Some Properties of the Catalytic Sites of Imidazoleglycerol Phosphate Dehydratase-Histidinol Phosphate Phosphatase, a Bifunctional Enzyme from Salmonella typhimurium*

SUMMARY Kinetic and aggregatory properties of a partially purified preparation of D-erythro-imidazoleglycerol phosphate dehy-dratase-histidinol phosphate phosphatase from a derepressed mutant of Salmonella typhimurium (his012$2) have been investigated. The molecular weight in crude extracts is 300,000. Purification of the enzyme causes disaggregation, the main component keing 75,000 in molecular weight. Addition of MnCIZ causes reeggregation resulting in a molecular weiglt greater than 300,000. Aminotriazole (K, = 3.2 PM) and phosphate ion (KI = 1.3 mu) competitively inhibited only the dehydratase activity. Zinc chloride, at micromolar concentrations, inhibits only the dehydratase activity. Both histidinol (KI = 52 pm) and histidine (Kr = 10 mM) are competitive inhibitors of only the phosphatase activity. Heating at 54” results in a very rapid loss of only the phosphatase activity, TI/~ = 2 the dehydratase activity is stable under these conditions.

Heating at 54" results in a very rapid loss of only the phosphatase activity, TI/~ = 2 min; the dehydratase activity is stable under these conditions. ImidazoIeglyceroI phosphate is not hydrolyzed and does not inhibit the phosphatase activity.
Histidinol phosphate does not affect the dehydratase activity. We conclude that the dehydratase and pkosphatase active sites are separate and distinct, although they appear to reside in a single protein.
The energies of activation for both substrates were determined to be 15,100 cal per mole for the hydrolysis of histidinol phosphate and 14,700 cal per mole for the dehydration of imidazoleglycerol phosphate. The optimum pH for phosphatase activity was 6.5 to 7 and was near pH 7.5 for the dehydratase activity.
The histidine biosynthetic enzymes are encoded by an opcron iu Salmonella typhi~~uriu~n and have been the subject of maq * This investigation was supported by Public Health Service (1 FO 2 GM 50952). elegant genetic studies in several laboratories (l-5). Fig. 1 shows the enzymatic reactions of the terminal part of the pathway and the genes that code for the enzyme catalyzing reactions 7 to 10 in the pathway.
Two activities, imidazoleglycerol phosphate dehydratase and histidinol phosphate phosphatase are shown to be coded by the same gene, the hisB gene.
The hisB gene product appears to be a bifunctional enzyme; a single protein responsible for two enzymatic activities, the dehydration of D-er#hro-imidazoleglycerol phosphate to produce imidazoleacetol phosphate and Hz0 and the hydrolysis of histidinol phosphate to histidinol and phosphate. This conclusion is supported by both genetic and biochemical studies. The hisB gene is characterized by four complement'ation groups (6,7) each of which is distributed throughout the hisB gene. Mutants exist which lack only dehydratase activity; however, all mutants lacking phosphatase activity also lack dehydratase activity (4), some of those being single site mutations involving one DNA base pair (5,7,8). Phosphatase activity cannot be restored by in vitro complementation (8). Noncomplementing mutants are distributed throughout the gene (8). Antisera towards wild type enzyme reacts with enzyme isolated from phosphatase+-dehydratase-mutants (7). No procedure has yet been found to physically separate the two activities during attempted purification of the enzyme (4,(8)(9)(10)(11). The enzyme has been reported to bc difficult to purify (4) and as a result, little information concerning the structure and mechanism of action of this c~lzyl~e is available. It is Lissumed that two catalytic sites are responsible for the act,ivities since aminotriazolc was shown to iuhibit ouly the dehydratasc activit>- (12), aud the nature of the reactions that arc catalyzed are quite different.
Because of the complex patterns of aggrc,gation observed (10, II), a multimeric subunit structure has bcrn proposed (8,11). Roth 150,000 and 75,000 molrcular weight components have been reported (8,10,11). It has been suggested that the enzyme consists of subunits with phosphatase activity which when aggregat,ed yield the drhydratase activity (11).
