ENZYMATIC SYNTHESIS OF THIAMINE

Transcription

1 THE JOURNAL OF VITAMINOLOGY 7, (1961) ENZYMATIC SYNTHESIS OF THIAMINE U. THE THIAMINE SYNTHESIS FROM PYRIMIDINE AND THIAZOLE PHOSPHATES AND THE ENZYMATIC SYN THESIS OF PYRIMIDINE MONO- AND DIPHOSPHATE AND THIAZOLE MONOPHOSPHATE1 YOSHITSUGU NOSE, KIYOSHI UEDA, TAKASHI KAWASAKI, AKIO IWASHIMA AND TETSURO FUJITA Biochemical Institute, Kyoto Prefectural University of Medicine, Kawaramachi, Nishijin, Kyoto (Received November 20, 1960) In the previous paper (1), the conditions for enzymatic synthesis of thiamine from the pyrimidine and thiazole moieties of thiamine were described. It was found that the activations of both OMP and Th by ATP occurred prior to the condensation of pyrimidine and thiazole moieties of thiamine, followed by an enzymatic condensation of the two active compounds to form the thia mine molecule. The active compounds were expected to be some phosphate esters of OMP and Th which had been phosphorylated by ATP at the hy droxymethyl group of OMP and the hydroxyethyl group of Th respectively. Therefore, in order to clarify the nature of the active compounds, phosphate esters of OMP and Th were prepared chemically and the activities as the sub strates for thiamine synthesis without ATP were examined before the isola tion of the active compounds from the enzyme reaction mixture. The present paper first describes the results which indicate that the most active com pounds of OMP and Th were OMP-PP and Th-P respectively, as already re ported (2). These conclusions have already been reported from the laboratory of Brown (3) and Leder (4). The data of isolating P32-labeled OMP-P and OMP-PP from the enzyme reaction mixtures are also presented in this paper which indicate that the enzymatic synthesis of OMP-PP might occur by a two step reaction, first the formation of OMP-P from OMP, followed by a second phosphorylation of OMP-P to OMP-PP. The formation of Th-P has already been reported (3) and the isolation from the enzyme reaction mixture was carried out by Suzuoki and Kobata (5) using S35-thiazole. The authors have demonstrated the formation of Th-P by bioautography with E. coli mutant. 1 Abbreviations: OMP, 2-methyl-4-amino-5-hydroxymethyl-pyrimidine; OMP-P, OMP monophosphate; OMP-PP, OMP diphosphate; Th, 4-methyl-5-ƒÀ-hydroxyethyl thia zole; Th-P, Th monophosphate; Th-PP, Th diphosphate; TMP, thiamine monophos phate; TPP, thiamine pyrophosphate; ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; Tris, tris (hydroxymethyl) aminome thane; EDTA, ethylendiaminetetraacetate. 98

2 Vol. 7 ENZYMATIC SYNTHESIS OF THIAMINE. U 99 EXPERIMENTAL 1. Materials OMP and Th were obtained as previously described (1). TMP and Th-P were kindly supplied by Dr. J. Suzuoki, Research Laboratories, Takeda Phar maceutical Industries Ltd. ATP and ADP were purchased from the Cali fornia Corporation, TPP from the Takeda Pharmaceutical Industries Ltd., Dowex 1 from the Dow Chemical Company. 2. Preparation of OMP Diphosphate OMP-PP was prepared by heating OMP with metaphosphoric acid, fol lowed by isolation with Dowex 1 as follows: 5g P2O5 and 0.7ml cold water were mixed under cooling, and the mixture was heated at for 1.5hr to form metaphosphoric acid. After cooling, 500mg of OMP was added to the mixture at 100, followed by heating for 1-1.5hr with gentle stirring. The reaction product was tested on a paper chromatogram which revealed that the majority of the OMP added had been converted to OMP-P, OMP-PP and OMP polyphosphate. A solution of the products in 50ml cold water was allowed to adsorb on a column consisting of 5g charcoal and an equal volume of Hyflosupercel. After washing with 20ml water, 100ml 0.5N HCl and 100ml water successively, it was eluted with 200ml strong ammonia water-ethanol (20:80) or 200ml isoamyl alcohol-ethanol-water (1:4:5). Re coveries of the products through this procedure were nearly complete. The eluate was concentrated under reduc ed pressure below 50. An aliquot of the solution was adjusted to ph 7 and the OMP-PP was separated from other OMP phosphates by passing through a Dowex 1-formate column (1 ~28cm). After the adsorption step, the column was washed with 100ml water, followed by eluting FIG. 1 Elution of OMP Phosphates Chemi cally Synthesized Methods of preparation of OMP-PP are described in the text. P-1, OMP-P; P-2, OMP-PP; P-3, OMP-poly phosphates. gradiently from an initial concentra tion of 0 to a final concentration of 2M ammonium formate using a mix ing chamber containing water. The eluate was collected in each 10ml fraction. The elution pattern of the fractions was shown in Fig. 1. Each fraction was examined photometri cally at 245mƒÊ and by paper chro matography detecting the absorption spots by Mineralight2. The eluate corresponding to OMP-PP was col 2 Mineralight sold by Ultra at 254mƒÊ. violet Prod. Inc., San Francisco with a transmittance peak

