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The biosynthesis of thiamine. IV. Inhibition by vitamin B6 compounds
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 101 , 1 9 7 - 2 0 3
(1963)
The Biosynthesis of Thiamine. IV. 1 Inhibition by Vitamin Be Compounds L A W R E N C E M. L E W I N AND G E N E M. B R O W N From the Division of Biochemistry, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts Received January 24, 1963 A novel method for the synthesis of tile mono- and pyrophosphate esters of 2-methyl-4-amino-5-hydroxymethylpyrimidine has been described. The inhibitory effect of vitamin Be compounds on the biosynthesis of thiamine has been shown to be due primarily to the inhibition by pyridoxal phosphate of the enzymic reaction whereby thiamine monophosphate is formed from the monophosphate ester of 4methyl-5-(2-hydroxyethyl)thiazole and the pyrophosphate ester of 2-methyl-4-amino-5-hydroxymethylpyrimidine. Kinetic experiments indicated that the inhibition is of a "mixed" type, i.e., a mixture of a purely noncompetitive type and a partially competitive type (partially competitive with the pyrophosphate ester of 2-methyl-4-amino-5-hydroxymethylpyrimidine). INTRODUCTION Harris has reported that an excess of vitamin B6 inhibits the growth of Neurospora (2) and the net synthesis of thiamine by this organism (3) apparently by interfering with the enzymic system whereby hydroxymethylpyrimidine2 and thiazole ~ are utilized to form thiamine (4). The inhibition appeared to be competitive between vitamin B6 and hydroxymethylpyrimidine, although the author was careful to point out t h a t this conclusion could be only a tentative one since the experimental data were obtained by using whole cells (4). 1 This work was supported by grants A-3442 from the National Institutes of Health and G-4580 from the National Science Foundation. For Paper III of this series see Ref. (1). 2Abbreviations used are: hydroxymethylpyrimidine, hydroxymethylpyrimidine-P, and hydroxymethylpyrimidine-PP for 2-methyl-4amino-5-hydroxymethylpyrimidine, the monophosphate ester of this compound, and the pyrophosphate, respectively; thiazole and thiazole-P for 4-methyl-5-(2-hydroxyethyl)thiazole and the monophosphate ester of this compound, respectively; thiamine-P for thiamine monophosphate; bromomethylpyrimidine for 2-methyl-4-amino-5bromomethylpyrimidine; and ATP for adenosine triphosphate.
The results of the work of several groups of investigators have been instrumental in establishing that the enzymic synthesis of thiamine from hydroxymethylpyrimidine and thiazole proceeds as follows: (a) hydroxymethylpyrimidine
ATP
)
hydroxymethylpyrimidine-P (1, 5-7) (b) hydroxymethylpyrimidine-P
ATP
>
hydroxymethylpyrimidine-PP (1, 5-7) (e) thiazole
ATP
) thiazole-P (8-11)
(d) hydroxymethylpyrimidine-PP + thiazole-P ~- thiamine-P + PPI (8-13) (e) thiamine-P H20) thiamine + Pi (8-13) The enzymes that catalyze reactions (a-e) have been named, respectively, hydroxymethylpyrimidine kinase (1), hydroxymethylpyrimidine phosphokinase (1), thiazole kinase (11), thiamine-P synthase (11) or thiamine-P pyrophosphorylase (12) (since the reaction is reversible), and thiamine-P phosphatase. There is no evidence that a specific phosphatase is involved in reaction e. I n fact, it would appear that,
197
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LEWIN AND BROWN
in yeast, this r e a c t i o n is c a t a l y z e d o n l y by nonspecific p h o s p h a t a s e s (14). T h e e l u c i d a t i o n of t h e e n z y m i c steps i n v o l v e d in t h i a m i n e synthesis p r o v i d e d t h e o p p o r t u n i t y to i n v e s t i g a t e t h e effects of v i t a m i n B6 c o m p o u n d s on each of these e n z y m i c reactions. T h e results p r e s e n t e d i n d i c a t e t h a t t h e i n h i b i t i o n of t h i a m i n e synthesis b y v i t a m i n B6 c o m p o u n d s c a n be ascribed p r i m a r i l y t o a n iHhibitory effect of p y r i d o x a l - P on t h e r e a c t i o n c a t a l y z e d b y t h i a m i n e - P s y n t h a s e (reaction d, a b o v e ) . MATERIALS AND METHODS -MATERIALS Generous amounts of hydroxymethylpyrimidine, bromomethylpyrimidine dihydrobromide, and thiazole were kindly supplied by Merck and Co., Inc. Crystalline ATP was purchased from Pabst Laboratories; Dowex 1 from Dow Chemical Company; intestinal alkMine phosphatase from Pentex, Inc.; thiamine hydrochloride, pyridoxal hydrochloride, pyridoxal phosphate, and pyridoxamine dihydrochloride from Nutritional Biochemicals Corporation; pyridoxamine phosphate from California Corporation for Biochemical Research; and pyridoxine hydroehloride from Hoffmann-LaRoche, Inc. Thiazole-P was prepared as described earlier (11). PREPARATION OF PHOSPHATE AND PYROPHOSPHATE ESTERS OF HYDROXYMETHYLPYRIMIDINE The pyrophosphate ester of hydroxymethylpyrimidine was prepared from bromomethylpyrimidine in a novel way. Bromomethylpyrimidine dihydrobromide (80 rag.) was dissolved in 1 ml. of a saturated solution of sodium pyrophosphate and allowed to stand at room temperature for 4 hr. The pH of the solution was approximately 7.5. A saturated solution of barium hydroxide was then added until the pH had risen to 9.0. The white precipitate of barium pyrophosphate was separated from the solution by centrifugation. Analysis of the supernatant fluid by paper chromatographic and bioautographie methods indicated the presence of hydroxymethylpyrimidine, hydroxymethylpyrimidine-PP, and a somewhat smaller amount of hydroxymethylpyrimidine-P. The pyrophosphate ester was separated from the other compounds by chromatography on Dowex 1-formate (20(~400 mesh, 10X). For this purpose, a column of Dowex 1-formate was prepared 2.0 cm. in diameter and 28 cm. long. The solution contain-
