Composition of the First Enzyme of Histidine Biosynthesis Isolated ...

JOURNAL OF BACTERIOLOGY, Feb. 1975, p. 485-490 Copyright 0 1975 American Society for Microbiology

Vol. 121, No. 2 Printed in U.S.A.

Composition of the First Enzyme of Histidine Biosynthesis Isolated from Wild-Type and Mutant Operator Strains of Salmonella typhimurium STANLEY M. PARSONS* AND MARTIN LIPSKY Department of Chemistry, University of California, Santa Barbara, California 93106 Received for publication 7 October 1974

The first enzyme of histidine biosynthesis in Salmonella typhimurium, adenosine triphosphate phosphoribosyltransferase (EC 2.4.2.17), has been purified from two bacterial strains containing histidine operator deletions and compared to the enzyme from a strain that has a normal operator. The enzymes isolated in different ways also are compared. Evidence as to the separateness of the operator and first structural gene or covalent modification of the first enzyme was sought. Specific activity, histidine feedback inhibition, amino acid analysis, discontinuous-gel electrophoresis and gel filtration of the native enzyme, and Ouchterlony double-immunodiffusion tests were carried out. The purified enzyme contains little phosphorous and has five cysteine residues per subunit, which all are readily titratable. No evidence for differences in the enzyme preparations was obtained. Thus, no evidence for overlap of the histidine operator with the first structural gene was obtained. The first enzyme of histidine biosynthesis, N '-(5'-phosphoribosyl)adenosine triphosphate: pyrophosphate phosphoribosyltransferase (EC 2.4.2.17), or adenosine triphosphate phos-

phoribosyltransferase (ATP-PRT), has been implicated in control of expression of the histidine operon in Salmonella typhimurium (4, 10, 12). The enzyme was formerly, although incorrectly, called PR-ATP synthetase. The histidine feedback-sensitive site on the enzyme subunit appears to be critical to gene regulation, although the precise role of the enzyme in this function remains unclear. In addition to the enzyme, wild-type mature histidyl-transfer ribonucleic acid is known to be indispensible to repression (13). A combination of positive and negative regulation of the histidine operon has been proposed (11). The gene for ATP-PRT is called hisG and is contiguous with the operator-promotor region (5). Most operator mutations have been selected on the basis of resistance to histidine analogues under conditions that required the

presence of functional ATP-PRT (20, 21). Since the product of the G gene appears to be involved in control of the histidine operon, an ambiguity could arise as to the nature of "operator" mutations. For example, an N-terminal region of the structural gene for the enzyme might be critical for normal repression, but not severely affect enzymatic function. A similar situation has been shown to be the case in the lac

485

repressor, where the N terminus specifies the deoxyribonucleic acid interaction and a different, nearly independent region of the repressor binds inducers (17, 18). The selections utilized for the histidine operator could favor expression of non-chain-terminating mutations of the N terminus of the G enzyme if it were acting

similarly. Thus, it is desirable in the histidine system to independently confirm the separateness of the operator region from the first structural gene. ATP-PRT has been purified by using standard procedures from the operator mutants hisO1242 and his03156 and from strain hisEll, which is wild type in the operator-promotor region. Both hisO1242 and hisO3156 are short deletions that have been mapped very close to the first mutation known to affect ATP-PRT activity (8, 9, 20). hisO3156 has been mapped over hisO1242 and hisO1830 by Philip Hartman and Bert Ely of Johns Hopkins University (unpublished observations). The enzymes are compared as to histidine inhibition, specific activity, molecular weights of denatured subunits and native enzyme, electrophoretic mobility of native enzyme, amino acid composition, and immunological cross-reactivity. Also, the hisEll enzyme isolated by avoiding the usual heat step was compared to the conventionally isolated enzyme. Previous work indicated that the purified enzyme differed from the enzyme in a gel-filtered crude extract by