3 100 NOSE, UEDA, KAWASAKI, IWASHIMA AND FUJITA 1961 lected and the NH4+ in the eluate was removed by adding Amberlite IR-120-H+ form until the ph of the solution gave 2. After filtration, the filtrate was shaken with ether to remove formic acid. The aqueous layer was lyophi lized. Partially crystallized precipitate was obtained by adding ether to an ammonia-alkaline ethanol solution of the residue. After recrystallization, white crystals [ T] were obtained which coincided with a 2 molar ammonium salt of OMP-PP containing 3 molar water by analysis. By further recrystal lization, the crystals [ U] were obtained which coincided with a one molar ammonium salt of OMP-PP containing 3 molar water. Both crystals were very hygroscopic and had no inorganic phosphate. Crystals [ T]: sample 0.703mg/ml, found: P 16.4; Amm-N 6.54: calc. for C6H8N3 P2O7H 2NH3 E3H2O: P 16.3; Amm-N 7.24%. Crystals [ U]: sample 0.590mg/ml, found: P 16.9: Amm-N 3.27: calc. for C6H8N3 P2O7H2 ENH3 E3H2O: P 16.8: Amm-N 3.79%. Absorption maxima and molar absorbances: ~104 at 245mƒÊ at ph 1, ~104 and ~104 at 245mƒÊ and 271mƒÊ at ph 7.4, respectively. 3. Preparation of OMP-monophosphate and P32-labeled OMP-monophosphate OMP-P was prepared by a slight modification of the method of thiamine phosphorylation by Weijlard (6), followed by isolation with Dowex 1 formate column according to the OMP-PP preparation. 10ml o-phosphoric acid and 0.1g anhydrous sodium pyrophosphate were mixed and the whole was heated at 300 for 1hr. After cooling to 100, 500mg OMP was added and allowed to react with gentle stirring at for 1hr. After standing at room temperature, the reaction products were dissolved in 150ml cold water, fol lowed by adding 10g charcoal and stirring for 1hr to adsorb the pyrimidine compounds in the solution. A column consisting of charcoal mixed with cellulose powder was washed with 100ml 0.5N HCl and 100ml water, follow ed by elution with 100ml strong ammonia water-ethanol (10:90). After the eluate was concentrated under reduced pressure, 3 volumes of acetone were added. The precipitate thus appeared was collected and washed with a mix ture of acetone and ether. The residue was dissolved in a small amount of water and crystallized from ethanol. 200mg of the crystals were obtained which coincided with a one molar ammonium salt of OMP-P containing 3 molar water. Found: N 18.13; P 10.81; Amm-N 4.76, calc. for C6H8N3 PO4H NH3 E3H2O: N 19.32; P 10.68; Amm-N 4.83%. Absorption maximum was the same as OMP-PP. Molar absorbances: ~104 at ph 1, ~104 and ~104 at ph 7.4. P32-labeled OMP-P was prepared from OMP, anhydrous sodium pyrophos phate and a mixture of H3P32O4 in dilute HCl solution containing 6.5 mc and 3ml H3PO4, according to the preparation method described above. 70mg OMP-P32 crystals thus obtained had a specific radioactivity of 13.1 ~103c.p.m./ mole. 4. Preparation of Th-diphosphate Th-PP was prepared by a similar method to OMP-P preparation, followed by isolation with Dowex 1-formate column similarly to OMP-PP isolation.

4 Vol. 7 ENZYMATIC SYNTHESIS OF THIAMINE. U mg Th was allowed to react with a mixture of o-phosphoric acid and an hydrous sodium pyrophosphate. The reaction product was treated similarly, and the eluaee from charcoal column was concentrated under reduced pres sure. By adding ether, about 75mg precipitate, a mixture of Th-phosphates, was obtained. The compound dissolved in 10ml water was subjected to ad sorption on a column of Dowex 1-formate (1 ~20cm). The fractions eluted similarly to OMP-PP were examined by absorbance at 245mƒÊ and paper chro matography. The fractions corresponding to Th-PP were collected, and NH4+ and formic acid were removed. The Th-PP lyophilized was not obtained as pure crystals, but it showed a single spot on paper chromatogram. Preparation of APP-ƒÁ-P323 ATP-ƒÁ-P32 (P32-labeled in the terminal phosphate of ATP) was prepared according to Tanaka (7). The enzyme which catalyses the synthesis of ATP from carbamyl phosphate and ADP, was obtained from the extract of Strepto coccus faecalis ATCC The ATP-ƒÁ-P32 isolated with Dowex 1-chloride column chromatography was obtained as 111mg of Ba salt. It had a specific radioactivity of 26 ~103c.p.m./ƒÊmole and partial hydrolysis with acid treat ment showed that ƒ -P was not labeled with P32 and the ratio of specific ac tivities of,ƒà-p to ƒá-p was 1:7. 5. Paper Chromatography and Bioautography For detection of the OMP-, Th- and thiamine phosphates, paper chromato graphic and bioautographic techniques were used. Paper chromatograms (Toyo Roshi No. 50) were developed by an ascending method. The solvents used and the RF values of these compounds chemically synthesized are given in Table T. For detection of OMP, Th and their phosphates, the ultraviolet absorption by Mineralight, and for that of thiamine and its phosphates thio chrome fluorescence produced with alkaline ferricyanide solution were used. TABLE Values of Authentic Thiamine, OMP and Th Derivatives A, Isopropanol-isoamyl alcohol-h2o-n-butyric acid-28% NH4OH (7.5:2.5:7.5:12.0:0.2) B, Isopropanol-0.5M acetic acid buffer, ph 4.5-H2O (65:15:20) C, n-propanol-0.5m acetic acid buffer, ph 4.5 (60:40) Toyo Roshi, No. 50 (1 ~20cm), ascending method, 4 hours, room temperature. Detection of P32-containing OMP compounds on paper chromatogram was carried out by Actigraph U, Nuclear Chicago (kindly performed by Dr. O. 3 The three phosphor atoms of adenosine triphosphate are designated as ƒ, ƒà and ƒá.