ing the pyrimidine compounds was applied to the column, and a linear gradient elution of the column was immediately begun. The eluting solution was scaled to proceed from 0 to 0.6 M ammonium formate at a rate of increase of 0.6 M/1. The effluent liquid from the column was collected in 20-ml. fractions, and each fraction was analyzed for ultraviolet light-absorbing material at 245 mg. A large amount of such material was found in the fractions that were eluted from the column with 0-0.4 M an~monium formate; another compound began to elute with approximately 0.55 M ammonium formate. In order to elute all of the latter compound from the resin, an exti-a 200 ml. of 0.6 M ammonium formate was passed through the column. The fast-eluting material proved to be a mixture of hydroxymethylpyrimidine and hydroxymethylpyrimidine-P, and the compound that eluted with 0.6 M ammonium formate was hydroxymethylpyrimidine-PP. The latter compound was identified by: (a) its absorption spectrum, which was identical with hydroxymethylpyrimidine both in acid (pH 3.0) and alkali (pH 9.0); (b) a ratio of phosphate to pyrimidine of 2 [phosphate was determined by the method of Fiske and SubbaRow (15) and pyrimidine was estimated spectrophotometrically at 245 mg; molar extinction coefficient = 9.6 X 103]; (c) paper chromatographic evidence also showed that its migration characteristics were identical with those of enzymically formed hydroxymethylpyrimidinePP; and (d) enzymic evidence that the compound could be converted to thiamine-P in the presence of thiazole-P, Mg ++, and an enzyme preparation from yeast. The amount of pyrophosphate ester recovered from the column has varied in different individual preparations from 15 to 25% of the total bromomethylpyrimidine used as reactant. Hydroxymethylpyrimidine-P was prepared in a similar way by dissolving the bromomethylpyrimidine in a saturated solution of Na~HPO,. The same isolation procedure was followed except that the size of the column was 2.0 cm. X 20 cm., and a linear gradient elution scaled to increase from 0 to 0.4 M ammonium formate at a rate of 0.4 M~ 500 nil. was used. Hydroxymethylpyrimidine-P was eluted with approximately 0.4 M ammonium formate and was found by paper chromatographic techniques to be a single component. The idea for this method of preparation of these phosphate esters originated from the suspicion that bromomethylpyrimidine, when dissolved in water, was being hydrolyzed to hydroxymethylpyrimidine. It was felt that in the presence of large amounts of phosphate or pyrophosphate the compound might be phosphorylyzed or pyrophosphorolyzed.
BIOSYNTHESIS OF THIAMINE. IV
199
MICROBIOLOGICAL METHODS
TABLE I
Thiamine was determined by microbiological assay with Lactobacillus viridescens (A.T.C.C. No. 12706) according to the nlethods described previously (7). Thiamine-P is also active in this assay, but neither hydroxymethylpyrimidine nor thiazole nor a mixture of the two is active. For the sake of convenience, data will be presented as "thiamine produced"; however, in all instances most of the "thiamine" formed enzymically was actually thiamine-P. Paper chromatographic separation of hydroxymethylpyrimidine, hydroxymethylpyrimidine-P, and hydroxymethylpyrimidine-PP was accomplished by the use of isobutyric acid-NH,OHwater (198:3:99,v/v/v) as the solvent system. The ascending technique of developing the ehromatograms was used in all experiments to be described. Zones of migration were located by bioautography with a hydroxymethylpyrimidine-requiring mutant of Aerobacter aerogenes PD-1 as the test organism. Since this organism cannot utilize the phosphate esters of hydroxymethylpyrimidine for growth, the ehromatogram first had to be sprayed with a phosphatase preparation (intestinal alkaline phosphatase) to dephosphorylate the esters. Specific directions for carrying out this operation as well as the details of preparation of the bioautograms are described in earlier publications (1, 7). The details of the bioautographie methods used to distinguish between thiazole and thiazole-P have also been published (11).
INHIBITION OF ENZYMIC SYNTHESIS OF THIAMINE BY VITAMIN B6 COMPOUNDS
Reaction mixtures contained in 1 ml. : hydroxymethylpyrimidine, 58 uM; thiazole, 36 ~M; ATP, 5 mM; MgC12, 0.01 M; vitamin B6 compound, 10 mM; 0.1 M phosphate buffer; and extract of yeast equivalent to 4 mg. protein. Reaction mixtures were incubated for 2 hr. at 37 ~ heated at 100~ (water bath) for 5 min., centrifuged to eliminate precipitated protein, and analyzed for thiamine by microbiological assay with L. viridescens. Vitamin Be compound added to reaction mixture
mttmole$
None Pyridoxal Pyridoxine Pyridoxamine Pyridoxal-P Pyridoxamine-P
RESULTS T h e effects of v i t a m i n Be c o m p o u n d s o n t h i a m i n e synthesis b y extracts of y e a s t are s h o w n i n T a b l e I. P y r i d o x a l - P i n h i b i t e d synthesis significantly; p y r i d o x a l a p p e a r e d to i n h i b i t to a lesser extent, a n d all o t h e r v i t a m i n Be c o m p o u n d s exhibited no inh i b i t o r y properties. E a c h i n h i b i t o r y comp o u n d was t h e n tested a t v a r i o u s concent r a t i o n s . T h e d a t a of T a b l e I I d e m o n s t r a t e t h a t m a x i m u m i n h i b i t i o n is achieved a t a c o n c e n t r a t i o n of a p p r o x i m a t e l y 20 m M . T h e e x p e r i m e n t a l d a t a of T a b l e s I a n d I I were o b t a i n e d b y using h y d r o x y m e t h y l p y r i m i d i n e , thiazole, a n d A T P as s u b s t r a t e s for t h i a m i n e synthesis. However, t h i a m i n e synthesis f r o m these s u b s t r a t e s is k n o w n to be d e p e n d e n t on the a c t i o n of a t least four
7.8 6.0 8.4 8.0 4.0 8.4
TABLE II AMOUNT OF VITAMIN B6 COMPOUND NEEDED TO INHIBIT ENZYMIC SYNTHESIS OF THIAMINE
Reaction mixtures were prepared as described in Table I except that the vitamin B6 conlpound was added in the amounts shown. Incubation conditions and analytical procedure were as described in Table I.
ENZYME PREPARATION An extract of baker's yeast was prepared by an autolysis procedure described earlier (10).