The work reported here was undertaken in an attempt to more clearly define the identity of the two sites as well as to characterize some kinetic properties of the c~nzymc from Salmonella typhimurium hisOl$?/tW. Yreparation of Cell Extract of Salmonella typhimurium--Tithe derepressed mutant hisOl%$ was grown on Vogel-Bonner (13) minimal medium in 0.2% glucose in 15-liter carboys by the method of Ames et al. (14). The cells were vigorously aerated for 20 hours and the harvested cells were stored as a frozen paste at -20" until needed.
Cell breakage was accomplished by sonification of a 3346 ye weight to volume suspension of frozen cells in 0.05 M Tris chloride-O.01 M mercaptoethanol at pH 8.5. A Branson sonifier was used to deliver three l-mm bursts.
After sonification in a Rosett cell at O", the extract was centrifuged at 40,000 X g for 30 min at 4". TEAE-cellulose1 Chromatography-TEAE-cellulose was prepared for chromatography by the procedure of Peterson and Sober (15) and equilibrated with buffer. The extract was applied to a column (2.5 x 80 cm for the extract from 25 g of cells), washed with 1 to 2 volumes of buffer, and developed with a linear gradient using 500 ml of 0.05 M ammonium sulfate in buffer versus 500 ml of 0.3 M ammonium sulfate in buffer.
Sephadex G-ZOO Gel Filtration-A pool of the active fractions from TEAE-cellulose chromatography was concentrated on an Amicon Diaflo using a PM-10 membrane and applied to a column (6.0 x 42 cm) of Sephadex G-200 at 4". The pH of the elution buffer was adjusted to 7.5 for this and subsequent steps.
The fractions containing activity were pooled and concentrated as described above. The concentrate was made 1 mM in MnC12 and heated at 37" for 10 min. Following cooling to 4", the COIIcentrate was chromatographed on the same G-200 column after equilibration with fresh buffer containing 1 mM MnC12. Columns were usually developed at 20 to 30 ml per hour. The fractions containing dehydratase and phosphatase activity were pooled and concentrated.
Unless otherwise stated, the enzyme preparation used for all assays was the one obtained from this second passage through Sephadex G-200.
Enzymatic Assays-Imidazoleglycerol phosphate dehydratase was assayed by a modification of the method used by Ames (16). The assay was performed at 37" in a final volume of 100 ~1 which contained 25 RIM triethanolamine hydrochloride at pH 7.5, 50 pM MnC12, 18 mivr mercaptoethanol, 1 mM imidazoleglycerol phosphate and 5 to 10 ~1 of enzyme.
The reaction was initiated by the addition of enzyme and was terminated with 200 ~1 of 1.43 N NaOH.
After further incubation at 37" for 30 min, the absorbance at 290 nm was determined against a blank containing all react.ion components except imidazoleglycerol phosphate using a Zeiss PMQ II spectrophotometer and a microcell with a l-cm path length.
The molar extinction coefficient at 290 nm of imidazoleacetol phosphate is 5100 as calculated from the data of Ames (16).
Histidinol phosphate phosphatase was assayed by a modification of the procedure of Ames et al. (14). The rcartion was carried out at 37" in a final volume of 100 ~1 containing 25 nlM triethanolamine hydrochloride at pH 7.5, 1 mM histidinol phosphate, and 5 to 10 ~1 of enzyme.
The reaction was terminated by the addition of 200 ~1 of molybdate-ascorbate reagent. After 20 min incubation at 45", the absorbance was read at 820 nm against a blank using all reaction components but with 10 ~1 of water in place of histidinol phosphate. For these conditions, a molar extinction coefficient at 820 nm of 2.60 x lo4 was calculated from the data of Ames et al. (14) and was verified in this laboratory.
Protein concent.rations were measurrd by the proccdure of Lowry et al. (17). All inhibitors used were buffered to pH 7.5.

RESULTS
Enzyme Isolation-The results of the chromatography of a crude extract of Xalmonella typhimurium hisOl%'@? 011 TEAEcellulose can be seen in Fig. 2. The phosphatase and dehydratase activity chromatograph together as a single, rather broad peak toward the end of the ammonium sulfate gradient. The activities appear to be inseparable on this column.