5 102 NOSE, UEDA, KAWASAKI, IWASHIMA AND FUJITA 1961 Hayaishi, Kyoto University). Bioautography was mostly used for detection of the small quantities of these compounds formed enzymatically. The micro organisms used for bioautography were three mutants of E. coli ATCC 9637, i.e., OMP-less (70-17), Th-less (26-43) and thiamine-less (70-23) strains which were kindly supplied by Dr. R. Hayashi, Yamaguchi Medical College. The growth responses of these microorganisms showed fairly high sensitivities. The minimal amounts of OMP, OMP-P and OMP-PP detectable on the bioauto gram for the growth response of strain was 1 ~10-3, 1 and 1 ~10-1 mƒêmole respectively, whereas those of Th, Th-P and Th-PP for strain was 1 ~10-4, 1 ~10-3 and 1 ~10-1mƒÊmole respectively and those of thiamine, TMP and TPP for strain were all 1 ~10-3mƒÊmole. For bioautography, the developed chromatogram was placed on the surface of a solid medium containing each microorganism spread on a Pyrex dish. The solid medium consisted of the basal medium by Davis and Mingioli, solidified with 1.5% agar, triphenyltetrazolium chloride (0.02mg/ml) and an inoculated suspension of the microorganism. The suspension was prepared by collecting the cells grown for 24hr in 10ml of the basal medium and washing twice with saline solution About 5min after placing on the agar plate the paper was re moved. After covering the plate it was incubated at 37 for 16hr. The resulting growth zones corresponding to the RF values of the compounds to be tested were used for detection. 6. Enzyme Preparation The enzyme was prepared by the method reported in the previous paper (1) except for a following point. The supernatant of the extract from frozen yeast was treated with 1/10 volume of 2% protamine sulfate solution to remove nucleic acids before addition of solid ammonium sulfate. Then the succeeding steps followed the method described previously. The enzymatic activity for thiamine synthesis was determined by estimating the thiamine synthesized by the thiochrome method described previously. 7. Determinations The nucleotides, OMP-P and OMP-PP were determined spectrophotometri cally. OMP-PP was also determined enzymatically as described below. Radioactivities were determined with a Geiger-Muller Counter (The Kobe Kogyo Co. Ltd.) after the aliquots of the P32-containing solution were evap orated to dryness on counting discs. Total thiamine was measured by the thiochrome assay after Takadiastase hydrolysis and free thiamine was deter mined without enzyme treatment. Protein was measured by the method of Stadtman et al. (8). Enzymatic Assay for OMP-PP-OMP-PP was determined from the amount of thiamine synthesized in the presence of Th-P by the enzymatic reaction responsible for the following reaction E. OMP-PP+Th-P Thiamine When a fairly large amount of Th-P as compared with OMP-PP was used, the reaction rate was rapid and amount of the thiamine formed is propor tional to the amount of OMP-PP used. Though the enzyme prepared as (E)