Thiamine produced
Vitamin B6 compound added
Experiment 1 None Pyridoxal Pyridoxal Pyridoxal Experiment 2 None Pyridoxal-P Pyridoxal-P Pyridoxal-P Pyridoxal-P
Concentration of vitamin Be compound
Thiamine produced
mM
m~moles
0 10 20 30
6.2 4.6 4.2 4.0
0 10 20 30 40
5.2 2.5 1.3 1.3 1.4
e n z y m e s : h y d r o x y m e t h y l p y r i m i d i n e kinase, h y d r o x y m e t h y l p y r i m i d i n e phosphokinase, thiazole kinase, a n d t h i a m i n e - P synthase. F r o m the e x p e r i m e n t s s u m m a r i z e d i n T a b l e s I a n d I I , it is n o t possible to decide which of the four e n z y m i c reactions m i g h t
200
LEWIN AND BROWN
be inhibited by pyridoxal and pyridoxal-P. Of the four enzymes listed above, thiamine-P synthase is the easiest to use to obtain quantitative data, since a reliable assay has been developed to measure thiamine-P formation; namely the microbiological assay with L. viridescens. When hydroxymethylpyrimidine-PP and thiazole-P are added to reaction mixtures as suhstrates, thiamine (as thiamine-P) synthesis becomes dependent only on the action of thiamine-P synthase. It is then possible to examine the effects of various vitamin Be compounds on the specific reaction catalyzed by this enzyme. The results given in Table I I I show that only pyridoxal-P had an inhibitory effect on the formation of thiamine. The other compounds appeared to have no significant effects on the reaction. Two sets of data are shown; one for a reaction time of 120 rain. and the other for a reaction time of 20 min. The two are given as representative sets of data (a) at an incubation time when thiamine synthesis is maximal (120 rain.) and (b) at a time when thiamine synthesis is proceeding linearly (20 min.) with respect to time (see Fig. 2 in a later section of this paper). No reliable methods have been devised TABLE III INHIBITION OF ENZYMIC SYNTHESIS OF THIAMINE BY VITAMIN B6 COMPOUNDS: USE OF HYDROXYMETHyLPYRIMIDINE-PP AS SUBSTRATE R e a c t i o n mixtures c o n t a i n e d per 1 ml.: hyd r o x y m e t h y l p y r i m i d i n e - P P , 78 uM; thiazole-P, 36 uM; MgCl~, 0.01 M: v i t a m i n Be compound, 10 raM; p h o s p h a t e buffer (pH 7.4), 0.1 M; and yea,st e x t r a c t equivalent to 4 mg. protein. I n c u b a t i o n was for 20 min. (Expt. 1), and 120 min. (Expt. 2). O t h e r i n c u b a t i o n conditions and t h e analytical procedure were as described in T a b l e I. Thiamine produced Vitamin Be compound added
None Pyridoxal Pyridoxine Pyridoxamine Pyridoxal-P Pyridoxamine-P
Experiment 1
Experiment 2
m~moles
m~moles
13.0 13.5 12.1 12.1 3.9 12.1
35.0 36.0 32.2 36.1 16.2 39.6
to measure quantitatively the action of hydroxymethylpyrimidine kinase, hydroxymethylpyrimidine phosphokinase, and thiazole kinase. However, it is possible to observe the formation of the compounds produced by the action of each of these enzymes by paper chromatographic and bioautographic techniques, and, from the sizes of the growth zones on the bioautograms, it is possible to estimate differences of approximately twofold or more in the amounts of products formed. The action of hydroxymethylpyrimidine kinase and the effects of vitamin B6 compounds on the reaction were observed as follows: Hydroxymethylpyrimidine, ATP, and MgC12 were incubated with yeast extract in the presence and absence of the various vitamin Be compounds (the concentrations of these components of the reaction mixture were as given in Table I), and a 0.005-ml. aliquot of each reaction mixture was spotted on paper. After development, the paper chromatogram was sprayed with a phosphatase and then used to prepare a bioautogram with the hydroxymethylpyrimidine-requiring mutant of A. aerogenes as the test organism. From examination of the growth zones on the bioautogram it appeared that none of the vitamin Be compounds listed in Table I had any perceptible effect on the formation of hydroxymethylpyrimidine-P. Since this compound serves as substrate for hydroxymethylpyrimidine phosphokinase, small amounts of hydroxymethylpyrimidine-PP were also found in these reaction mixtures, and the amounts formed appeared not to be diminished in the presence of vitamin Be compounds. In order to test more directly the effects of vitamin Be compounds on the formation of hydroxymethylpyrimidine-PP, hydroxymethylpyrimidine-P was incubated with ATP and the yeast enzyme preparation in the presence and absence of vitamin Be compounds, and the reaction mixtures were analyzed bioautographically as described above. Again, it appeared that none of the vitamin Be compounds inhibited the formation of the pyrophosphate ester. Similar experiments have revealed that the vitamin B6 compounds also have no apparent effect on the formation of thiazole-P
201
B I O S Y N T H E S I S OF T H I A M I N E . IV
from thiazole and ATP. In the latter experiments, a thiazole-requiring mutant of E. coli was used as the test organism in the preparation of the bioautograms (11). It would appear from the results cited above that the major, if not the only, effect of pyridoxal-P on thiamine synthesis is to inhibit the enzymic conversion of hydroxymethylpyrimidine-PP and thiazole-P to thiamine-P. The amounts of pyridoxal-P needed to inhibit this reaction are shown in Fig. 1. It should be noted that the concentration of pyridoxal-P needed to exhibit significant inhibitory properties is at least 25 times the concentration of the substrate (hydroxymethylpyrimidine-PP). In order to establish whether the inhibition was of the competitive or noncompetitive type, it was necessary to perform some kinetic experiments. The data plotted in Fig. 2 show that thiamine synthesis is linear with time up to 30 rain. Therefore, the measurement of thiamine produced for any incubation period up to 30 rain. could be defined also as the measurement of the velocity of the reaction. In the kinetic experiments to be described below, incubation periods of 20 rain. were used. For the kinetic experiments to be valid, it was necessary to show that in the prescribed incubation period the reactants were not degraded by contaminating enzymes. Bioautographic studies established that, under the conditions used
2ol
0
/
I0
20
.
.
.
.
.
:50 40 50 60 TIME,MINUTES
.
70
80
90
FIG. 2. Enzymic synthesis of t h i a m i n e as a f u n c t i o n of the time of i n c u b a t i o n , Reaction mixtures were p r e p a r e d a n d analyzed as described in T a b l e III, except t h a t no v i t a m i n B0 compound was added. o
~
0.6 0.5
PLUS P Y R I ~
0.4. Q3
/ ~ I/K.
0.2 ~ , / /
/ o
MENUS PYRID~P
.
~ , /
,...._......-"./ o 6 21o io 4o I/(S)xl0-3
so do
F]6. 3. A Lineweaver-Bnrk plot showing the
15 I
".
effect of t h e c o n c e n t r a t i o n of s u b s t r a t e (hydroxym e t h y l p y r i m i d i n e - P P ) on t h e enzymic synthesis of t h i a m i n e in t h e presence a n d absence of pyridoxal-P. R e a c t i o n mixtures were p r e p a r e d a n d analyzed as described in Table I I I , except t h a t t h e c o n c e n t r a t i o n of h y d r o x y m e t h y l p y r i m i d i n e was varied as shown.
