TlIE JOURNAL OF UroLoomnL ClrEallsTnY Vol. 244, No. 3, Issue of February 10, pp. 551650, lOGO

Printed in U.S.A.

The Purification and Characterization of L-Histidine

Ammonia-lyase (Pseudomoaas)*

(Received for publktion, September 20, 1968)

MATTHEW M. RECHLER$

From the Laboratory of Biochemical Pharmacology, National Institute of Arthritis and Metabolic Diseases,

National Institutes of Health, Bethesda, Maryland 2001~

SUMMARY

Histidine ammonia-lyase was purified from a pseudo- monad grown on L-histidine. Enzyme preparations appeared homogeneous in the ultracentrifuge and by analytical disc electrophoresis. Native enzyme had an s&,,~ of 11.1 S and a D2~,w of 0.50 X low6 cm2 see-l. A molecular weight of 214,000 was obtained from sedimentation velocity and diffu- sion and 211,000 from sedimentation equilibrium. The enzyme was dissociated by 6 M guanidine hydrochloride-O.1 M

mercaptoethanol into subunits of molecular weight 35,000. Mercaptoethanol both activates and inhibits the native en- zyme.

L-Histidine is nonoxidntively deaminated to trans-urocanate and ammonium ion in enzymat.ic reaction is (EC 4.3.1.3, formerly as follows (2-4).1

H

animals and many bacteria (1). The catalyzed by histidine ammonia-lyase called histidase) and may be denoted

H I I

Imidazole-CH?-C-COO- -+ Imidazole-C=C-COO- + NH*+ I

NHs+ Ii

This is the first step in a degradative pathway which ultimately yields formatc and glutamate. Histidine ammonia-lyase from Pseudomonas has previously been partially purified (4-7) and some aspects of its reaction mechanism studied (4, 5, 7, 8). The present investigation describes physical and chemical properties of the homogeneous enzyme and its dissolution into subunitas.

* A preliminary account of this work has been presented (I~EcH-

LER. M. M.. AND TABOR. H.. Fed. Proc. 27. 586 (1968)). I , I I

$ Present address, Laboratory of Molecular Biology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland 20014.

1 Reversibility of the enzymatic reaction after a l-week incuba- tion has been recently reported (5).

MATERIALS AND METHODS

Reagents-Commercial reagents were used throughout. Urea (Baker) was recrystallized from water and dissolved immediately prior to use. Recrystallized guanidine monohydrochloride was purchased from Aldridge Associates and Company (Washington, D. C. 20009). Iodoacetic acid (Eastman) was twice recrys- tallized from petroleum ether-ether.

Xtandard Assay-Histidine ammonia-lyase was assayed spectrophotometrically, taking advantage of the extinction coefficient of Iruns-urocanate, 1.88 X lo4 M-' cm-l at 277 rnp (9). In a 2.9.ml volume, the reaction cuvette (l-cm light path) contained enzyme, 0.133 M diethanolamine-Cl (pH Q.O), 1.33 mM mercaptoethanol, and 0.1 mM MnCl*. This mixture was incubated at 25” for 15 min, after which the reaction was initiated with 0.1 ml of L-histidine.HC1.HzO, giving a final substrate concentration of 3.3 mM. The absorbance at 277 rnp was moni- tored with a Gilford spectrophotometer. At low enzyme con- centrations, the reaction could be observed for at least 1 hour, and the absorbance increased linearly with time. Reaction rate was proportional to enzyme concentration from 5 to 1000 units of enzyme. One unit (6) of enzyme caused an increase in absorb- ance of 0.001 per min at 277 rnp and 25”. Specific activity refers to the number of enzyme units per mg of protein.

