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Effect of antivitamins on the microbiological biosynthesis of vitamines. IV. Effect of oxythiamine on the thiamine phosphorylation by the thiamine pyrophosphokinase of yeast
207
SHORT COMMUNICATIONS BBA 23515
Effect of antivitamins on the microbiological biosynthesis of vitamins. IV. Effect of oxythiamine on the thiamine phosphorylation by the thiamine pyrophosphokinase of yeast When studying the effect of oxythiamine on thiamine biosynthesis by Saccharomyces cerevisiae it was found that in a definite concentration range, oxythiamine stimulated the thiamine biosynthesis 1. This is probably due to the effect of certain controlling systems on the inhibition of thiamine diphosphate biosynthesis or the inhibition of thiamine-dependent enzymes by oxythiamine. Although the total amount of thiamine increased 3-fold during the stimulation of thiamine biosynthesis by oxythiamine, the amount of thiamine diphosphate did not change significantly ~. This, of course, leads one to assume that oxythiamine probably inhibits the phosphorylation of thiamine. It is generally thought that thiamine pyrophosphokinase (ATP: thiamine pyrophosphotransferase, EC 2.7.6.2) from rat liver and rat intestine is not inhibited by oxythiamine ~,4. In the case of yeast thiamine pyrophosphokinase, however, an inhibitory effect of oxythiamine has been observed 5. The aim of the present study was to clarify the effect of oxythiamine. The sodium salt of ATP was a product of Reanal, Hungary, and had a molecular weight of 623.23 and declared purity of minimal 95 %. Thiamine was the product of L6~iva, Prague. Dowex I-X8 (200/400 mesh) was the product of Fluka, Chemische Fabrik, Buchs. Oxythiamine was prepared from thiamine according to RYI)ON6. Concentrated thianline pyrophosphokinase was prepared from brewer's yeast from the Smichov Brewery (Prague) according tO the method of WEIL-MALHERBE7. The inhibition constant of oxythiamine for yeast thiamine pyrophosphokinase was to be determined, therefore, the Michaelis constant (Kin) for yeast thiamine pyrophosphokinase was first determined and then the apparent Michaelis constant (Kp) for the same enzyme in the presence of oxythiamine. From the values obtained the inhibition constant was calculated according to the following equationS: K~
[I]Km K~--Km
(I)
The thiamine pyrophosphokinase activity was measured in terms of the amount of thiamine diphosphate formed in I h. Thiamine diphosphate was determined after separatinn on the column of Dowex 9 by the thiochrome method l°. For the separation the acetate form of Dowex was used in order to retain only thiamine diphosphate on the column. The Michaelis constant was determined graphically by reciprocal plotting s. The determination of the thiamine pyrophosphokinase activity was carried out in the solution of the following composition : 3 ml of 0.067 M phosphate buffer (pH 6.2) ; 3 ml of 333 mg thiamine pyrophosphokinase concentrate per IOO ml phosphate buffer v (IO mg pyrophosphokinase) ; 2 ml of IOO mg ATP per IOO ml distilled water (2 mg ATP); and I ml of thiamine solution (resulting concentration of thiamine might be between 8.72 ttM and o.872 mM). The volume of the mixture was brought to IO ml with distilled water. The Biochim. Biophys. Acta, 184 (1969) 2o7-2o9
208
SHORT COMMUNICATIONS
reaction mixture was incubated for i h at 27 ° in a H6ppler ultrathermostat. After I h the sample was removed from the mixture (1- 4 ml, according to the concentration of thiamine) and put into 8 ml boiling 0.05 M HC1 to stop the enzymatic reaction. Thiamine diphosphate was separated on the column and determined as described above. The inhibition was determined graphically by the method of reciprocal plottings. The reaction mixture and reaction conditions were the same as those used for the determination of the Michaelis constant of thiaminokinase, the only difference being that oxythiamine was added to the reaction mixture. The apparent Michaelis constant was measured for the different concentrations of oxythiamine (1.73, 5.19 and 34.6/~M). After incubation and interruption of the enzymatic reaction the amount of the thiamine diphosphate was determined as described above.