Changing the steepness of the salt gradient, adjusting the pH from 8.5 to 7.5, or using a NaCl gradient had 110 effect. upon the coincident appearance of both activities within the peak. The COJKCJItrated eluant from TEAE-cellulose chromatography was passed through a Sephadex G-200 column resulting in the appearance of a complex activity profile with at least two peaks of activity (Fig. 3A).
Both activities were found in each of the peaks; however, the ratio of phosphatase activity to dehydratase activity was different between Fraction I and Fraction II. The peaks The buffer used was 0.05 M Tris chloride-O.01 M mercaptoethanol, pH 7.5. Phosphatase activity (0) was measured on lo-p1 aliquots for 10 min and dehydrase activity ( l ) was measured on 25-~1 aliquots for 15 min. B, Fraction II was concentrated, made to a final concentration of 1 mM MnC12, and incubated at 37" for 20 min before Sephadex chromatography at 4'. Histidinol phosphate phosphatase activity (0) was measured on 25-~1 aliquots for 10 min and imidazoleglycerol phosphate dehydratase activity (0) was measured on 25-J aliquots for 30 min. The same elution buffer at pH 7.5 was used and contained 1 mM MnCL.
Fractions of 9.5 ml were collected.
were not sharp and the profile may represent the sum of several peaks. The ratio of phosphatase activity to dehydratase activity iu Fraction I was 1.5 and it changed to 3.3 in Fraction II. The addition of manganese chloride at a final concentration of 1 1x1~ to Fraction II affected the elution profile of the two activities quite dramatically (Fig. 3B). The dehydratase activity was now found to elute in a sharp peak at a new position in the profile (Fraction III). In contrast to this, the phosphatase activity was distributed throughout the fractions. Much of the activity appeared in Fraction III in association with the dehydratase activity, although a large portion still resided in the lower molecular weight fractions.
The ratio of phosphatase activity to dehydratase activity was now changed from 3.3 in Fraction II to 1.6 in Fraction III, nearly the same as in Fraction I. A similar shift in the elution profile was demonstrated with the addition of manganese chloride to Fraction I. In this case, essentially all of both the activities was shifted to a smaller elution volume identical with Fraction III; no smaller molecular weight species were observed.
The molecular weights of the enzyme present in crude extract and Fractions I and II were estimated on standardized Sephadex G-200 columns.
A column ( The buffer for the phosphatase assay &as 0.1 M tri-ethanoIamine adiusted to nH with succinic acid and for the dehydratase assay,"adjusted &th HCI.
aliquot of the concentrate of the TEAE-cellulose pool was determ'ned in the absence of the MnC$.
Fraction II was treated with 1 mM MnClz and run on the same column equilibrated with buffer containing 1 rnM MnC&.
Fractions I and II had molecular weights of 300,000 and 75,000, respectively.
The elution volume of Fraction III was very close to the void volume of the column and its molecular weight was not determined.
The enzyme had a molecular weight of 300,000 in the crude extract.
Fraction III had a specific activity which was 5-to lo-fold higher than that of the crude extract resulting from sonification. All the sample remained at the interface of the stacking and separating gel on analytical polyacrylamide gel electrophoresis (18,19). Sodium dodecyl sulfate disc gel electrophoresis (20) of this final preparation showed the enzyme to be only partially purified.
Two intense bands accompanied by six less intense bands were observed.
Further attempts to purify the enzyme have not been successful.
The presence of MnCl~ in the buffer has a stabilizing influence on the enzyme.
Daily freezing and thawing a crude extract for 50 times in 0.05 RI Tris chloride-10 mM mercaptoethanol-1 mM MnClz at pH 7.5 resulted in no loss of phosphatase activity and no decrease in molecular weight.
Effect of pH-The effect of pH on both dehydratase and phosphatase activities is shown in Fig. 4. For the phosphatase assay, buffers were made to a final concentration of 0.1 M in triethanolamine and titrated with succinic acid to the desired pH. Hydrochloric acid was used to adjust the pH for the buffers used in the dehydratase assay. Maximal dehydratase activity occurs near pH 7.5, dropping off sharply below that pH and more slowly at a more alkaline pH.