O
~_so
1-
O0
46 8 I0 12 PYRIDOXAL-P,m M FIG. 1. T h e a m o u n t of pyridoxal-P needed to i n h i b i t the enzymic synthesis of thiamine. Reaction mi• were prepared a n d analyzed as described in T a b l e III. I n c u b a t i o n was for 120
min.
2
in the kinetic experiment to be described, only a trace (estimated to be less than 1%) of hydroxymethylpyrimidine-PP was degraded to the monophosphate. None of the thiazole-P and pyridoxal-P could be shown to be degraded. The rate of formation of thiamine-P was determined at several different levels of substrate (hydroxymethylpyrimidine-PP) concentration in the presence and absence of pyridoxal-P. Line-
202
LEWIN AN]) BROWN
weaver-Burk plots of the data (Fig. 3) indicate that the inhibition corresponds to a "mixed" type i.e., a mixture of competitive and noncompetitive effects. The K~ for hydroxymethylpyrimidine-PP can be shown from the data plotted in Fig. 3 to be 3.7 X 10-5 M. This value is greater than the 1 X 10-B M value reported by Leder (12); however, it should be pointed out that the determinations were made under different sets of conditions. DISCUSSION The results of the present investigation suggest that the in vivo inhibition of thiamine synthesis by vitamin B6 noted by Harris (2, 3) can be explained as an inhibition by pyridoxal-P of the enzymic conversion of hydroxymethylpyrimidine-FP and thiazoleP to thiamine-P. Pyridoxal was also somewhat inhibitory when hydroxymethylpyrimidine, thiazole, and ATP were used as substrates, but this compound appeared not to be inhibitory when hydroxymethylpyrimidine-PP and thiazole-P were used as substrates; hence, the possibility remains that pyridoxal might exert a low-level inhibitory effect on one or both of the enzymic reactions required to convert hydroxymethylpyrimidine to its pyrophosphate ester. The bioautographic method used to detect differences in formation of these esters is not sensitive enough to detect subtle differences in the amounts of the phosphate esters formed. The data indicate, however, that the mQor effect of vitamin Be on thiamine biosynthesis appears to be an inhibition by pyridoxal-P of the reaction catalyzed by thiamine-P synthase. Other compounds that have been tested in this laboratory and found to be ineffective as inhibitors of any of the enzymic reactions concerned with the synthesis of thiamine are: hydroxymethylcytosine, 2-methyl-4amino-5-aminomethylpyrimidine, and 2methyl - 4 - amino - 5 - methoxymethylpyrimidine. Also, hydroxymethylpyrimidine and hydroxymethylpyrimidine-P have been found to have no effect on the utilization of hydroxymethylpyrimidine-PP in the reaction catalyzed by thiamine-P synthase. The nature of the types of inhibition which
could theoretically function to give the type of "mixed" inhibition noted in the present study deserves some consideration. Dixon and Webb (16) have considered that this type of inhibition phenomenon is likely to be a resultant of two types of inhibition occurring simultaneously: namely, a partially competitive type and a purely noncompetitive type. In both of these kinds of inhibition, the inhibitor probably combines with the enzyme-substrate complex (ES ~ EIS) at a site on the protein different from the site of attachment of the substrate. In the partially competitive type, EIS decomposes at a definite rate to give the enzymeinhibitor complex (EI) plus product, whereas in the noncompetitive type no such decomposition is thought to occur (16). Since nothing is known about the chemical and physical structure of thiamine-P synthase, no data can yet be offered which might shed light on the possible sites of attachment of either the substrate or pyridoxal-P. Harris (2-4) has suggested that a vitamin B6 compound might actually react enzymically in the thiamine-synthesizing system to yield a product which would contain thiazole joined to vitamin B6 in place of the pyrimidine. From what is known of the reactions involved in the biosynthesis of thiamine, it seems reasonable to suppose that if this suggestion is correct, the pyrophosphate ester of pyridoxal would have to be formed in order to react with thiazole-P. We obtained no evidence for the formation of such a pyrophosphate ester of pyridoxal under any condition (i.e., in the presence and absence of ATP and/or hydroxymethylpyrimidine-PP); thus, it appears unlikely that a pyridoxal-thiazole compound is formed, although the present state of knowledge does not conclusively rule out the possibility (e.g., pyridoxal-P possibly could react with thiazole-P to yield such a compound). Since the concentration of pyridoxal-P required to inhibit thiamine synthesis is relatively high, it appears unlikely that the intracellular concentration of this substance would ever be great enough to cause a significant decrease of thiamine formation except under contrived conditions, such as
BIOSYNTHESIS OF THIAMINE. IV t h e case in w h i c h a large a m o u n t of v i t a m i n B6 is a d d e d to t h e g r o w t h m e d i u m of t h e microorganism. ACKNOWLEDGMENT We wish to thank Mrs. Chai Ho Lo for her skillful technical assistance during the course of these investigations. REFERENCES 1. LEWlN, L. M., AND BROWN, G. M., J. Biol. Chem. 236, 2768 (1961). 2. HARRIS, D. L., Federation Proc. 11,226 (1952). 3. H.~Rms, D. L., Arch. Biochern. Biophys. 41, 294 (1952). 4. HARRIS, D. L., Arch. Biochem. Biophys. 60, 35 (1956). 5. KAWASAKI,T., and FUJITA, T., Seikagaku 33, 737 (1961); C. A. 56, 7676 (1962). 6. KAWASAKI,T., AND FUJITA, T., Seikagaku 33, 742 (1962); C. A. 56, 7676 (1962).
203
7. CAMIENER, G. W., ANn BROWN, G. M., J. Biol. Chem. 235, 2404 (1960). 8. LEDER, I. G., Biochem. Biophys. Res. Commun. 1, 63 (1959). 9. NOSE, Y., I~EDA, K., AND KAWASAKI, T., Biochim. Biophys. Acta 34,277 (1959). 10. CAMIENER, G. W., AND BROWN, G. M., J. Am. Chem. Soe. 81, 3800 (1959). 11. CAMIENER,G. W., AND BROWN, G, M., J. Biol. Chem. 9.35, 2411 (1960). 12. LEDER, I. G., J. Biol. Chem. 236, 3066 (1961). 13. NOSE, T., UEDA, I~., KAWASAKI, T., IWASHIMK, i . , ANn FUJITA, T., J. Vitaminol. (Kyoto) 7, 98 (1962).
14. LATtIAM, I-L, Master's Thesis, Massachusetts Institute of Technology, Cambridge, 1961. 15. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 66, 375 (1925). 16. DIXON, M., AND WEBB, E. C., "Enzymes," p. 178. Academic Press, New York, 1958.