6 Vol. 7 ENZYMATIC SYNTHESIS OF THIAMINE. U 103 described above catalyses effectively the condensation of OMP-PP and Th-P without ATP to form thiamine as given in results, the enzyme for OMP PP assay was reduced in the activity of OMP phosphorylation by heating at 55 for 2min before the protamine sulfate treatment in the preparation procedure. The activity of the en zyme responsible for the reaction E remained unchanged after the above FIG. 2 Standard Curve of OMP-PP for Enzymatic Assay The reaction mixture consisted of 200 moles tris buffer (ph 7.0), 2-10mƒÊmoles OMP-PP, 1ƒÊmole Th-P and 4.3mg enzyme protein in a total volume of 10ml. In cubation: 37 K, 1hr. treatment, giving reliable results. The reaction mixture for OMP-PP assay contained 200ƒÊmoles tris buffer (ph 7.0), 1ƒÊmole Th-P, diluted sam ple of OMP-PP (containing 1 to 10 mƒêmoles OMP-PP) and the enzyme solution thus treated in a final volume of 10ml. After incubating at 37 for 1hr, the thiamine synthesized was estimated. The concentration of OMP-PP was calculated from the linear standard curve using the same en zyme preparation as shown in Fig. 2. RESULTS 1. The Enzymatic Synthesis of Thiamine from OMP- and Th-Phosphates The chemically synthesized OMP and Th-phosphate were examined as substrates for the enzymatic synthesis of thiamine without ATP and Mg2+ in various combinations. As shown in Table U, Reaction E gave the best result of thiamine synthesis, i.e., 63.2mƒÊmoles per 2hr. It was 5 times as TABLE U Thiamine Synthesis from OMP- and Th-Phosphates without ATP The complete system contained 200ƒÊmoles tris buffer, ph 7.0, 10ƒÊmoles cysteine, 0.1ƒÊmoles OMP-P or OMP-PP, 0.1ƒÊmole Th, Th-P or Th-PP, and 5mg enzyme protein. a O riginal complete system containing 10ƒÊmoles ATP and 10ƒÊmoles MgCl2.

7 104 NOSE, UDA, KAWASAKI, IWASHIMA AND FUJITA 1961 much as the complete system containing ATP and Mg2 (Reaction G). The time course of Reaction E (Table ) was estimated at various time inter vals. A maximum synthesis of thiamine was obtained 4hr after incubation and the amount of thiamine corresponded to 69% of each substrate added. Reaction F (Table ) had also a fair activity; it was 2 times as much as that of the complete system. Though Th-PP was active as a substrate by the extracted enzyme and the thiamine formed contained TPP on paper chro matogram, as shown later, Th-PP could not be detected as an active inter mediate from the enzyme reaction mixture. On the other hand, Reactions A, B, C and D (Table ) showed far less thiamine synthesis than the com plete system. In addition, when Reaction C was performed by adding ATP and Mg2, the reaction rate of thiamine synthesis was larger than that of the complete system, the difference being larger in short incubation as shown in Table. This suggested that OMP-P might be further phosphorylated TABLE Reaction Rate of OMP and OMP-P as Substrates for Thiamine Synthesis with ATP The reaction mixture: 200moles tris buffer (ph 7.0), 10moles ATP, 10moles MgC2, 10moles cysteine, 0.1mole OMP or OMP-P, 0.1mole Th, and 5mg enzyme protein. Incubation 38 by ATP to form OMP-PP. From these findings it is considered that the most active intermediates of OMP and Th are OMP-PP and Th-P and the formation of these compounds requires ATP and Mg2 as the first step reac tion, then as the second step the two intermediates condense to form thia mine molecule.. Nature of Enzymatically Formed Thiamine If thiamine was synthesized by Reaction E, thiamine formed was ex pected to be TMP. However, when each thiamine form synthesized by four reactions was estimated by chemical assay, all reaction mixtures contained considerable amount of free thiamine. In order to clarify this point, the activities of phosphatases (TMPase and TPPase) contaminated in the en zyme preparation were determined by estimating the free thiamine liberated after incubation of the mixture containing 3mmoles TMP or TPP, 200 moles tris buffer (ph 7.0) and the enzyme solution at 37 for 1hr. The hydrolysis of TMP and TPP was 78 and 30 respectively. Further, for identification of esterified thiamine, paper chromatography was carried out, whereby the spot was located by the thiochrome fluorescence after spraying alkaline ferricyanide. Though the reactions A and F did not show distinct

8 Vol. 7 ENZYMATIC SYNTHESIS OF THIAMINE. U 105 results, Reaction E gave two spots of TMP and free thiamine, while Reaction F gave three spots of TPP, TMP and free thiamine. However, as will be shown later in the succeeding paper, when the enzyme catalyzing Reaction E is purified, it gives a spot of TMP alone. 3. Isolation of OMP-PP Enzymatically Formed from P32-Labeled OMP-P In the earlier stage of this study, efforts were made to isolate a possible active intermediate of OMP (OMP-PP) from the enzyme reaction mixture in cubated with OMP, ATP and Mg2+ without Th. However, OMP-PP obtained was too small a quantity to identify it as OMP-PP clearly. This is proba bly due to the lability of OMP-phosphorylating enzyme (OMP-kinase) which is completely inactivated by heating at 55 for several min. Therefore, for isolation of the compound, OMP-P32 was used as a substrate which caused much more synthesis of thiamine than OMP and in addition it was convenient for tracing P32-labeled compound. A 25ml reaction mixture was prepared with 1mmole tris buffer (ph 7.2), 34ƒÊmoles OMP-P32 (445 ~103c.p.m.), 200 moles ATP, 200ƒÊmoles MgCl2, 500moles NaF and 105mg enzyme protein. The mixture was incubated at 37 for 2hr. To stop the reaction and de proteinize, 25ml 10% perchloric acid was added with stirring and the preci pitate was removed by centrifugation. The supernatant was neutralized FIG. 3 Elution Diagram of the Reaction Mixture Containing OMP-P32 and ATP The mixture is composed of and treated as given in the text. : Fractions which give the enzymatic synthesis of thiamine with Th-P without ATP and MgCl2. with 5M KOH, followed by the re moval of the excessive acid. The supernatant, after diluting with water was adsorbed on a column of Dowex 1-formate (1 ~20cm). The adsorbed column was washed with 100ml water, followed by gradient elution with ammonium formate solu tions from an initial concentration of 0 to a final concentration of 1M using a mixing chamber. A flow rate was 0.7ml per min and each 6.5 ml fraction was collected. The ab sorbance of each fraction was meas ured at 245 and 257mƒÊ. As shown in Fig. 3, three peaks containing ab sorbing materials at 245mƒÊ were obtained. However, the compound which gave the enzymatic synthesis of thiamine with Th-P without ATP and Mg2+ was found only in No. 35 and 36 fractions of peak 2. From the ratio of the absorbance at 245 and 257mƒÊ, No. 35 fraction was proved to contain a pyrimidine compound, while No. 36 fraction was mixed with adenine derivatives. The radioacti