Phosphatase activity was maximal in the region of pH 6.5 to 7. The inclusion of 1 mM MnClz in the phosphatase assay had no effect on the pH profile.
A compromise pH was chosen, pH 7.5, so that both assays could be done under conditions that were as similar as possible.
E$'ect of ilfanganese-Manganese is known to be required for imidazoleglycerol phosphate dehydratase activity in Neurospora (16) and it is used in the assay for the Salmonella enzyme (14). Using the TEAE-cellulose preparation, the requirement for Mns+ was shown for the Salmonella enzyme and a K, for MnClz of 7.3 pM was determined.
Aminotriazole Inhibition-Aminotriazole has been demonstrated to be an inhibitor of imidazoleglycerol phosphate de-hydratase in yeast (21) and Salmonella typhimurium (12). We have examined the effect of aminotriazole on our preparation by the use of a Dixon plot and have found the inhibition of the dehydratase activity to be competitive (Fig. 5A). The Kr was determined to be 3.2 PM.
The effect of aminotriazole on phosphatase activity was also evaluated on the same preparation.
No inhibition of phosphatase was observed at concentrations of aminotriazole as high as 20 rnM.
Phosphate Inhibition- Klopotowski and Wiater (21) have shown that phosphate inhibits imidazoleglycerol phosphate dehydratase from yeast. We have found that the dehydratase from Salmonella typhimurium is also inhibited competitively by the presence of phosphate (Fig. 5B) with a K1 of 1.3 InM.
Phosphate inhibition of the phosphatase was also examined, but because the assay depends upon the measurement of phosphate released from histidinol phosphate, attempting to assess phosphate inhibition using this assay presented obvious difficulties. Therefore, an assay was used in which the phosphatase was coupled to histidinol dehydrogenase, and the reduction of NAD+ was followed (22). Using this assay, phosphate was found not to inhibit the phosphatase at concentrations up to 25 mM. Histidinol dehydrogenase activity was unaffected by phosphate over the same concentration range. Arsenate has been reported to be an inhibitor of phosphatase activity (7,8), but in our hands proved to be a poor inhibitor Inhibition-Histidinol is one of the product,s of dephosphorylation of histidinol phosphate and it is, therefore, not surprising that inhibition of the phosphatase by histidinol was observed.
A Dixon plot of that data revealed the inhibition to be competitive with an inhibition constant of 52 PM (Fig. 5C). On the other hand, dehydratase activity, using 1 mM imidazoleglycerol phosphate, was not affected by the presence of histidinol at concentrations some 500 times (25 mM) as high as those giving 50% inhibition of the phosphatase. Histidine Inhibition-A Dixon plot of the histidine inhibition of phosphatase activity (Fig. 50) shows that changing the terminal alcoholic function to the charged carboxyl group results in a much weaker competitive inhibitor of the enzyme.2 The K1 for histidine was found to be 10 mM or about 200 times larger than the Kr for histidinol.
Dehydratase activity is not affected by histidine up to a concentration of 20 mM. Zn2f Inhibition-During the course of examining the effect of other metal ions, it was found that zinc inhibits imidazoleglycerol phosphate dehydration.
At a ZnCb concentration of 1.1 PM, dehydration of imidazoleglycerol phosphat'c is inhibited by about 50%, even though Mn2f is present at a concentration seven times its K,.
No inhibition of phosphatase activity by zinc ion could bc detected over the concentration range that resulted in inhibiting 80% of the dehydratase activity. Inhibition by Heat-Heat treatment of the dehydratasr-phosphatase enzyme allowed an additional means of showing a differential effect on the two activities.
Heating at 54" at pH 7.5 results in a very rapid first order loss of phosphatase activity with a half-life of about 2 min. In contrast to the marked sensitivity of the phosphatase, no loss of dehydratase activity was detected upon heating for up to 16 min. Loss of dehydratase activity showed some variability, but the rate of loss for dehydratase activity was always greatly different (at least 15 times slower) from that for phosphatase activity.