4r 31-
o
o
1
,.,L ~
0
:~
,~
~
4
,,6
12
,~s] ,,lo-4 (M -1) 1
Fig. I. D e t e r m i n a t i o n of t h e t y p e of inhibition of y e a s t t h i a m i n e p y r o p h o s p h o k i n a s e b y o x y t h i a m i n e . D e t e r m i n a t i o n of Michaelis c o n s t a n t a n d a p p a r e n t Michaelis c o n s t a n t . T h e t h i a m i n e p y r o p h o s p h o k i n a s e a c t i v i t y was m e a s u r e d in t e r m s of t h e a m o u n t of t h i a m i n e d i p h o s p h a t e f o r m e d a t 27 ° in I h. R e a c t i o n m i x t u r e : 3 ml of 0.067 M p h o s p h a t e buffer (pH 6.2) ; IO m g t h i a m i n o k i n a s e c o n c e n t r a t e ; 2 m g A T P ; t h i a m i n e (resulting c o n c e n t r a t i o n of t h i a m i n e b e t w e e n 8 . 7 2 / , M a n d 0.872 mM). T h e v o l u m e of t h e m i x t u r e w a s m a d e to io ml w i t h distilled water. After i n c u b a t i o n aliquots of t h e s a m p l e were p u t into 8 ml boiling 0.o5 M HC1. T h e t h i a m i n e d i p h o s p h a t e w a s s e p a r a t e d on t h e c o l u m n of D o w e x a n d d e t e r m i n e d b y t h e t h i o c h r o m e m e t h o d . O - - O , d e p e n d e n c e of i / t h i a m i n e d i p h o s p h a t e on I/[S] in t h e absence of o x y t h i a m i n e ; & - - & , d e p e n d e n c e of 1/thia m i n e d i p h o s p h a t e on I[[S 1 in t h e presence of 1.73/~M o x y t h i a m i n e ; 0 - - 0 , d e p e n d e n c e of I / t h i a m i n e d i p h o s p h a t e on I/[S] in t h e presence of 5.19/zM o x y t h i a m i n e ; × - × , d e p e n d e n c e of i / t h i a m i n e d i p h o s p h a t e on I/[S] in t h e presence of 34.6/zM o x y t h i a m i n e .
The value of Km was determined graphically by the method of reciprocal plotting. The thiamine pyrophosphokinase activity was measured as the concentration of the resulting thiamine diphosphate. Km w a s determined from the results shown in Fig. I. K m = 1.35 #M. The inhibitory effect of oxythiamine was measured at three different concentrations of oxythiamine: 1.73, 5.19 and 34.6 #M. The average values of four K~ and K, determinations for the different concentrations of oxythiamine are shown in Table I and were determined from the results shown in Fig. I. As may be seen from the results obtained (Fig: i), oxythiamine proved to be Biochim. Biophys. Acta, 184 (1969) 2o7-2o9
SHORT COMMUNICATIONS
209
an inhibitor of yeast t h i a m i n e pyrophosphokinase. At lower concentrations of i n h i b i t o r we could not exclude the competitive effect, while at higher concentrations the m e c h a n i s m of i n h i b i t i o n seemed to be more complicated. TABLE I INHIBITION
CONSTANTS,
/~l,
OF OXY'THIAMINE
FOR YEAST
THIAMINE
PYROPHOSPHOKINASE
For the determination of the apparent Michaelis constant see Fig. i. From the values obtained the inhibition constants were calculated according to Eqn. i (see text).