9 106 NOSE, UEDA, KAWASAKI, IWASHIMA AND FUJITA 1961 vity of P32 was found in all fractions of peak 1 (unreacted OMP-P32) while in peak 2 P32 was found in No. 35 and 36 fractions. 4. Identification of OMP-PP For identification of OMP-PP the No. 35 fraction expected to be pure OMP-PP was used. To the fraction Amberlite IR-120 H+ form was added until the ph of the solution became to 2. By filtration NH4+ was removed, and the filtrate was washed with ether to remove formic acid. The aqueous layer was lyophilized and the residue was dissolved in a small amount of water. Paper Chromatography-A small aliquot of the sample was applied to paper chromatography. The only one spot which gave the same RF value (0.15) as authentic OMP-PP was found by Mineralight as shown in Fig. 4. When No. 36 fraction was similarly tested, two spots were detected and prov ed to be OMP-PP and AMP. Moreover, it was certified on paper chromato gram that the fractions of peak 2 besides No. 35 and 36 contained AMP, the peak 1 corresponded to unreacted OMP-P32 and the peak 3 to ADP. Absorption Spectrum-The absorption spectrum of the sample in 0.1N HCl showed a maximum at 245mƒÊ, agreeing with that of pyrimidine com pound. Bioautography-As described earlier, the developed paper chromatogram was applied to bioautography with three kinds of E. coli mutants. As given in Fig. 4, two growth zones of OMP-less strain (70-17) were found. One coincided with OMP-PP and the other with OMP probably produced by de FIG. 4 Paper Chromatography and Bio autography of Fraction No. 35, paper chromatogram. a, fr. no. 35; b, fr. no. 35 plus authentic OMP-PP; c, fr. no. 35 plus authentic OMP-P., bio autogram. Ua, OMP-less; Ub, Th-less; U c, thiamine-less strain of E. coli. Sol vent used for paper chromatography is given as solvent A in Table T. UV ab sorption spots are detected by Minera light. FIG. 5 Detection of P32 Corresponding to the Spot of OMP-PP on the Paper Chromatogram The same paper chromatogram as that shown in Fig. 4- Ta was applied on Acti graph.

10 Vol. 7 ENZYMATIC SYNTHESIS OF THIAMINE. U 107 composition of OMP-PP during the treatment. On the other hand, no growth zone of Th-less (26-43) and thiamine-less (70-23) strains was found. Actigraphy-Fig. 5 shows the radioactivity of P32 corresponding to the spot of OMP-PP which was determined by using Actigraph. 5. Specific Radioactivity of OMP-PP Formed Though OMP-P was more active as substrate than OMP and the reaction mixture contained NaF to inhibit phosphatase activity, the formation of OMP PP can not be decided whether it is formed by a direct phosphorylation of OMP-P or by a pyrophosphorylation of OMP produced after hydrolysis of OMP-P. To clarify this question, the specific activities of OMP-P32 and OMP PP were compared. As obvious from the actigram of fraction No. 35, the radioactivity of the spot was derived from the P32 of OMP-PP and a contami nation of that of inorganic phosphate (RF 0.23) and pyrophosphate (RF 0.03) is excluded. As shown in Table W, the specific activity of OMP-PP was 6,470 TABLE W Specific Radioactivity of OMP-PP Formed from OMP-P32 a Calculated from enzymatically synthesized amount of thiamine with Th-P without ATP and MgCl2. Others were obtained from the molar absorbance of each sub stance. when calculated from the amount of OMP-PP determined by the absorbance at 245mƒÊ and 6,920 when calculated from the amount determined by the en zymatic assay. Consequently 66% of the P32 of the OMP-P32 added has been transfered to OMP-PP. However, the radioactivity of the isolated OMP-P32 after incubation has been diluted to 67% of that of OMP-P32 added. It is not clear whether such a dilution is due to an exchange of phosphate of OMP-P32 with some phosphate compounds such as ATP, ADP, etc., or due to phosphorylation of OMP produced after hydrolysis of OMP-P32. However, it is concluded that most of OMP-PP has been formed by a direct phosphoryla tion of added OMP-P32. In this experiment, the total amount of OMP-PP formed including the fraction No. 36 was 1.44ƒÊmoles, corresponding to 4.2% of added OMP-P32.6. Isolation of OMP-P and OMP-PP Formed from OMP and ATP-ƒÁ-P32 The formation of OMP-PP from OMP-P requires the confirmation that the OMP-P is a real intermediate of OMP-PP formation in the enzyme reaction,