Protein Determination-Protein was quantified by the colori- metric assay of Lowry et al. (10) after precipitation with 10% trichloracetic acid. Bovine serum albumin was the reference standard. The nitrogen content of purified enzyme samples was determined with Nessler’s reagent, following Kjeldahl digestion and distillation, or microdiffusion (11). Purified enzyme, at a concentration of 1.0 mg per ml by the method of Lowry et al., had a nitrogen content of 0.20 mg per ml and an absorbance of 0.50 at 280 rnp in 0.1 M potassium phosphate (pH 7.2).

Analytical Disc Electrophoresis-Polyacrylamide gel clectro- phoresis was performed at pH 9.5 and 0” as described by Davis (12), with apparatus and reagents purchased from Canalco. The protein solution was layered above the gels in 25 To glycerol.

2 Nitrogen determinations were kindly performed by Dr. Wil- liam C. Alford of the Microanalytical Chemistry Laboratory, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health.

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552 Hi&dine Anzmonia-lyase Vol. 244, No. 3

After electrophoresis, the gels were stained for protein with Amido black. Duplicate gels were sectioned horizontally into 3-mm slices. These were incubated overnight at 0” in 0.3 ml of 0.1 M potassium phosphate buffer (pH 7.2) and the eluate was examined for enzyme activity.

Hydrodynamic ilfeasurements-The sedimentation velocity of histidine ammonia-lyase was studied in the Spinco model E ultracentrifuge at protein concentrations of 1.5 to 10 mg per ml. A 4” single sector cell with a 12-mm filled Epon centerpiece was centrifuged at 60,000 rpm in an An D rotor at 2-5”. Schlieren patterns were photographed at intervals of 16 or 32 min through- out the centrifugation and the photographic plates were read on a Nikon microcomparator. Correction of the sedimentation coefficient to 25” and calculation of the molecular weight were according to Schachman (13).

The diffusion coefficient was determined by the rnet,hod of Longsworth (14), adapted for the ultracentrifuge by Dr. William R. Carroll. The separate chambers of a double sector synthetic boundary cell were loaded with 0.15 ml of enzyme solution (3 to 6 mg per ml) and 0.45 ml of buffer, respectively. The cell was accelerated t,o 10,000 rpm in an An D rotor for sharp boundary formation and then maintained at 5,000 rpm and 5”. Boundary spreading was observed with Raylcigh interference optics and was photographed at approximately 30.min intervals for 3 hours.

Meniscus depletion sedimentation equilibrium was performed according to Yphantis (15). Buffer, 0.12 ml, and 0.10 ml of a 0.1 to 1.0 mg per ml solution of enzyme were introduced into the compartments of a double sector cell, yielding a column of approximately 3 mm of fluid. Native enzyme was centrifuged in an An J rotor at 16,000 rpm for 36 hours at approximately 5”. For subunit studies in 6 M guanidine hydrochloride-O.1 M mer- captoethanol, the centrifugation was conducted in an An D rotor at 25” for 25 hours at approximately 40,000 rpm. The Rayleigh interference fringe pattern was constant for at least 1 hour before terminating the centrifugation. In all experiments, at least 35 y0 of the fluid column had been depleted of protein.

Before ultracentrifugation, native enzyme was dialyzed against four daily changes of 4000 volumes of 0.1 M potassium phosphate (pH 7.2).0.1 mM MnCl,. Buffer viscosity and density at 25” were determined experimentally in an Ubbeholde viscosimeter and by pycnometry, respectively.

Amino Acid Analysis-Three 0.5-mg samples of purified cn- zyme were each dialyzed against 1000 volumes of distilled water, which were changed daily for 4 days. Each sample was trans- ferred to a vacuum hydrolysis tube (Kontes Glass, No. K-896850- 0010) and lyophilized. Three milliliters of constant boiling (6 N) hydrochloric acid were added to each tube. The tubes were evacuated, sealed, and placed in an oven at 105”. After 24 hours, one tube was withdrawn and its hydrochloric acid was removed by lyophilization. The remaining tubes were similarly trrated at 48 and 72 hours, respectively. Amino acid analysis of two aliquots of each hydrolysate was performed on a Beckman model 120C amino acid analyzer (16). The nitrogen content of t,he hydrolysates was also determined (11).