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being cold sensitive and immunologically differ- with 1:50 cross-linker was used. Phosphorous content ent, and that these differences were related to was determined by a modification for micro-quantity the heat step (15). Evidence was sought here by the method of Sumner (22). Protein was deterfor a covalent difference that would explain the mined by the method of Lowry et al. (14), with bovine albumin as a standard. Sulfhydryl groups apparent change in properties associated with serum accessible to DTNB were determined for native enheating. Phosphorous content and accessible zyme in a solution of 0.10 M NaCl, 0.01 M tris(hysulfhydryls were determined for the hisEl 1 droxymethyl)aminomethane, 0.5 mM ethylenediaenzyme as well. minetetraacetic acid, and 0.4 mM histidine adjusted to pH 8.5 with HCI. Dithiothreitol-treated hisEll enzyme was chromatographed on Sephadex G50 MATERIALS AND METHODS equilibrated with the above buffer which had been deoxygenated by sparging with nitrogen. Carnosine, dithiothreitol, bis-(5-carboxyl-4- was determined by assay and absorbance at Enzyme 280 nm. nitrophenyl)-disulfide (DTNB), and bis-(2-hydrox- The reaction of the enzyme with DTNB produced were from yethyl)-imino-tris(hydroxymethyl)methane an absorbance increase which was measured at 412 Sigma Chemical Co. Electrophoresis gel materials nm. The following parameters have been assumed: were from Bio-Rad Laboratories. Cacodylic acid an extinction coefficient of 13,600 cm-' M-(Baker grade) was from J. T. Baker Chemical Co. sulfhydryl reacted (7), 2,400 U of enzyme per mg (15), lonagar No. 2S was from Wilson Diagnostics. N-tris- 0.75 mg of enzyme/ml per absorbance at 280 nm (hydroxymethyl)methyl-2-aminomethane sulfonic and a subunit molecular weight of 35,000 (23). (15), acid was from Calbiochem. Other chemicals were from usual commercial sources. S. typhimurium LT2 histidine auxotroph hisEll RESULTS was grown on minimal salts medium supplemented with 0.5% glycerol, 0.4 mM adenine, 20 ug of L-carnoAn abbreviated genetic map of the histidine sine per ml, and 0.3 yg of L-histidine per ml (15). operon is presented in Fig. 1. The operator Bacteria were harvested in early stationary phase. deletion mutants studied here have been Prototroph SB2246, which carries his03156, was mapped to the right of numerous others, but do grown as above without carnosine and histidine. Cells were harvested when growth reached an absorbancy not overlap the first known G mutation. The at 600 nm of 1.2, where growth slowed considerably hisEll strain was utilized as the source of the G due to an unknown limitation. ATP-PRT was isolated enzyme from a wild-type operator, since it is from these mutants as described previously (15), derepressed for the enzymes of the histidine except that the recycle steps were omitted and the pathway when grown on limiting histidine. enzyme was chromatographed on diethylaminoethylATP-PRTs rapidly isolated from all three Sephadex A50 at pH 8.0 instead (3). The hisEll strains by utilizing the usual 61 C heat step enzyme was also isolated by a previous procedure (3) were identical, within experimental error, in utilizing three chromatography steps with the follow- specific activity and histidine sensitivity (Table ing changes. The 61 C heat step was omitted, and a pH 4.7 precipitation step was added (15). The enzyme 1). The hisEll enzyme isolated without the previously isolated from histidine auxotroph TA2165 heat step had a lower specific activity and (15) was utilized as the source of the enzyme from the decreased histidine sensitivity (Table 1) and gave an ultraviolet difference spectrum, with his01242 mutation. The enzyme specific activity and histidine inhibi- peaks at 265 and 291 nm and troughs at 280 and tion studies were performed on enzyme preincubated 303 nm, when compared to the heated hisEll in standard buffer (pH 7.5) containing 0.4 mM enzyme. However, these differences probably histidine by using the standard assay (1). Amino acid can be attributed to partial denaturation of the analyses were performed with a Beckman model 120C analyzer on reduced and carboxymethylated (2) unheated enzyme. The isolation procedure utihis03156 enzyme hydrolyzed for 24, 48 and 72 h in 1202 3 56 constant-boiling HCl at 110 C after vacuum freeze1828 2321 1242 1830 200 I thaw deoxygenation. Other analyses were on samples hydrolyzed for 24 h only. Antiserum against very pure Operotor - promoter 0G DCBHAFI I E hisEll enzyme, obtained by sequentially subjecting the enzyme to two different purification schemes (3, FIG. 1. Abbreviated genetic map of the S. typhi15), was prepared by standard adjuvant technique murium histidine operon. The gene for ATP-PRT, from six New Zealand rabbits and pooled (6a). hisG, is contiguous with the operator-promotor. Antiserum against pure his01242 enzyme has been his 01828 is the left most operator-type mutation, described (15). Ouchterlony double-immunodiffusion his2321 is a promotor-type mutation, and hisG200 is was carried out in 1% agar in borate-saline (6b). the first mutation affecting ATP-PRT activity. The Disc-gel electrophoresis was performed in sodium hisO3156 deletion occurs closest to hisG200 of any dodecyl sulfate and on native enzyme in the pH 7.8 known operator mutation. The hisO1242 deletion system of Rodbard and Chrambach (19), except that overlaps hisO3156 and is used in many studies. The 0.5 mM histidine, 1 mM disodium ATP, and 2 mM hisEll mutation is an ochre occurring near the end of MgCl2 were added to all buffers. A 6% running gel the operon. I