11 108 NOSE, UEDA, KAWASAKI, IWASHIMA AND FUJITA 1961 The reaction mixture contained in ƒêmoles: 500 tris buffer (ph 7.2), 15 OMP, 50 ATP-ƒÁ-P32 (7,800 ~103c.p.m.) with 250 ATP as carrier, 300 MgCl2, 250 NaF and 69mg enzyme protein in a final volume of 25ml. After incubating at 37 for 1.5hr 25ml 10% perchloric acid was added with stirring to stop the reaction. After centrifugation, excess of the perchloric acid was removed by adding 5M KOH. The neutralized supernatant was diluted with water, and was applied to a Dowex 1-chloride column (1 ~20cm). The chloride form was found effective for separating OMP-PP and AMP. The adsorbed column was eluted gradiently with NaC HCl system from a initial concentration of 0 to a final concentration of 0.2M NaCl in 0.01N HCl. Each 6.5 ml fraction was collected at a flow rate of 0.7ml per min. The absorbance of each frac tion was measured similarly to the earlier experiment. As shown in Fig. 6, four peaks containing absorbing materials besides percolated OMP were obtained. It was determined on paper chromatogram that the three major peaks, 1, 2 and 4, corresponded to OMP P, AMP and ADP respectively. Only the minor peak 3 had the enzymatic activity of thiamine synthesis with Th-P and it was expected to be OMP-PP. 7. Identification of OMP-P and OMP-PP FIG. 6 Elution of OMP-P and OMP-PP from the Reaction Mixture The composition of the mixture is shown in the text. _??_ Fractions showing enzymatic synthesis of thiamine with ThP without ATP and MgCl2. Fig. 7 shows the absorption spectra of fraction no. 56 of peak 1 and frac tion no. 83 of peak 3 in acid and alkaline solution. Both compounds had the maximal absorbances at 245m at ph 2 and at 270m at alkaline ph, in dicating the compounds to be pyrimidine derivatives. The fraction nos. 56 and 57 of peak 1 and nos. 83 and 84 of peak 3 were separately collected and lyophilized. The two samples obtained were applied to paper chromatogra phy. As shown in Fig. 8 only one spot was detected from each sample and the spot obtained from the peak 1 coincided with the authentic OMP-P, the one from peak 3 with the OMP-PP. These compounds were also identified as OMP-P and OMP-PP bioautographically as described earlier. Further, Fig. 9 shows the radioactivities detected from the spots on actigram. From these results it is clear that OMP-P and OMP-PP were isolated from the enzyme

12 Vol. 7 ENZYMATIC SYNTHESIS OF THIAMINE. U 109 FIG. 7 Absorption Spectra of P-1 and P-3 7- T, fraction no. 56; 7- U, fraction no. 83. œ, ph 2;, ph 7.2 Beckman Spectrophotometer Model DU was used. FIG. 8 Paper Chromatography of Peak 1 and 3 a, concentrate of fraction nos. 56 and 57; Tb, the concentrate plus authentic OMP-P. Ua, concentrate of fraction nos. 83 and 84; Ub, the concentrate plus au thentic OMP-PP. Solvent used is given as solvent A in Table T. Toyo Roshi, No. 50 (1 ~20cm); ascending method at room temperature for 4hr. Detection of UV absorption spots was carried out by Mineralight. reaction mixture with no appreciable contamination and the labeled phos phates of the compounds have been derived from the labeled ATP. 8. Specific Radioactivities of OMP-P and OMP-PP As shown in Table X, the specific activities of OMP-P and OMP-PP were TABLE X Specific Activities of OMP-P and OMP-PP Formed from OMP with ATP-ƒÁ-P32 a Calculated f rom the thiamine enzymatically synthesized with Th-P. Others were given from the molar absorbance of each substance.