Another 0.5mg protein sample was treated with performic acid. The dialyzed and lyophilized protein was reacted with 2.0 ml of freshly generated performic acid (17) at 0” for 2.5 hours and then immediately frozen in a Dry Ice-acetone bath. After lyophilization, the samples were hydrolyzed for 24 hours as described above and cysteic acid was determined on the amino acid analyzer.

Carboxymethylated histidine ammonia-lyase was prepared for amino acid analysis as follows. A Thunbcrg tube containing 2 mg per ml of enzyme in a solution of 8 M urea-O.1 M Tris-Cl (pH 8.6)-0.11 M mercaptoethanol was flushed thoroughly with nitrogen and incubated at 37” for 8.5 hours. Alkylation with iodoacetate was performed as described by Craven, Steers, and Anfinsen (18). S-Carboxymethylcysteine was determined by amino acid analysis of a 24-hour hydrolysate of the alkylated enzyme.

RESULTS

Purification

Step 1: Growth and Extraction-A Pseudomonas species (ATCC l1,299b), 3 grown with L-histidine as sole carbon and nitrogen source, produces high levels of histidine ammonia-lyase (19). Three hundred liters of the medium of Tabor and Mehler (6), prepared in distilled water, were inoculated with 15 liters of a logarithmically growing culture of the bacterium. Growth for 17 hours at 25-30” with forced aeration yielded 1 kg (wet weight) of cells. These could be stored at -20” for at least 7 months without loss of enzyme activity.

Thawed cells were suspended in 4 volumes (milliliters per g) of 0.05 M potassium phosphate (pH 7.2) at 2” and disrupted by compression in a Gaulin Laboratory Homogenizer.4 Two passages at 9,000 to 11,000 p.s.i. were required. This and all subsequent operations were performed at 2-4” unless otherwise specified. After 15 min of centrifugation at 27,300 X g, the supernatant fraction was retained.

Step 6: Heat Treatment-The enzyme solut.ion was transferred to 50-ml test tubes, heated to 78-83” in a water bath for 15 min, and then chilled to 0”. The precipitate was discarded after centrifugat,ion for 15 min at 27,300 x g.

Step 3: Protamine Sulfate-Protamine sulfate,5 0.026 ml of a 2 g/100 ml solution, was added per mg of protein in the super- natant fraction obtained from Step 2. After 15 min, the sus- pension was centrifuged at 27,300 x g. To the supernatant solution, 20 ml of glycerol were added per 100 ml.

Step 4: Ammonium sulfate-Ammonium sulfate (Mann, enzyme grade), 209 g, was dissolved in each liter of glycerol- enzyme solution. After 15 min, the precipitate was removed by centrifugation at 27,300 x g for 15 min. The supernatant portion, containing 85% of the enzyme activity, was treated with an additional 75 g of ammonium sulfate per liter. After 15 min, another centrifugation was performed and the supernatant solu- tion was discarded. The pellet, which contained the enzyme, was dissolved in 25 to 50 ml of 0.02 M potassium phosphate (pH 7.2)-0.1 mM MnC12-30% (v/v) glycerol (Buffer A) and dialyzed for 12 to 36 hours against two 4-liter volumes of this buffer.

Xtep 5: DEAE-cellulose Chromatography-Microgranular DEAE-cellulose6 was equilibrated with 0.02 M potassium phos- phate (pH 7.2)O.l mM MnClz-25% (v/v) glycerol (Buffer B) and packed in a column, 60 x 3 cm, at atmospheric pressure. Enzyme was applied to the column and the latter was washed with 400 to 600 ml of Buffer B. Histidine ammonia-lyase was

3 American Type Cull;ure Collection Catalogue, 19G8. 4 Manton-Gaulin Manufacturing Company, Everett, Mas-

sachusetts. 5 Protamine sulfate was obtained from Nutritional Biochemi-

cals. The solution was adjusted to pH 7 with NaOH. 0 Whatman nE52, supplied by H. Reeve Angel, Inc., Clifton,

New Jersey.