Nonreciprocal Inhibition by Substrates--If mutually escluaive and independent sites existed for the two activities, then no reciprocal inhibition would be observed between imidazoleglycerol phosphate and histidinol phosphate. Accordingly, it was first demonstrated that no phosphate was released from imidazoleglycerol phosphate when varied from 5 to 50 11131. To determine whether imidazoleglycerol phosphate could inhibit phosphatase activity, concentrations from 5 to 50 rnbf were added to the enzyme in 6.2 mM histidinol phosphate. The activity of the phosphatase was not affected under these conditions.
MnC12 was at a concentration of 60 pM. In order to investigate the converse effect, histidinol phosphate inhibition of dehydratase act.ivity, it was first necessary to inhibit the phosphatase activity since continual release of phosphate from histidinol phosphate would in itself result in an observed inhibition of the dehydratase. Therefore, the experiment was carried out using a concentration of 50 mM histidinol which inhibits the phosphatase activity, but not the dehydratase activity.
Even at this high histidinol concentration, it was not possible to completely inhibit the phosphatase activit'y and some phosphate was released at high histidinol phosphate concentrations.
However, the presence of histidinol phosphate up to a concentration of 20 mM had no significant effect on the dehydratase activity.
B slight decrease in velocity (15%) of

DISCUSSION
The data presented here on the inhibition patterns of the bifunctional hisB enzyme using a variety of different inhibitors indicate that the dehydratasc and phosphatase sites are separate and distinct.
The dehydratase activity is inhibited by aminotriazole and phosphate (Fig. 5) as well as by Znzf. The phosphatase activity is inhibited by none of these, but rather is inhibited by heating at 54", histidinol and histidine (Fig. 5). Dehydratase activity is not inhibited by the latter two and is considerably more stable to heat. Aminotriazole and phosphate act (Lompetitively with imidazoleglycerol phosphate and, therefore, presumably bind at the active site. Since all of these inhibitors differ widely in either charge, size, or structure, and each one inhibits competitively only one of the two activities, and in view of the dissimilarity of the two reactions, it seems reasonable to conclude that two separate sites on the protein must be responsible for the two activities. IIowever, as Dison and Webb (23) point out, competitive inhibition cau occur by two possible modes of binding; at the active site itself (fully competitive inhibition), or at some other site which affects catalytic activity in a partially competitive maluler.
If the inhibitors used in this study are binding at the active site, then the lack of inhibition of one of the two activities speaks strongly for there being two separate sites. If, however, binding of the inhibitors occurs at a site other than the active site, the possibility then exists that parts of the two active sites may overlap.
This possibility was ruled out, however, by observing the effects of incubating both substrates together. Uecause no reciprocal inhibitory effects were observed, it (aan be concluded that there is no overlap between the two sites; that the sites are separate and do not share common catalytic residues.
Our data on the molecular weight forms are somewhat at variance with those found by others.
We have not observed any distinct species with a molecular weight of 150,000 which has been previously reported (10,11). Rather, we observe 300.000 and 75.000 molecular weight forms as the major species under the conditions used as well as larger aggregates much higher than 300,000. Vasington and LeBeau (11) report a 75,000 molecular weight species upon either purification of the enzyme (without MI?+) or freezing and thawing of a crude preparation. Our data indicate that the 75,000 molecular weight unit is capable of aggregation to produce species of molecular weight greater than 150,000.
The numerical values for these molecular weight forms must be considered tentative in light of the limitations of the method used to determine molecular weights.
However, aggregation does indeed appear to occur. Although Mn2+ influences this aggregation, other factors may be operative as well, since further attempts to purify the enzyme by ion exchange chromatography or preparative disc gel electrophoresis even with Mn2f present resulted in broad peaks or multiple bands of activity.3 Acknowledgments-The excellent technical assistance of Mary Richards in the early part of this work and Margaret Graham in the latter stages is greatly appreciated.
A generous and timely gift of histidinol phosphate by Dr. Robert Goldberger was very welcome.