13 110 NOSE, UEDA, KAWASAKI, IWASHIMA AND FUJITA ~103, and 21 ~103 respectively, as calculated by ultraviolet absorbances. As the specific activity of ATP32 was 26 ~103 and the ratio of specific acti vities of Ĉ-p to ć-p was 1:7, those of Ĉ- and ć-p were 3.2 ~103, and 22.8 ~ 1 03 respectively. If OMP-PP had been formed from OMP by one step reaction, i.e., by py rophosphorylation from ATP and OMP-P had been produced by hydrol ysis of OMP-PP, OMP-P obtained might have a specific activity nearly corresponding to that of Ĉ-p of ATP. However, the specific activity of the isolated OMP-P was 13.5 ~103 which was 4.2 times as much as that of Ĉ-p of ATP. This difference indi cates that the formation of OMP-P was not due to the hydrolysis of OMP-PP formed. Therefore, the for mation of OMP-P might be due to a direct phosphorylation of OMP and the specific activity of OMP-P was expected to be the same as that of ć-p of ATP. However, in fact the specific activity of OMP-P isolated was 13.5 ~103, only 59% of that of ć-p of ATP. The cause of such a dilu tion is unknown, but the specific ac tivity of ADP isolated after the FIG. 9 Detection of P32 from the Spots of OMP-P and OMP-PP on Paper Chro matograms Paper chromatograms T and U used were the same as those shown in Fig. 8- Ta and 8- Ua respectively. Detection was carried out by Actigraph. reaction showed a relatively high increase as compared with that of Ĉ-p of ATP, although ATP isolated had no appreciable change in radioactivity. Probably this is one factor of the dilution of P32 of OMP-P. The specific activity of OMP-PP was also determined from the amount obtained by the enzymatic assay. The two values of OMP-PP were nearly the same in two fractions of peak 3. Considering that OMP-PP synthesis is a successive phosphorylation by ć-p of ATP via the intermediate formation of OMP-P, the specific activity of OMP-PP was very low as compared with that of the duplicate of ć-p. However, the fact that the radioactivity of OMP- P32 added was diluted during incubation and that of OMP-PP formed from OMP-P32 contained only 66% of the radioactivity of OMP-P32 added as des cribed earlier, the low specific activity of OMP-PP obtained in this experiment can be admitted. From these results, it is assumed that OMP-PP formation might be a two step phosphorylation of OMP by ATP. Further, the total amount of OMP-P and OMP-PP synthesized were esti

14 Vol. 7 ENZYMATIC SYNTHESIS OF THIAMINE. U 111 mated. As shown in Table Y, the amount of OMP-P was 1.72ƒÊmole, cor responding to 11.5% of the added OMP, while the amount of OMP-PP was 0.24ƒÊmole, corresponding to 1.6% of the added OMP. The amount of OMP- PP calculated by the enzymatic assay gave a little higher value. TABLE Y T otal Amount of OMP-P and OMP-PP Formed from OMP Composition of the reaction mixture is given in the text. a Calculated from the thiamine enzymatically f ormed with Th-P. Others were de termined by the molar absorbances of the products. 9. Enzymatic Formation of an Activated Th Compound Though Th-PP beside Th-P was fairly active as a substrate for thiamine synthsis as shown in Table U, Suzuoki and Kobata (5) isolated Th-P alone as an activated Th compound from the incubation mixture of S35-labeled Th with ATP, Mg2+ and the similar enzyme preparation from bakers' yeast. Camiener and Brown (3) also detected Th-P on a bioautogram with a Th-requir ing E. coli mutant. The authors also confirmed Th-P formation, but failed to detect Th-PP by using a similar bioautographic technique. The reac tion mixture contained, in ƒêmoles, 200 tris buffer (ph 7.2), 0.1 Th, 10 ATP, 10 MgCl2 and 0.2ml enzyme solution in a total volume of 1.0ml. After incubating at 37 for 1hr, the reaction was stopped by heating at 90 for 2min, followed by deproteini zation. As a control, a boiled en zyme was used. An aliquot of the FIG. 10 Bioautography of the Reaction supernatant was applied to bioauto Mixture for Detection of Th-P graphy and examined with three Th-less, Pm-less and thiamine-less strains kinds of E. coli mutants, OMP-, Thand were used for T, U and V respectively. thiamine-less strains. As shown a, Ua and Va, complete reaction mixture in given in the text; Tb, complete reaction Fig. 10, four kinds of growth zones mixture containing the boiled enzyme; were found on a bioautogram using a and Va, the enzyme solution alone. the Th-less strain. The two major Solvent used is shown as solvent C in Table T. Toyo Roshi No. 50 paper (1 growth zones correspond to Th and 20cm); ascending method at room tem Th-P in the RF values of the authentic perature for 4hr. compounds added. The compounds