TABLE 1. Kinetics and chemical properties of ATP-PRT

EnzyATP-PRT matic orgn sp originl aCta

Histidine inhibition constants ill K

Phos- Accessible

phomus

sulfhydrylsc

(m)coeffi(m)cient hisEll

3,100

0.05

2.4

his01242 hisO3156

3,200 3,160 985

0.04 0.05 0.18d

2.8 2.4 (3)

hisEll

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(unheated)

0.047 0.16

5.0

+

0.2

0.081

0.21

a Units/absorbancy

at 280 nm per ml. 'Moles per 35,000 molecular weight; 3.8 and 1.9 mg of unheated hisEll enzyme and 8.4 and 3.4 mg of heated hisEll enzyme were analyzed. The smaller relative phosphorous content occurred in the larger enzyme samples in both cases. c Moles per 35,000 molecular weight. Average of four determinations on 2.6 x 10-' M to 5.4 x 10-' M subunits reacted with 2.16 x 10-° M to 5.24 x 10-5 M DTNB held at approximately twofold excess over sulfhydryl. "Inhibition at concentrations of histidine greater than 1 mM was incomplete with about 10% residual activity. The Hill coefficient was not accurately determined.

lizing the heat step can be completed in 1.5 days, whereas the procedure for the unheated enzyme requires about 4 days, much of which is under conditions where the enzyme lacks stability (15). Thus, there is no kinetic evidence that the enzyme from the three bacterial strains differs. Amino acid analyses of the enzymes obtained from the three strains are the same, within experimental error, for a 24-h hydrolysis (Table 2). Averaged or extrapolated data were obtained for the reduced and carboxymethylated enzyme from hisO3156. This analysis differs substantially in some residues from that published previously on the enzyme which was approximately 85% pure, as judged by specific activity and gel electrophoresis (23). Discontinuous-gel electrophoresis of native enzyme as a single, sharp band was accomplished in the presence of histidine, sodium ion, and magnesium ATP (Fig. 2). These components have all been shown to stabilize the hexamer form of the enzyme (16). Histidine alone led to streaking of the enzyme. hisEll that was heated or unheated during isolation and hisO3156 migrated as single, sharp bands with traces of faster-moving components. The hisO1242 enzyme migrated as two sharp bands, with a more intense one corresponding in position to the hisEll and hisO3156 enzymes and a less intense one corresponding to one of the minor bands in the other samples. Mixtures of the enzymes showed that the major band in all four was electrophoretically identical. The fast-

er-moving minor components may be oxidized or deamidated ATP-PRT. In view of the identity of the major component of the hisO1242 enzyme with the others, no particular genetic significance is attached to the more intense faster-running band found in that sample. Electrophoresis at pH 10.2 in a glycine-tris(hydroxymethyl)aminomethane-phosphate system resulted in a single diffuse band of protein for all samples, and all were electrophoretically identical. Electrophoresis in sodium dodecyl sulfate gave identical single bands. Also, gel filtration of heated hisEll and hisO3156 enzymes in standard buffer with histidine (15) separate and mixed resulted in indistinguishable elution volumes (last three experiments not shown). Thus, there is no electrophoretic or chromatographic evidence for a difference among the four preparations in charge characteristics or molecular volume. Pairwise comparison of all four enzyme preparations by Ouchterlony double-immunodiffusion using antiserum directed against heated hisEll enzyme indicated immunological identity for all (Fig. 3). A similar experiment utilizing antiserum prepared against heated TABLE 2. Amino acid analyses of ATP-PRT

Amino acid acid Amino

Average or extrapolated values for

24-h Valuesa

hisO31566

hisEll hisO1242 hisO3156

Asp Thr Ser Glu

Gly Ala Val Met Ilu Leu Tyr Phe Lys

His Arg Pro

Carboxy-

1.31 0.552 0.661 1.74 1.00 1.16 0.731

1.23 0.529 0.655 1.76 1.00 1.20 0.787

1.23 0.470 0.664 1.67 1.00 1.22 0.753

0.927 2.03 0.219 0.183 0.588 0.190 1.06 0.823

0.900 1.93 0.227 0.162 0.655 0.223 1.08 0.592

0.862 1.92 0.216 0.147 0.520 0.163 1.05 0.585

A

B

1.27 0.532 0.728 1.77 1.00 1.23 0.83 0.362 1.00 1.97 0.254 0.182 0.488 0.163 0.96 0.579 0.221

29.9 12.5 17.1 41.6 23.6 28.9 19.5 16.9 23.6 46.2 6.0 4.3 11.4 3.9 22.6 13.6 5.0

methyl Cys

__

a Ratio relative tL glycine _ 1.00. Heated hisElI enzyme was analyzed. b Reduced carboxymethylated enzyme was analyzed. Thr and Ser extrapolated to 0 h; Val, Leu, and Ilu extrapolated to Xc h; the others averaged. (A) Ratio relative to glycine - 1.00. (B) Number of residues per 35,000 molecular weight, which does not include tryptophan.