(1963)
The Biosynthesis of Thiamine. IV. 1 Inhibition by Vitamin Be Compounds L A W R E N C E M. L E W I N AND G E N E M. B R O W N From the Division of Biochemistry, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts Received January 24, 1963 A novel method for the synthesis of tile mono- and pyrophosphate esters of 2-methyl-4-amino-5-hydroxymethylpyrimidine has been described. The inhibitory effect of vitamin Be compounds on the biosynthesis of thiamine has been shown to be due primarily to the inhibition by pyridoxal phosphate of the enzymic reaction whereby thiamine monophosphate is formed from the monophosphate ester of 4methyl-5-(2-hydroxyethyl)thiazole and the pyrophosphate ester of 2-methyl-4-amino-5-hydroxymethylpyrimidine. Kinetic experiments indicated that the inhibition is of a "mixed" type, i.e., a mixture of a purely noncompetitive type and a partially competitive type (partially competitive with the pyrophosphate ester of 2-methyl-4-amino-5-hydroxymethylpyrimidine). INTRODUCTION Harris has reported that an excess of vitamin B6 inhibits the growth of Neurospora (2) and the net synthesis of thiamine by this organism (3) apparently by interfering with the enzymic system whereby hydroxymethylpyrimidine2 and thiazole ~ are utilized to form thiamine (4). The inhibition appeared to be competitive between vitamin B6 and hydroxymethylpyrimidine, although the author was careful to point out t h a t this conclusion could be only a tentative one since the experimental data were obtained by using whole cells (4). 1 This work was supported by grants A-3442 from the National Institutes of Health and G-4580 from the National Science Foundation. For Paper III of this series see Ref. (1). 2Abbreviations used are: hydroxymethylpyrimidine, hydroxymethylpyrimidine-P, and hydroxymethylpyrimidine-PP for 2-methyl-4amino-5-hydroxymethylpyrimidine, the monophosphate ester of this compound, and the pyrophosphate, respectively; thiazole and thiazole-P for 4-methyl-5-(2-hydroxyethyl)thiazole and the monophosphate ester of this compound, respectively; thiamine-P for thiamine monophosphate; bromomethylpyrimidine for 2-methyl-4-amino-5bromomethylpyrimidine; and ATP for adenosine triphosphate.
The results of the work of several groups of investigators have been instrumental in establishing that the enzymic synthesis of thiamine from hydroxymethylpyrimidine and thiazole proceeds as follows: (a) hydroxymethylpyrimidine
ATP
)
hydroxymethylpyrimidine-P (1, 5-7) (b) hydroxymethylpyrimidine-P
ATP
>
hydroxymethylpyrimidine-PP (1, 5-7) (e) thiazole
ATP
) thiazole-P (8-11)
(d) hydroxymethylpyrimidine-PP + thiazole-P ~- thiamine-P + PPI (8-13) (e) thiamine-P H20) thiamine + Pi (8-13) The enzymes that catalyze reactions (a-e) have been named, respectively, hydroxymethylpyrimidine kinase (1), hydroxymethylpyrimidine phosphokinase (1), thiazole kinase (11), thiamine-P synthase (11) or thiamine-P pyrophosphorylase (12) (since the reaction is reversible), and thiamine-P phosphatase. There is no evidence that a specific phosphatase is involved in reaction e. I n fact, it would appear that,
197
198
LEWIN AND BROWN
in yeast, this r e a c t i o n is c a t a l y z e d o n l y by nonspecific p h o s p h a t a s e s (14). T h e e l u c i d a t i o n of t h e e n z y m i c steps i n v o l v e d in t h i a m i n e synthesis p r o v i d e d t h e o p p o r t u n i t y to i n v e s t i g a t e t h e effects of v i t a m i n B6 c o m p o u n d s on each of these e n z y m i c reactions. T h e results p r e s e n t e d i n d i c a t e t h a t t h e i n h i b i t i o n of t h i a m i n e synthesis b y v i t a m i n B6 c o m p o u n d s c a n be ascribed p r i m a r i l y t o a n iHhibitory effect of p y r i d o x a l - P on t h e r e a c t i o n c a t a l y z e d b y t h i a m i n e - P s y n t h a s e (reaction d, a b o v e ) . MATERIALS AND METHODS -MATERIALS Generous amounts of hydroxymethylpyrimidine, bromomethylpyrimidine dihydrobromide, and thiazole were kindly supplied by Merck and Co., Inc. Crystalline ATP was purchased from Pabst Laboratories; Dowex 1 from Dow Chemical Company; intestinal alkMine phosphatase from Pentex, Inc.; thiamine hydrochloride, pyridoxal hydrochloride, pyridoxal phosphate, and pyridoxamine dihydrochloride from Nutritional Biochemicals Corporation; pyridoxamine phosphate from California Corporation for Biochemical Research; and pyridoxine hydroehloride from Hoffmann-LaRoche, Inc. Thiazole-P was prepared as described earlier (11). PREPARATION OF PHOSPHATE AND PYROPHOSPHATE ESTERS OF HYDROXYMETHYLPYRIMIDINE The pyrophosphate ester of hydroxymethylpyrimidine was prepared from bromomethylpyrimidine in a novel way. Bromomethylpyrimidine dihydrobromide (80 rag.) was dissolved in 1 ml. of a saturated solution of sodium pyrophosphate and allowed to stand at room temperature for 4 hr. The pH of the solution was approximately 7.5. A saturated solution of barium hydroxide was then added until the pH had risen to 9.0. The white precipitate of barium pyrophosphate was separated from the solution by centrifugation. Analysis of the supernatant fluid by paper chromatographic and bioautographie methods indicated the presence of hydroxymethylpyrimidine, hydroxymethylpyrimidine-PP, and a somewhat smaller amount of hydroxymethylpyrimidine-P. The pyrophosphate ester was separated from the other compounds by chromatography on Dowex 1-formate (20(~400 mesh, 10X). For this purpose, a column of Dowex 1-formate was prepared 2.0 cm. in diameter and 28 cm. long. The solution contain-
ing the pyrimidine compounds was applied to the column, and a linear gradient elution of the column was immediately begun. The eluting solution was scaled to proceed from 0 to 0.6 M ammonium formate at a rate of increase of 0.6 M/1. The effluent liquid from the column was collected in 20-ml. fractions, and each fraction was analyzed for ultraviolet light-absorbing material at 245 mg. A large amount of such material was found in the fractions that were eluted from the column with 0-0.4 M an~monium formate; another compound began to elute with approximately 0.55 M ammonium formate. In order to elute all of the latter compound from the resin, an exti-a 200 ml. of 0.6 M ammonium formate was passed through the column. The fast-eluting material proved to be a mixture of hydroxymethylpyrimidine and hydroxymethylpyrimidine-P, and the compound that eluted with 0.6 M ammonium formate was hydroxymethylpyrimidine-PP. The latter compound was identified by: (a) its absorption spectrum, which was identical with hydroxymethylpyrimidine both in acid (pH 3.0) and alkali (pH 9.0); (b) a ratio of phosphate to pyrimidine of 2 [phosphate was determined by the method of Fiske and SubbaRow (15) and pyrimidine was estimated spectrophotometrically at 245 mg; molar extinction coefficient = 9.6 X 103]; (c) paper chromatographic evidence also showed that its migration characteristics were identical with those of enzymically formed hydroxymethylpyrimidinePP; and (d) enzymic evidence that the compound could be converted to thiamine-P in the presence of thiazole-P, Mg ++, and an enzyme preparation from yeast. The amount of pyrophosphate ester recovered from the column has varied in different individual preparations from 15 to 25% of the total bromomethylpyrimidine used as reactant. Hydroxymethylpyrimidine-P was prepared in a similar way by dissolving the bromomethylpyrimidine in a saturated solution of Na~HPO,. The same isolation procedure was followed except that the size of the column was 2.0 cm. X 20 cm., and a linear gradient elution scaled to increase from 0 to 0.4 M ammonium formate at a rate of 0.4 M~ 500 nil. was used. Hydroxymethylpyrimidine-P was eluted with approximately 0.4 M ammonium formate and was found by paper chromatographic techniques to be a single component. The idea for this method of preparation of these phosphate esters originated from the suspicion that bromomethylpyrimidine, when dissolved in water, was being hydrolyzed to hydroxymethylpyrimidine. It was felt that in the presence of large amounts of phosphate or pyrophosphate the compound might be phosphorylyzed or pyrophosphorolyzed.