Oxythiamine conch. (t~M)
K~ (#M)
K~ (nM)
1.73 5.19 34.6
1.725 2.30 7.00
62. 4 73.5 82. 7
I n view of these results we m a y suppose t h a t d u r i n g the t h i a m i n e biosynthesis in the presence of oxythiamine, the phosphorylation of t h i a m i n e to t h i a m i n e diphosphate is inhibited. K n o w i n g from previous studies 2 t h a t the a m o u n t of t h i a m i n e diphosphate formed d u r i n g the c u l t i v a t i o n of S. cerevisiae is nearly the same both in the presence a n d absence of o x y t h i a m i n e , the authors assume t h a t due to the controlling system the biosynthesis of t h i a m i n e m o n o p h o s p h a t e a n d t h i a m i n e will be increased to such a value as to form the necessary a m o u n t of active t h i a m i n e diphosphate even if the phosphorylation is i n h i b i t e d b y oxythiamine. This controlling mechanism, of course, is active only when low concentrations of o x y t h i a m i n e are present 1. The authors wish to t h a n k Ing. Stanislav ProcMzka, from the Smlchov Brewery, who k i n d l y provided the brewer's yeast, a n d Miss J i f i n a OsnerovA for her technical assistance.
Department of Biochemistry, Institute of Chemical Technology, Prague (Czechoslovakia) i 2 3 4 5 6 7 8 9 IO
VLADISLAV ~ICHO BLANKA KR.~LOV.~
v. ~fcHo AND B. KR~LOV/~,Sci. Papers Inst. Chem. Technol. Prague, E 9 (1966) 75. V. ~fCHO AND B. KR~LOVA,Biochim. Biophys. Avta, 165 (1968) 459. S. EICH AND L. R. CERECEDO,J. Biol. Chem., 2o7 (1954) 295. L. R. CERECEDO, S. EICH AND E. BRESNICK,Biochim. Biophys. Acta, 15 (1954) 144. A. J. EusEEI AND L. R. CERECEDO, Federation Pro¢., 9 (195o) 169. A. N. RYDON, Biochem. J., 48 (1951) 383 . H. WEIL-MALHERBE,Biochem. J., 33 (1939) 1997, H. LINEWEAVERAND D. BURK,J. Am. Chem. Sot., 56 (1934) 658. L. DEGuIsEPPE AND G. RINDI, J. Chromatog., 1 (1958) 545Association of Vitamin Chemists, Methods of Vitamin Assay, Interscience, New York, 195o, p. lO7.
Received March 3rd, 1969
Biochim. Biophys. Acta, 184 (1969) 2o7-2o9
SHORT COMMUNICATIONS BBA 23515
Effect of antivitamins on the microbiological biosynthesis of vitamins. IV. Effect of oxythiamine on the thiamine phosphorylation by the thiamine pyrophosphokinase of yeast When studying the effect of oxythiamine on thiamine biosynthesis by Saccharomyces cerevisiae it was found that in a definite concentration range, oxythiamine stimulated the thiamine biosynthesis 1. This is probably due to the effect of certain controlling systems on the inhibition of thiamine diphosphate biosynthesis or the inhibition of thiamine-dependent enzymes by oxythiamine. Although the total amount of thiamine increased 3-fold during the stimulation of thiamine biosynthesis by oxythiamine, the amount of thiamine diphosphate did not change significantly ~. This, of course, leads one to assume that oxythiamine probably inhibits the phosphorylation of thiamine. It is generally thought that thiamine pyrophosphokinase (ATP: thiamine pyrophosphotransferase, EC 2.7.6.2) from rat liver and rat intestine is not inhibited by oxythiamine ~,4. In the case of yeast thiamine pyrophosphokinase, however, an inhibitory effect of oxythiamine has been observed 5. The aim of the present study was to clarify the effect of oxythiamine. The sodium salt of ATP was a product of Reanal, Hungary, and had a molecular weight of 623.23 and declared purity of minimal 95 %. Thiamine was the product of L6~iva, Prague. Dowex I-X8 (200/400 mesh) was the product of Fluka, Chemische Fabrik, Buchs. Oxythiamine was prepared from thiamine according to RYI)ON6. Concentrated thianline pyrophosphokinase was prepared from brewer's yeast from the Smichov Brewery (Prague) according tO the method of WEIL-MALHERBE7. The inhibition constant of oxythiamine for yeast thiamine pyrophosphokinase was to be determined, therefore, the Michaelis constant (Kin) for yeast thiamine pyrophosphokinase was first determined and then the apparent Michaelis constant (Kp) for the same enzyme in the presence of oxythiamine. From the values obtained the inhibition constant was calculated according to the following equationS: K~