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Issue of February 10, 1969 M. M. Rechler 553

TABLE I

Purijicalion of histidine ammonia-lyase

step VOlllIlE

1. Crude extract”. . 1,760 2. Heated. . . . . 1,400 3. Protamine sulfate.. 1,440 4. Ammonium sulfate 42 5. DEAE-cellulose.. . 229 6. Preparative disc gel . 275b 7. Sephadex G-150 50

ml

T

Total Total Speciiic activity protein activity

units x 10-s

276 237 195 143 115 18b 14

mg wtits lmg

36,000 770 3,360 7,050 2,950 6580

625 22,800 160 71,800 33b 54,600” 21 66,700

- a Obtained from 400 g (wet weight) of cells. b Combined totals from four separate electrophoreses c Glycine, a known competitive inhibitor of the enzyme (8), is

present in the electrophoresis buffer, and is partly responsible for the lower specific activity.

eluted with a linear gradient formed from 750 ml of Buffer B in the mixing bottle and 750 ml of Buffer B-l M NaCl in the reser- voir. The elution rate was 45 ml per hour, and IO-ml fractions were collected. The enzyme was eluted between 610 and 840 ml of the gradient.

Step 6: Preparative Polyacrylamide Gel Electrophoresis-The DEAE-cellulose eluate was concentrated in an Amicon ultm- filtration cell with a UM-1 membrane7 and was dialyzed over- night against Buffer A to remove NaCI. Electrophoresis was performed in a Canalco Preparative Electroghoresis apparatus, with upper column PD-2/320, the gel system of Davis (12), and the electrode buffers described by Buchler (20). The dialyzed protein, in less than 10 ml of 30% (v/v) glycerol, was layered above 10 ml of stacking and 10 ml of separating gels. Electrophoresis at 15.ma current proceeded for approsimately 24 hours, with circulating ice water to help maintain the gel temperature at 0”. Materials emerging from the gel during electrophoresis were eluted at a rate of 45 ml per hour with 250/, (v/v) glycerol-O.1 M Tris-Cl (pH 8.0); lo-ml fractions were collected. Enzymatically active fractions were examined by analytical disc electrophoresis, and those giving a single protein band were combined. DEAE-cellulose eluates of up to 50 mg of protein could be sharply resolved by this procedure.

Step 7: Sephadex G-150 Chromatography-The pooled prepara- tive electrophoresis fractions were concentrated to 6 ml in an Amicon ultrafiltration cell. The enzyme was applied to a column, 90 x 2.5 cm, of Sephadex G-150, previously equili- brated with Buffer B, and waseluted with this buffer at a rate of 10 ml per hour. Fractions of 3.0 ml were collected and those con- taining enzyme activity were combined.

A representative purification of histidine ammonia-lyase is summarized in Table I. Comparable results have been obtained with five different preparations.

Stability-Enzyme stored prior to the DEAE-cellulose purifi- cation step irreversibly lost up to 40% of its activity within several days. This loss could be prevented if the first four purification steps, from disruption of the cells through dialysis of the dissolved ammonium sulfate pellets, were performed without interruption. When delay was unavoidable, anaerobic storage

7 Amicon Corporation, Lexington, Massachusetts 02173. The UM-1 membrane retained materials with a molecular weight of

FIG. 1. Analytical disc electrophoresis at three stages of purification. Acrylamide gel electrophoresis (pH 9.5) was performed on enzyme obtained after the DEAE-cellulose, pre- parative electrophoresis, and Sephadex G-150 purification steps. Corresponding enzyme specific activities are shown in Table I. The respective samples contained 13, 14, and 6 pg of protein. Protein migrated to the anode (bottom) and was stained with Amido black. The lower gel margin represents the bromphenol blue marker dye position. The three electrophoreses were performed at different times. One protein band in the DEAE- cellulose sample possessed enzymatic activity and has been aligned with the single enzymatically active band in the Prep- gel and G-150 samples.