BIOSYNTHESIS OF THIAMINE. IV
199
MICROBIOLOGICAL METHODS
TABLE I
Thiamine was determined by microbiological assay with Lactobacillus viridescens (A.T.C.C. No. 12706) according to the nlethods described previously (7). Thiamine-P is also active in this assay, but neither hydroxymethylpyrimidine nor thiazole nor a mixture of the two is active. For the sake of convenience, data will be presented as "thiamine produced"; however, in all instances most of the "thiamine" formed enzymically was actually thiamine-P. Paper chromatographic separation of hydroxymethylpyrimidine, hydroxymethylpyrimidine-P, and hydroxymethylpyrimidine-PP was accomplished by the use of isobutyric acid-NH,OHwater (198:3:99,v/v/v) as the solvent system. The ascending technique of developing the ehromatograms was used in all experiments to be described. Zones of migration were located by bioautography with a hydroxymethylpyrimidine-requiring mutant of Aerobacter aerogenes PD-1 as the test organism. Since this organism cannot utilize the phosphate esters of hydroxymethylpyrimidine for growth, the ehromatogram first had to be sprayed with a phosphatase preparation (intestinal alkaline phosphatase) to dephosphorylate the esters. Specific directions for carrying out this operation as well as the details of preparation of the bioautograms are described in earlier publications (1, 7). The details of the bioautographie methods used to distinguish between thiazole and thiazole-P have also been published (11).
INHIBITION OF ENZYMIC SYNTHESIS OF THIAMINE BY VITAMIN B6 COMPOUNDS
Reaction mixtures contained in 1 ml. : hydroxymethylpyrimidine, 58 uM; thiazole, 36 ~M; ATP, 5 mM; MgC12, 0.01 M; vitamin B6 compound, 10 mM; 0.1 M phosphate buffer; and extract of yeast equivalent to 4 mg. protein. Reaction mixtures were incubated for 2 hr. at 37 ~ heated at 100~ (water bath) for 5 min., centrifuged to eliminate precipitated protein, and analyzed for thiamine by microbiological assay with L. viridescens. Vitamin Be compound added to reaction mixture
mttmole$
None Pyridoxal Pyridoxine Pyridoxamine Pyridoxal-P Pyridoxamine-P
RESULTS T h e effects of v i t a m i n Be c o m p o u n d s o n t h i a m i n e synthesis b y extracts of y e a s t are s h o w n i n T a b l e I. P y r i d o x a l - P i n h i b i t e d synthesis significantly; p y r i d o x a l a p p e a r e d to i n h i b i t to a lesser extent, a n d all o t h e r v i t a m i n Be c o m p o u n d s exhibited no inh i b i t o r y properties. E a c h i n h i b i t o r y comp o u n d was t h e n tested a t v a r i o u s concent r a t i o n s . T h e d a t a of T a b l e I I d e m o n s t r a t e t h a t m a x i m u m i n h i b i t i o n is achieved a t a c o n c e n t r a t i o n of a p p r o x i m a t e l y 20 m M . T h e e x p e r i m e n t a l d a t a of T a b l e s I a n d I I were o b t a i n e d b y using h y d r o x y m e t h y l p y r i m i d i n e , thiazole, a n d A T P as s u b s t r a t e s for t h i a m i n e synthesis. However, t h i a m i n e synthesis f r o m these s u b s t r a t e s is k n o w n to be d e p e n d e n t on the a c t i o n of a t least four
7.8 6.0 8.4 8.0 4.0 8.4
TABLE II AMOUNT OF VITAMIN B6 COMPOUND NEEDED TO INHIBIT ENZYMIC SYNTHESIS OF THIAMINE
Reaction mixtures were prepared as described in Table I except that the vitamin B6 conlpound was added in the amounts shown. Incubation conditions and analytical procedure were as described in Table I.
ENZYME PREPARATION An extract of baker's yeast was prepared by an autolysis procedure described earlier (10).