[I]Km K~--Km
(I)
The thiamine pyrophosphokinase activity was measured in terms of the amount of thiamine diphosphate formed in I h. Thiamine diphosphate was determined after separatinn on the column of Dowex 9 by the thiochrome method l°. For the separation the acetate form of Dowex was used in order to retain only thiamine diphosphate on the column. The Michaelis constant was determined graphically by reciprocal plotting s. The determination of the thiamine pyrophosphokinase activity was carried out in the solution of the following composition : 3 ml of 0.067 M phosphate buffer (pH 6.2) ; 3 ml of 333 mg thiamine pyrophosphokinase concentrate per IOO ml phosphate buffer v (IO mg pyrophosphokinase) ; 2 ml of IOO mg ATP per IOO ml distilled water (2 mg ATP); and I ml of thiamine solution (resulting concentration of thiamine might be between 8.72 ttM and o.872 mM). The volume of the mixture was brought to IO ml with distilled water. The Biochim. Biophys. Acta, 184 (1969) 2o7-2o9
208
SHORT COMMUNICATIONS
reaction mixture was incubated for i h at 27 ° in a H6ppler ultrathermostat. After I h the sample was removed from the mixture (1- 4 ml, according to the concentration of thiamine) and put into 8 ml boiling 0.05 M HC1 to stop the enzymatic reaction. Thiamine diphosphate was separated on the column and determined as described above. The inhibition was determined graphically by the method of reciprocal plottings. The reaction mixture and reaction conditions were the same as those used for the determination of the Michaelis constant of thiaminokinase, the only difference being that oxythiamine was added to the reaction mixture. The apparent Michaelis constant was measured for the different concentrations of oxythiamine (1.73, 5.19 and 34.6/~M). After incubation and interruption of the enzymatic reaction the amount of the thiamine diphosphate was determined as described above.
4r 31-
o
o
1
,.,L ~
0
:~
,~
~
4
,,6
12
,~s] ,,lo-4 (M -1) 1
Fig. I. D e t e r m i n a t i o n of t h e t y p e of inhibition of y e a s t t h i a m i n e p y r o p h o s p h o k i n a s e b y o x y t h i a m i n e . D e t e r m i n a t i o n of Michaelis c o n s t a n t a n d a p p a r e n t Michaelis c o n s t a n t . T h e t h i a m i n e p y r o p h o s p h o k i n a s e a c t i v i t y was m e a s u r e d in t e r m s of t h e a m o u n t of t h i a m i n e d i p h o s p h a t e f o r m e d a t 27 ° in I h. R e a c t i o n m i x t u r e : 3 ml of 0.067 M p h o s p h a t e buffer (pH 6.2) ; IO m g t h i a m i n o k i n a s e c o n c e n t r a t e ; 2 m g A T P ; t h i a m i n e (resulting c o n c e n t r a t i o n of t h i a m i n e b e t w e e n 8 . 7 2 / , M a n d 0.872 mM). T h e v o l u m e of t h e m i x t u r e w a s m a d e to io ml w i t h distilled water. After i n c u b a t i o n aliquots of t h e s a m p l e were p u t into 8 ml boiling 0.o5 M HC1. T h e t h i a m i n e d i p h o s p h a t e w a s s e p a r a t e d on t h e c o l u m n of D o w e x a n d d e t e r m i n e d b y t h e t h i o c h r o m e m e t h o d . O - - O , d e p e n d e n c e of i / t h i a m i n e d i p h o s p h a t e on I/[S] in t h e absence of o x y t h i a m i n e ; & - - & , d e p e n d e n c e of 1/thia m i n e d i p h o s p h a t e on I[[S 1 in t h e presence of 1.73/~M o x y t h i a m i n e ; 0 - - 0 , d e p e n d e n c e of I / t h i a m i n e d i p h o s p h a t e on I/[S] in t h e presence of 5.19/zM o x y t h i a m i n e ; × - × , d e p e n d e n c e of i / t h i a m i n e d i p h o s p h a t e on I/[S] in t h e presence of 34.6/zM o x y t h i a m i n e .