15 112 NOSE, UEDA, KAWASAKI, IWASHIMA AND FUJITA 1961 showed no response to the OMP- and thiamine-less strains. The two kinds of minor zones corresponded to thiamine (RF 0.63) and TPP (RF 0.13) which were contained in the enzyme preparation. The authentic Th-PP mixed with the samples showed a growth zone near TPP. Though the sensitivity of Th-PP was ten times less than that of Th-P, the Th-PP enzymatically formed could not be detected. The minor thiamine spot in the main test is considered to have been converted to TPP during incubation. DISCUSSION The most active phosphate esters of OMP and Th as substrates for thiamine synthesis without ATP were isolated from the enzyme reaction mixtures. The main enzymatic formation of OMP-PP was presumed to be a two step phosphorylation reaction of OMP via formation of an intermediate, OMP-P, even though a side way of OMP-PP formation, i.e., a pyrophosphorylation of OMP can not be completely excluded considering the decline of the specific radioactivity of the OMP-PP isolated. To clarify this point, the analysis of the radioactivities of two phosphates of OMP-PP was tried, but it failed ow ing to too small quantities of the compounds isolated. However, an acid labile phosphate group of the isolated compound was demonstrated bioauto graphically. Therefore the stability of OMP-PP chemically synthesized was investigated. Heating 0.1ƒÊmole OMP-PP at 100 for 10min in 1 N HCl solu tion caused a complete destruction of the enzymatic activity for thiamine synthesis, while the heat treatment in 1 N NaOH resulted in a loss of 76% of the enzymatic activity. On the other hand, at neutral ph in 0.02 M tris buffer only 2% was destroyed after the heat treatment. Consequently, OMP-PP has an acid- and alkaline-labile phosphate group which causes, in the presence of the enzyme, the condensation of pyrimidine and thiazole moie ties liberating pyrophosphate as follows: OMP-PP+Th-P TMP+PP The formation of TMP and the liberation of PP were demonstrated with a partially purified enzyme (TMP-synthetase) which will be reported later. The lability of the phosphate group of OMP-PP is similar to other examples of pyrophosphate ester, i.e., 5-phosphoribosyl-1-pyrophosphate (9) and iso pentenyl pyrophosphate (10) which are active intermediates in biosynthetic reactions. The phosphorylating enzyme of OMP (OMP-kinase) which might be a mixture of two enzymes was inactivated by heating at 55 for 2min. On the other hand, the phosphorylating enzyme of Th (Th-kinase) and the condensing activity of OMP-PP and Th-P (TMP-synthetase) remained unchang ed with this treatment as described before. Thus the extracted enzyme preparation might be a mixture of these enzymes including phosphatases. Separation of these enzymes and partial purification of TMP-synthetase will be reported later in the succeeding paper. A plausible pathway of enzymatic synthesis of thiamine and synthesis of TPP as an active coenzyme is present ed in the following scheme, which is similar to that proposed by Camiener and Brown (3).

16 TPP synthesis is expected to occur by a pyrophosphorylation reaction of free thiamine by ATP with the participation of thiaminokinase which is active in the enzyme preparation and has been purified by Simazono et al. (11) from the extract of baker's yeast. The strong phosphatase activities converting TMP to free thiamine contained in the enzyme preparation also supports the plausible pathway described above. However, no evidence can exclude an enzyme activity which catalyses a direct phosphorlyation of TMP to form TPP. SUMMARY 1. Phosphates of 2-methyl-4-amino-5-hydroxymethylpyrimidine (OMP) and 4-methyl-5-ƒÀ-hydroxymethyl thiazole (Th) were synthesized chemically and the activities of thiamine synthesis without ATP were examined. OMP pyro phosphate (OMP-PP) and Th phosphate (Th-P) were most active as substrates and had a reaction rate of 5 times as much as that of the thiamine synthesis from OMP and Th with ATP. OMP-PP and Th pyrophosphate (Th-PP) had also a fair activity as substrates. 2. The thiamine synthesized from OMP-PP and Th-P was proved to be a mixture of thiamine and thiamine monophosphate, while that from OMP-PP and Th-PP a mixture of thiamine, thiamine monophosphate and thiamine pyrophosphate. 3. The active compound of OMP was isolated by Dowex 1 column chro matography from the enzyme reaction mixture incubated with OMP-P32 or ATP-ƒÁ-P32. The isolated OMP-PP formed from OMP-P32 and ATP, and OMP-P and OMP-PP formed from OMP and ATP-ƒÁ-P32 were identified by using paper chromatography, bioautography with a E. coli mutant, actigraphy and absorption spectra. 4. Comparing the specific radioactivities of the OMP-P and OMP-PP isolated with those of OMP-P32 or ATP-ƒÁ-P32 added, OMP-PP is presumed to be mainly formed by successive phosphorylation of the ƒá-phosphate of ATP through OMP-P.

17 114 NOSE, UEDA, KAWASAKI, IWASHIMA AND FUJITA The active intermediate of Th enzymatically formed was identified bio autographically as Th-P and the mechanism of enzymatic synthesis of thia mine was discussed. REFERENCES 1. Nose, Y., Ueda, K., and Kawasaki, T., J. Vitaminol. 7, 99 (1961). 2. Nose, Y., Ueda, K., and Kawasaki, T., Biochim. Biophys. Acta 34, 277 (1959). 3. Camiener, G. W., and Brown, G. M., J. Biol. Chem. 235, 2404, 2411 (1960). 4. Leder, I. G., Biochem. Biophys. Research Communications 1, 63 (1959). 5. Suzuoki, Z., and Kobata, A., J. Biochem. 47, 262 (1960). 6. Weijlard, J., J. Am. Chem. Soc. 64, 207 (1960). 7. Tanaka, R., J. Biochem. 47, 207 (1960). 8. Stadtman, E. R., Novelli, G. D., and Lipmann, F., J. Biol. Chem. 191, 365 (1951). 9. Kornberg, A., Lieberman, I., and Simms, E. S., ibid. 83, 389 (1955). 10. Henning, U., Moslein, E. M., and Lynen, F., Arch. Biochem. Biophys. 83, 259 (1959). 11. Shimazono, N., Mano, Y., Tanaka, R. and Kaziro, Y., J. Biochem, 46, 959 (1959).