was found preferable. Glycerol and 0.1 mM MnClt were added as indicated to further prevent activity loss. In contrast, following DEAE-cellulose chromatography or complete purifica- tion, enzyme preparations remained fully active after storage for 9 months at 2” in either Buffer B or in 0.1 M potassium phosphate (pH 7.2)-0.1 mM MnC&, with chloroform as a preservative. Mercaptoethanol was not introduced during purification or storage; after several weeks of exposure to the thiol, enzyme preparations irreversibly lost activity.

Analytical Disc Gels-Acrylamide gel electrophoresis at pH 9.5 of enzyme eluted from DEAE-cellulose revealed one protein band which possessed enzyme activity and two to four enzymat- ically inactive protein bands (Fig. 1). The inactive species were removed by preparative gel electrophoresis. Fully purified enzyme, obtained after Sephadex G-150 chromatography, migrated as a single protein band in three different analytical disc gel systems, at pH 9.5,8.9,8 and 7.8.8 Enzyme activity coin-

8 These gel formulations were generously made available prior to publication by Dr. Andreas Chrambach, National Cancer

10,000. Helium was maintained at 50 to 100 p.s.i. Institute, National Institutes of Health.

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Ilistidine Ammonia-lyase Vol. 244, No. 3

FIG. 2. Sedimentation of histidine ammonia-lyase. The sedimentation velocity of 3.1 mg per ml of purified enzyme was examined in 0.1 M potassium phosphate (pH 7.2)-0.1 mM MnClt as described under “Materials and Methods.” The schlieren patterns, from left to right, were photographed 4,20,36, 52,84, and 116 min after a speed of 60,600 rpm was reached.

11.6 I I I I I -

II.4 -

II.2 - m

10.4 -

0 2.0 4.0 6.0 8.0 10.0

CONC.(mglml)

FIG. 3. Dependence of the sedimentation coefficient of histidine ammonia-lyase on protein concentration. Sedimentation velocity studies were performed as described under “Materials and Methods.” Protein was determined calorimetrically. The line drawn is a least squares fit of the experimental points. Buffers: n , 0.1 M potassium phosphate (pH 7.2)O.l mM MnClz; 0, 0.133 M diethanolamine-Cl (pH 9.0).

cident with the protein band could be eluted from the first two of these gels, but could not be recovered from the third.

Physical Properties

Sedimentation Coeficient-Fully purified histidine ammonia- lyase sedimented in the ultracentrifuge as a homogeneous protein (Fig. 2). Sedimentation coefficients determined at different protein concentrations are plotted in Fig. 3. These data are alsc represented by the equation: s = 11.12 (1 - O.O0817C), where s is the sedimentation coefficient (in S) and C is the protein con- centration (in milligrams per ml). Extrapolation to zero protein concentration gives an .s&,,~ of 11.12 S, with a standard deviation of 0.06 S.

Diffution CoeJicient-Diffusion coefficients of 0.52, 0.48, and 0.50 x 10m6 cm2 set-1 were determined in the analytical ultra- centrifuge at protein concentrations of 3.4,5.7, and 5.1 mg per ml, respectively. The distribution of enzyme was gaussian at all times of observation, as expected for homogeneous protein.

Molecular Weight-From the mean diffusion coefficient (0.50 x 10e6 cm2 see-I), the s&,~ of 11.1 S, and the partial specific volume (s) of 0.740 ml per g based on the amino acid composi- tion, a molecular weight of 214,000 was calculated.

Meniscus depletion sedimentation equilibrium further con- firmed that the enzyme preparations were homogeneous. In six experiments, plots of log C against R2 were linear (Fig. 4). The molecular weights obtained from these determinations are summarized in Table II, with a mean value of 211,000.

I I , I I , I I , I , ,

3.20 -

3.00 -

2.80 - /* .