The value of Km was determined graphically by the method of reciprocal plotting. The thiamine pyrophosphokinase activity was measured as the concentration of the resulting thiamine diphosphate. Km w a s determined from the results shown in Fig. I. K m = 1.35 #M. The inhibitory effect of oxythiamine was measured at three different concentrations of oxythiamine: 1.73, 5.19 and 34.6 #M. The average values of four K~ and K, determinations for the different concentrations of oxythiamine are shown in Table I and were determined from the results shown in Fig. I. As may be seen from the results obtained (Fig: i), oxythiamine proved to be Biochim. Biophys. Acta, 184 (1969) 2o7-2o9
SHORT COMMUNICATIONS
209
an inhibitor of yeast t h i a m i n e pyrophosphokinase. At lower concentrations of i n h i b i t o r we could not exclude the competitive effect, while at higher concentrations the m e c h a n i s m of i n h i b i t i o n seemed to be more complicated. TABLE I INHIBITION
CONSTANTS,
/~l,
OF OXY'THIAMINE
FOR YEAST
THIAMINE
PYROPHOSPHOKINASE
For the determination of the apparent Michaelis constant see Fig. i. From the values obtained the inhibition constants were calculated according to Eqn. i (see text).
Oxythiamine conch. (t~M)
K~ (#M)
K~ (nM)
1.73 5.19 34.6
1.725 2.30 7.00
62. 4 73.5 82. 7
I n view of these results we m a y suppose t h a t d u r i n g the t h i a m i n e biosynthesis in the presence of oxythiamine, the phosphorylation of t h i a m i n e to t h i a m i n e diphosphate is inhibited. K n o w i n g from previous studies 2 t h a t the a m o u n t of t h i a m i n e diphosphate formed d u r i n g the c u l t i v a t i o n of S. cerevisiae is nearly the same both in the presence a n d absence of o x y t h i a m i n e , the authors assume t h a t due to the controlling system the biosynthesis of t h i a m i n e m o n o p h o s p h a t e a n d t h i a m i n e will be increased to such a value as to form the necessary a m o u n t of active t h i a m i n e diphosphate even if the phosphorylation is i n h i b i t e d b y oxythiamine. This controlling mechanism, of course, is active only when low concentrations of o x y t h i a m i n e are present 1. The authors wish to t h a n k Ing. Stanislav ProcMzka, from the Smlchov Brewery, who k i n d l y provided the brewer's yeast, a n d Miss J i f i n a OsnerovA for her technical assistance.
Department of Biochemistry, Institute of Chemical Technology, Prague (Czechoslovakia) i 2 3 4 5 6 7 8 9 IO
VLADISLAV ~ICHO BLANKA KR.~LOV.~
v. ~fcHo AND B. KR~LOV/~,Sci. Papers Inst. Chem. Technol. Prague, E 9 (1966) 75. V. ~fCHO AND B. KR~LOVA,Biochim. Biophys. Avta, 165 (1968) 459. S. EICH AND L. R. CERECEDO,J. Biol. Chem., 2o7 (1954) 295. L. R. CERECEDO, S. EICH AND E. BRESNICK,Biochim. Biophys. Acta, 15 (1954) 144. A. J. EusEEI AND L. R. CERECEDO, Federation Pro¢., 9 (195o) 169. A. N. RYDON, Biochem. J., 48 (1951) 383 . H. WEIL-MALHERBE,Biochem. J., 33 (1939) 1997, H. LINEWEAVERAND D. BURK,J. Am. Chem. Sot., 56 (1934) 658. L. DEGuIsEPPE AND G. RINDI, J. Chromatog., 1 (1958) 545Association of Vitamin Chemists, Methods of Vitamin Assay, Interscience, New York, 195o, p. lO7.
Received March 3rd, 1969
Biochim. Biophys. Acta, 184 (1969) 2o7-2o9