Table I. Acetoin Formation by Thiamine and Cocarboxylase. The data shows that : (1) Acetoin was formed from pyruvic acid and acetaldehyde in the presence of thiamine or cocarboxylase, but the catalytic action of thiamine is stronger than that of cocarboxylase in vitro. (2) Concerning the decarboxylation of pyruvate, cocarboxylase is stronger than thiamine. Special attention should be directed to the fact that a greater quantity of acetoin was produced by adding two equivalents of alkali to the thiamine than by three equivalents of alkali. (II) Acetoin Formation under Aerobic and Anaerobic Condition: The above experiments were carried out in a vacuum, but these experiments were done also by using nitrogen and air as a gas phase.

4 No. 6.] On the Mechanism of Thiamine Action. II. 305 As recorded in Table II, under anaerobic conditions-in vacuum or in nitrogen-about the same amounts of acetoin were produced, but under aerobic conditions a small amount was formed. It is considered that under aerobic conditions thiamine is oxidized by oxygen, and, as the result of its destruction, the quantities of acetoin become smaller. Table Acetoin Formation under Aerobic and Anaerobic Conditions. 1 ml. M/10 pyruvic acid+0.3 ml. M-acetaldehyde+0.5 ml. M/10 B1+0.2 ml. n-naoh II. (III) Determination of Carbon Dioxide : If the reaction above described is performed by the following scheme, carbon dioxide should be produced : CH3 CO COOH + CHI CHO ---~ CH;; CO CH CH;, + CO dil For the determination of C02, warburg manometers with conical flasks provided with a side arm are used. Into the main chamber 1 ml. M/10 pyruvic acid, 0.3 ml. M-acetaldehyde, 0.2 ml. n-naoh and 0.5 ml. M/10 thiamine or cocarboxylase are measured ; 0.2 ml. 10 n-h~so4 are placed in the side arm, and nitrogen is used as the gas phase. Table Determination III. of C02 Table III shows the production of C02, the yields of acetoin and the amounts of remaining pyruvate after five hours operation at 37 C. The quantity of CO produced and pyruvic acid broken down in the presence of cocarboxylase were much larger than in the presence of thiamine, but the amounts of acetoin formed in the presence of cocarboxylase were smaller than in the presence of thiamine. (IV) Confirmation of Acetoin by Nickel-Dimethylglyoxime : For the confirmation of acetoin by nickel-dimethylglyoxime, Langenbecks method ~~ > was used.

5 306 S. MIZUHARA, R. TAMURA, and H. ARATA. [Vol. 27, The following mixture-10 ml. M/10 pyruvic acid, 5 ml. M-acetaldehyde, 2 ml. n-na4h and 5 ml. M/10 thiamine or cocarboxylasewas placed in a vacuum flask. After being evacuated and kept at 37 C for 18 hours, 20 ml. of the mixture were used to form the nickel-dimethylglyoxime and 1 ml. for the colorimetric determination of acetoin. The data are recorded in Table IV. About the same quantities of acetoin were obtained by these different methods. two (V) Acetoin Formation by Thiamine Analogues : Table V. Acetoin Formation by Thiamine Analogues at 37 C for 18 hours.

6 No. 6.] on the Mechanism of Thiamine Action. II. 307 Thiamine analogues described in Table V were used in M/10 aqueous solution. The other experimental procedures were the same as above mentioned (I-III). The datum shows that thiaminedisulfide and the other thiamine analogues having tertiary nitrogen in the thiazol-ring are not capable of catalysing the acyloin condensation. As noted in Table V *6), thiazol-5-carboxylic acid ester can catalyse this condensation even when one equivalent of alkali is added, so it is necessary to test whether or not thiamine can catalyse the acyloin condensation under the same conditions. The results are shown in Table VI. Table Relationship between Acetoin Formation and Molar Concentration of Alkali Added to Thiamine. Materials : the same as in the above experiments at 37 C for 20 hours. VI. The maximum yield of acetoin was obtained when two equivalents of alkali were added. When one mol. of alkali was added the amounts of acetoin produced was as small as when three mol. of alkali was used. So it is concluded that thiamine can not show its catalytic action until its hydrochloric acid and chloride ion are removed by alkali. Summary. (1) In aqueous solution acetoin and carbon dioxide are formed from pyruvic acid and acetaldehyde by the action of thiamine only when two equivalents of alkali are added to one molecule of thiamine. (2) Thiaminedisulfide and the other tertiary thiazol derivatives can not catalyse this acyloin condensation under the same conditions. (3) From these results and the other reports from our laboratory, we discussed the mechanism of thiamine action and presented a new reaction scheme for the acetoin formation and the decarboxylation of pyruvic acid. The authors wish to make a grateful acknowledgement to Professor Dr. T. Shimizu, under whose direction this investigation has been carried out, and also to Dr. T. Matsukawa in making available materials used.

7 308 S. MIZUHARA, R. TAMURA, and H. ARATA. [Vol. 27, References. (1) P. Karrer, M. Viscontini, Helv. Chim. Acta, 29, 711 (1946). (2) D. E. Green, W. W. Westerfeld, B. Vennesland, W. E. Knox, J. Biol. Chem., 145, 69 (1942). (3) T. Ugai, S. Tanaka, S. Dokawa, J. Pharmaceut. Soc., Japan, 63, 269 (1943). (4) S. Mizuhara, Reports from the Commission for Vitamin B Research, Japan, XXVII (1949). (5) H. Arata, Reports from the Commission for Vitamin B Research, Japan, XXXV (1949). (6) W. W. Westerfeld, J. Biol. Chem., 161, 495 (1945). (7) W. Langenbeck, H. Werde, and W. Schlockermann, Z. Physiol. Chem., 224, 263 (1934).