2.60

o 2.40

g 2.20

J 2.00 .

1.80 ./ .

1.60

; /

;/ 1.40 -

1.20 -

1.00 ' 1 ' 0 ' ' ' ' ' I ' ' 48.50 48.80 49.10 49.40 49.70

R2

FIG. 4. Sedimentation equilibrium of histidine ammonia- lyase. Meniscus depletion sedimentation equilibrium is de- scribed under “Materials and Methods.” After equilibration with 0.1 M potassium phosphate (pH 72-0.1 mM MnCla, 0.5 mg per ml of purified enzyme was centrifuged at 16,000 rpm and 6.6’for 36 hours. C is the ve t’ r ma 1 f ringe displacement in microns and R is the centimeter distance from the center of the rotor.

Absorption Spectrum-The absorption spectrum of purified histidine ammonia-lyase in potassium phosphate buffer (pH 7.2) is shown in Fig. 5. The only discrete spectral peak was at 280 rnp. There was no evidence for a prosthetic chromophore in the absorption spectra at pH 7.2, at pH 13, or after treatment of the enzyme with 10 mM sodium borohydride.

pH Optimum-The optimal pH for histidine ammonia-lyase activity under standard assay conditions was 9.0. Half-maximal activity was observed at pH 8.1 and pH 9.9.

Subunit Xtructure

Histidine ammonia-lyase was dissociated into smaller units by 6 M guanidine hydrochloride-O.1 M mercaptoethanol. Sedimen- tation velocity studies performed in this solvent revealed a single schlieren peak. The sedimentation coefficients at different pro- tein concentrations are plotted in Fig. 6. The subunit molecular weight was calculated from these data by the method of Tanford, Kawahara, and Lapanje (21). These authors observed that in 6 M guanidine hydrochloride-O.1 M mercaptoethanol a series of proteins behaved as a random coil. To correct for preferential solvent interactions, 0.01 ml per g was subtracted from the partial

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Issue of February 10, 1969 M. M. Reckler 555

BUfffX Protein concentration Molecular weighta

-.

w/ml

K-Phosphate ............... 0.1 229,500 K-Phosphate ............... 0.5 215,000 K-Phosphate. .............. 1.0 198,000 Diethanolamine-Cl ......... 0.07 195,000 Diethanolamine-Cl ......... 0.1 225,000 Diethanolamine-Cl.. ........ 0.5 203,000

TABLE II Sedimentation equilibrium

Meniscus depletion sedimentation equilibrium of purified histidine ammonia-lyase was performed as described under “Materials and Methods.” The buffers used were 0.1 M potassium phosphate (pH 7.2)-0.1 mm MnClz and 0.133 M diethanolamineC1 (pH 9.0).

a Mean molecular weight, 211,000 (f13,OOO S.D

specific volume (21). A molecular weight of 33,500 was cal- culated.

Meniscus depletion sedimentation equilibrium was also per- formed in 6 M guanidine hydrochloride-O.1 M mercaptoethanol. A molecular weight of 38,300 was calculated when 0.9 mg per ml of enzyme was centrifuged fcr 24 hours at 40,000 rpm and 25”, and observed with Rayleigh optics. When 0.3 mg per ml of protein was centrifuged at 36,000 rpm for 25 hours and examined with ultraviolet optics, a molecular weight of 35,000 was ob- tained. The three ultracentrifuge studies in 6 M guanidine hydrochloride-O.1 M mercaptoethanol gave a mean subunit molecular weight of 35,000.

Histidine ammonia-lyase was also dissociated into subunits by 8 M urea-O.28 M mercaptoethanol-0.1 M potassium phosphate (pH 7.2). Appearance of the enzymatically inactive subunits followed an exponential time course: 50% of native enzyme remained after 10 hours and less than 5% after 48 hours.