Thiamine produced
Vitamin B6 compound added
Experiment 1 None Pyridoxal Pyridoxal Pyridoxal Experiment 2 None Pyridoxal-P Pyridoxal-P Pyridoxal-P Pyridoxal-P
Concentration of vitamin Be compound
Thiamine produced
mM
m~moles
0 10 20 30
6.2 4.6 4.2 4.0
0 10 20 30 40
5.2 2.5 1.3 1.3 1.4
e n z y m e s : h y d r o x y m e t h y l p y r i m i d i n e kinase, h y d r o x y m e t h y l p y r i m i d i n e phosphokinase, thiazole kinase, a n d t h i a m i n e - P synthase. F r o m the e x p e r i m e n t s s u m m a r i z e d i n T a b l e s I a n d I I , it is n o t possible to decide which of the four e n z y m i c reactions m i g h t
200
LEWIN AND BROWN
be inhibited by pyridoxal and pyridoxal-P. Of the four enzymes listed above, thiamine-P synthase is the easiest to use to obtain quantitative data, since a reliable assay has been developed to measure thiamine-P formation; namely the microbiological assay with L. viridescens. When hydroxymethylpyrimidine-PP and thiazole-P are added to reaction mixtures as suhstrates, thiamine (as thiamine-P) synthesis becomes dependent only on the action of thiamine-P synthase. It is then possible to examine the effects of various vitamin Be compounds on the specific reaction catalyzed by this enzyme. The results given in Table I I I show that only pyridoxal-P had an inhibitory effect on the formation of thiamine. The other compounds appeared to have no significant effects on the reaction. Two sets of data are shown; one for a reaction time of 120 rain. and the other for a reaction time of 20 min. The two are given as representative sets of data (a) at an incubation time when thiamine synthesis is maximal (120 rain.) and (b) at a time when thiamine synthesis is proceeding linearly (20 min.) with respect to time (see Fig. 2 in a later section of this paper). No reliable methods have been devised TABLE III INHIBITION OF ENZYMIC SYNTHESIS OF THIAMINE BY VITAMIN B6 COMPOUNDS: USE OF HYDROXYMETHyLPYRIMIDINE-PP AS SUBSTRATE R e a c t i o n mixtures c o n t a i n e d per 1 ml.: hyd r o x y m e t h y l p y r i m i d i n e - P P , 78 uM; thiazole-P, 36 uM; MgCl~, 0.01 M: v i t a m i n Be compound, 10 raM; p h o s p h a t e buffer (pH 7.4), 0.1 M; and yea,st e x t r a c t equivalent to 4 mg. protein. I n c u b a t i o n was for 20 min. (Expt. 1), and 120 min. (Expt. 2). O t h e r i n c u b a t i o n conditions and t h e analytical procedure were as described in T a b l e I. Thiamine produced Vitamin Be compound added
None Pyridoxal Pyridoxine Pyridoxamine Pyridoxal-P Pyridoxamine-P
Experiment 1
Experiment 2
m~moles
m~moles
13.0 13.5 12.1 12.1 3.9 12.1
35.0 36.0 32.2 36.1 16.2 39.6
to measure quantitatively the action of hydroxymethylpyrimidine kinase, hydroxymethylpyrimidine phosphokinase, and thiazole kinase. However, it is possible to observe the formation of the compounds produced by the action of each of these enzymes by paper chromatographic and bioautographic techniques, and, from the sizes of the growth zones on the bioautograms, it is possible to estimate differences of approximately twofold or more in the amounts of products formed. The action of hydroxymethylpyrimidine kinase and the effects of vitamin B6 compounds on the reaction were observed as follows: Hydroxymethylpyrimidine, ATP, and MgC12 were incubated with yeast extract in the presence and absence of the various vitamin Be compounds (the concentrations of these components of the reaction mixture were as given in Table I), and a 0.005-ml. aliquot of each reaction mixture was spotted on paper. After development, the paper chromatogram was sprayed with a phosphatase and then used to prepare a bioautogram with the hydroxymethylpyrimidine-requiring mutant of A. aerogenes as the test organism. From examination of the growth zones on the bioautogram it appeared that none of the vitamin Be compounds listed in Table I had any perceptible effect on the formation of hydroxymethylpyrimidine-P. Since this compound serves as substrate for hydroxymethylpyrimidine phosphokinase, small amounts of hydroxymethylpyrimidine-PP were also found in these reaction mixtures, and the amounts formed appeared not to be diminished in the presence of vitamin Be compounds. In order to test more directly the effects of vitamin Be compounds on the formation of hydroxymethylpyrimidine-PP, hydroxymethylpyrimidine-P was incubated with ATP and the yeast enzyme preparation in the presence and absence of vitamin Be compounds, and the reaction mixtures were analyzed bioautographically as described above. Again, it appeared that none of the vitamin Be compounds inhibited the formation of the pyrophosphate ester. Similar experiments have revealed that the vitamin B6 compounds also have no apparent effect on the formation of thiazole-P
201
B I O S Y N T H E S I S OF T H I A M I N E . IV
from thiazole and ATP. In the latter experiments, a thiazole-requiring mutant of E. coli was used as the test organism in the preparation of the bioautograms (11). It would appear from the results cited above that the major, if not the only, effect of pyridoxal-P on thiamine synthesis is to inhibit the enzymic conversion of hydroxymethylpyrimidine-PP and thiazole-P to thiamine-P. The amounts of pyridoxal-P needed to inhibit this reaction are shown in Fig. 1. It should be noted that the concentration of pyridoxal-P needed to exhibit significant inhibitory properties is at least 25 times the concentration of the substrate (hydroxymethylpyrimidine-PP). In order to establish whether the inhibition was of the competitive or noncompetitive type, it was necessary to perform some kinetic experiments. The data plotted in Fig. 2 show that thiamine synthesis is linear with time up to 30 rain. Therefore, the measurement of thiamine produced for any incubation period up to 30 rain. could be defined also as the measurement of the velocity of the reaction. In the kinetic experiments to be described below, incubation periods of 20 rain. were used. For the kinetic experiments to be valid, it was necessary to show that in the prescribed incubation period the reactants were not degraded by contaminating enzymes. Bioautographic studies established that, under the conditions used
2ol
0
/
I0
20
.
.
.
.
.
:50 40 50 60 TIME,MINUTES
.
70
80
90
FIG. 2. Enzymic synthesis of t h i a m i n e as a f u n c t i o n of the time of i n c u b a t i o n , Reaction mixtures were p r e p a r e d a n d analyzed as described in T a b l e III, except t h a t no v i t a m i n B0 compound was added. o
~
0.6 0.5
PLUS P Y R I ~
0.4. Q3
/ ~ I/K.
0.2 ~ , / /
/ o
MENUS PYRID~P
.
~ , /
,...._......-"./ o 6 21o io 4o I/(S)xl0-3
so do
F]6. 3. A Lineweaver-Bnrk plot showing the
15 I
".
effect of t h e c o n c e n t r a t i o n of s u b s t r a t e (hydroxym e t h y l p y r i m i d i n e - P P ) on t h e enzymic synthesis of t h i a m i n e in t h e presence a n d absence of pyridoxal-P. R e a c t i o n mixtures were p r e p a r e d a n d analyzed as described in Table I I I , except t h a t t h e c o n c e n t r a t i o n of h y d r o x y m e t h y l p y r i m i d i n e was varied as shown.