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I.

la

i

it

1

2

3

4

5

6

7

8

FIG. 2. Discontinuous-gel electrophoresis of native ATP-PRT. Approximately 10 ug total enzyme was subjected to electrophoresis at pH 7.8 and 0 C. Enzyme was protected from oxidatior. by 5% (vol/vol) saturated sodium thioglycolate added to each sample in the stacking buffer containing approximately 18% glycerol before electrophoresis at 1 mA per tube, until a bromophenol blue marker reached the bottom of the gel. Enzymes were stained in Coomassie brilliant blue in aqueous acetic acid-methanol and destained electrophoretically (24). The enzyme preparations were: 1, heated hisEll; 2, unheated hisEll; 3, mixed hisEll enzymes; 4, hisO3156; 5, mixed hisO3156 and heated hisEll; 6, hisO1242; 7, hisO1242 and heated hisEll; 8, mixed hisO3156, hisO1242 and heated hisEll. The faster band in the hisO1242 enzyme preparation was approximately one-fifth as intense as the slower band by visual estimation. Higher background light intensity clearly showed the difference in band intensities in gel 6 but washed out the trace bands in gels 1, 2, and 3.

his01242 enzyme also gave lines of identity (not shown). Thus, all four preparations of enzyme are immunologically identical. The hisEll enzyme was found to have 5.0 sulfhydryl groups per subunit accessible in the native enzyme (Table 1). The enzyme was inactivated immediately upon exposure to DTNB, but the activity was fully recovered after treatment with dithiothreitol (data not shown). Figure 4 shows a second-order plot for release of the 5-carboxy-4-nitrothiophenolate ion during reaction. An initial burst reaction occurred, followed by a final phase which was approximately 14 times slower. The relatively rapid reversible reaction of all five cysteine residues per subunit (Table 2) indicates that all are probably on the surface. The great instability of ATP-PRT synthetase toward air oxidation (15) probably is attributable to this large number of exposed sulfhydryls. Both heated and unheated hisEll enzyme was found to contain little phosphorous (Table 1). Since the phosphorous content appeared to decrease when larger samples of protein were analyzed, it is probable that the detected phos-

phorous was present as a contaminant. Phosphorylation, adenylation, or other similar phosphorous containing modifications are not present in the isolated hisEll enzyme.

DISCUSSION There is no evidence that either of the two histidine operator mutations affect the structure of purified ATP-PRT. Thus, there is no reason to suspect from these data that the operator region overlaps the first structural gene. Also, the enzyme isolated from a histidine prototroph was apparently identical to that isolated from bacteria grown under conditions of histidine limitation. There is no evidence that the enzyme undergoes stable modification in response to different states of histidine nutrition. However, the data presented here do not rule out the possibility that the operator is translated as part of the first structural gene and is cleaved off in a maturation step leading to active enzyme. Immature, inactive precursors would not have been isolated in the approach used here.

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nected with any charge difference. Thus, deamidation and other major primary sequence changes in the enzyme as a direct result of the heat step are precluded. However, aging of the enzyme at 4 C for about 8 h also induces these changes (15), and any labile covalent modification that controlled the differences in properties would not have been detected due to its loss during isolation of the enzyme. If ATP-PRT is involved directly in control of the histidine operon, it is probable that its interaction is modulated through a conformational change or a very labile covalent modification. ACKNOWLEDGMENTS We thank Mark Meyers and Tom Dixon for technical assistance, and Nancy Lee for amino acid analyses. This work was supported by a grant from the Cancer Research Funds of the University of California (JB-73-2) and by Public Health Service Biomedical Science Support Grant

RR-07099-06.

FiG. 3. Ouchterlony double-immunodiffusion of four preparations of ATP-PRT. Antiserum (12 uliters) prepared against very pure heated hisEll enzyme was placed in the center well, and 12 uliters each of the enzyme preparations, at 1 mg/ml, were placed in the following wells: 1, heated hisEll; 2, his01242; 3, heated hisEll; 4, unheated hisEll; 5, his01242; 6, hisO3156. Diffusion was at room temperature at pH 8.05 in the absence of any ligands known to stabilize the hexameric form of ATP-PRT. A well-defined line of identity formed. 20.0 In U. LnIe

.,.I

5.0

Z