O
~_so
1-
O0
46 8 I0 12 PYRIDOXAL-P,m M FIG. 1. T h e a m o u n t of pyridoxal-P needed to i n h i b i t the enzymic synthesis of thiamine. Reaction mi• were prepared a n d analyzed as described in T a b l e III. I n c u b a t i o n was for 120
min.
2
in the kinetic experiment to be described, only a trace (estimated to be less than 1%) of hydroxymethylpyrimidine-PP was degraded to the monophosphate. None of the thiazole-P and pyridoxal-P could be shown to be degraded. The rate of formation of thiamine-P was determined at several different levels of substrate (hydroxymethylpyrimidine-PP) concentration in the presence and absence of pyridoxal-P. Line-
202
LEWIN AN]) BROWN
weaver-Burk plots of the data (Fig. 3) indicate that the inhibition corresponds to a "mixed" type i.e., a mixture of competitive and noncompetitive effects. The K~ for hydroxymethylpyrimidine-PP can be shown from the data plotted in Fig. 3 to be 3.7 X 10-5 M. This value is greater than the 1 X 10-B M value reported by Leder (12); however, it should be pointed out that the determinations were made under different sets of conditions. DISCUSSION The results of the present investigation suggest that the in vivo inhibition of thiamine synthesis by vitamin B6 noted by Harris (2, 3) can be explained as an inhibition by pyridoxal-P of the enzymic conversion of hydroxymethylpyrimidine-FP and thiazoleP to thiamine-P. Pyridoxal was also somewhat inhibitory when hydroxymethylpyrimidine, thiazole, and ATP were used as substrates, but this compound appeared not to be inhibitory when hydroxymethylpyrimidine-PP and thiazole-P were used as substrates; hence, the possibility remains that pyridoxal might exert a low-level inhibitory effect on one or both of the enzymic reactions required to convert hydroxymethylpyrimidine to its pyrophosphate ester. The bioautographic method used to detect differences in formation of these esters is not sensitive enough to detect subtle differences in the amounts of the phosphate esters formed. The data indicate, however, that the mQor effect of vitamin Be on thiamine biosynthesis appears to be an inhibition by pyridoxal-P of the reaction catalyzed by thiamine-P synthase. Other compounds that have been tested in this laboratory and found to be ineffective as inhibitors of any of the enzymic reactions concerned with the synthesis of thiamine are: hydroxymethylcytosine, 2-methyl-4amino-5-aminomethylpyrimidine, and 2methyl - 4 - amino - 5 - methoxymethylpyrimidine. Also, hydroxymethylpyrimidine and hydroxymethylpyrimidine-P have been found to have no effect on the utilization of hydroxymethylpyrimidine-PP in the reaction catalyzed by thiamine-P synthase. The nature of the types of inhibition which
could theoretically function to give the type of "mixed" inhibition noted in the present study deserves some consideration. Dixon and Webb (16) have considered that this type of inhibition phenomenon is likely to be a resultant of two types of inhibition occurring simultaneously: namely, a partially competitive type and a purely noncompetitive type. In both of these kinds of inhibition, the inhibitor probably combines with the enzyme-substrate complex (ES ~ EIS) at a site on the protein different from the site of attachment of the substrate. In the partially competitive type, EIS decomposes at a definite rate to give the enzymeinhibitor complex (EI) plus product, whereas in the noncompetitive type no such decomposition is thought to occur (16). Since nothing is known about the chemical and physical structure of thiamine-P synthase, no data can yet be offered which might shed light on the possible sites of attachment of either the substrate or pyridoxal-P. Harris (2-4) has suggested that a vitamin B6 compound might actually react enzymically in the thiamine-synthesizing system to yield a product which would contain thiazole joined to vitamin B6 in place of the pyrimidine. From what is known of the reactions involved in the biosynthesis of thiamine, it seems reasonable to suppose that if this suggestion is correct, the pyrophosphate ester of pyridoxal would have to be formed in order to react with thiazole-P. We obtained no evidence for the formation of such a pyrophosphate ester of pyridoxal under any condition (i.e., in the presence and absence of ATP and/or hydroxymethylpyrimidine-PP); thus, it appears unlikely that a pyridoxal-thiazole compound is formed, although the present state of knowledge does not conclusively rule out the possibility (e.g., pyridoxal-P possibly could react with thiazole-P to yield such a compound). Since the concentration of pyridoxal-P required to inhibit thiamine synthesis is relatively high, it appears unlikely that the intracellular concentration of this substance would ever be great enough to cause a significant decrease of thiamine formation except under contrived conditions, such as
BIOSYNTHESIS OF THIAMINE. IV t h e case in w h i c h a large a m o u n t of v i t a m i n B6 is a d d e d to t h e g r o w t h m e d i u m of t h e microorganism. ACKNOWLEDGMENT We wish to thank Mrs. Chai Ho Lo for her skillful technical assistance during the course of these investigations. REFERENCES 1. LEWlN, L. M., AND BROWN, G. M., J. Biol. Chem. 236, 2768 (1961). 2. HARRIS, D. L., Federation Proc. 11,226 (1952). 3. H.~Rms, D. L., Arch. Biochern. Biophys. 41, 294 (1952). 4. HARRIS, D. L., Arch. Biochem. Biophys. 60, 35 (1956). 5. KAWASAKI,T., and FUJITA, T., Seikagaku 33, 737 (1961); C. A. 56, 7676 (1962). 6. KAWASAKI,T., AND FUJITA, T., Seikagaku 33, 742 (1962); C. A. 56, 7676 (1962).
203
7. CAMIENER, G. W., ANn BROWN, G. M., J. Biol. Chem. 235, 2404 (1960). 8. LEDER, I. G., Biochem. Biophys. Res. Commun. 1, 63 (1959). 9. NOSE, Y., I~EDA, K., AND KAWASAKI, T., Biochim. Biophys. Acta 34,277 (1959). 10. CAMIENER, G. W., AND BROWN, G. M., J. Am. Chem. Soe. 81, 3800 (1959). 11. CAMIENER,G. W., AND BROWN, G, M., J. Biol. Chem. 9.35, 2411 (1960). 12. LEDER, I. G., J. Biol. Chem. 236, 3066 (1961). 13. NOSE, T., UEDA, I~., KAWASAKI, T., IWASHIMK, i . , ANn FUJITA, T., J. Vitaminol. (Kyoto) 7, 98 (1962).
14. LATtIAM, I-L, Master's Thesis, Massachusetts Institute of Technology, Cambridge, 1961. 15. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 66, 375 (1925). 16. DIXON, M., AND WEBB, E. C., "Enzymes," p. 178. Academic Press, New York, 1958.