Amino Acid Analysis

The results of amino acid analyses of purified histidine am- monia-lyase are presented in Table III. Residues per 210,000 g of enzyme are the mean of the three timed hydrolyses, with the following exceptions. Serine and tyrosine were extrapolated to zero hydrolysis time. Values of leucine, isoleucine, and valine from the 72-hour hydrolysates were used. Cystine content was determined both as cysteic acid after performic acid treatment and as X-carboxymethylcysteine after alkylation with iodoacetate. Tryptophan was est’imated spectrophoto- metrically (23). From the integral amino acid composition, a mean residue weight of 122 g, a partial specific volume of 0.740 ml per g (24) and a molecular weight of 196,000 were obtained.

Effect of Thiol Compounds

Mehler and Tabor (9) observed that Pseudomonas histidine ammonia-lyase lost activity during purification or aging, but that this could be restored by glutathione or thioglycolate. Consequently, glutathione (6)) mercaptoethanol (4)) or dithio- threitol (7) have been included in the standard enzyme assay. Reactivation by glutathione seemed to be associated with a “lag” in the enzymatic reaction (4, 6). Cysteine, on the other hand, was a potent competitive inhibitor of the enzyme (8).

The present investigation confirmed the activating and

0.4

0.3 --

!.

z z z 0.2 - =: z

0.1 -

o-

I I I I I I I I

’ --I

i

240 280 320 360 400 440 480 520 560 WAVELENGTH imp)

FIG. 5. The absorption spectrum of histidine ammonia-lyase. The absorption spectrum of purified enzyme was examined in a Carey 14 spectrophotometer, with 0.78 mg per ml of enzyme in 0.1 M potassium phosphate (pH 7.2)-0.1 mM MnC12. No addi- tional absorption peaks were observed at 5.7 mg per ml of protein.

,700 I 1 I

PROTEIN (mg/ml)

FIG. 6. Sedimentation velocity of histidine ammonia-lyase in 6 M guanidine hydrochloride-O.1 M mercaptoethanol at different protein concentrations. Purified enzyme was made approxi- mately 6 M in guanidine hydrochloride and then dialyzed for 48 hours at 25” against several exchanges of 6 M guanidine hydro- chloride-0.1 M mercaptoethanol. Ultracentrifugation was per- formed in a single sector valve type synthetic boundary cell at 60,000 rpm and 25”. The apparent sedimentation coefficient (in S) is plotted without correction for bLtier composition.

inhibitory effects of thiols. For 8 hours after it was released from the cells, crude or partially purified histidine ammonia-lyase showed the same enzymatic activity when assayed under standard conditions as when assayed without added mercapto- ethanol. Thereafter, the activity determined without including mercaptoethanol in the assay mixture was consistently lower than that obtained in its presence. Several days to 7 months after extraction from the cells, enzyme activity determined in

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556 Histidine Ammonia-lyase Vol. 244, No. 3

Amino acid

Lysine. IIistidine Arginine Aspartic acid. Threonine Serine Glutamic acid.. Proline Glycine Alanine. Half-cystine . . . Valine . . . . . Methionine Isoleucine. Leucine Tyrosine. Phenylalanine. Tryptophan.

TABLE III

Amino acid ardysis

Hydrolysis time

24 hrs , 48 hrs / 72 hrs

~moles/?lzg protein molesjZ10,000 g enzyme

0.146 0.178 0.173 34.8 0.204 0.231 0.232 46.7 0.396 0.416 0.420 86.2 0.596 0.632 0.617 129.1 0.2GQ 0.278 0.264 56.8 0.434 0.424 0.394 96.4 0.749 0.782 0.768 161.1 0.340 0.345 0.364 73.5 0.673 0.706 0.714 146.5 1.249 1.286 1.198 261.3

0.629 0.674 0.679 142.7 0.173 0.178 0.168 36.3 0.341 0.369 0.373 78.4 0.813 0.849 0.851 178.6 0.091 0.089 0.083 20.2 0.159 0.171 0.161 34.4

-

Residuesa Mean integr& residues

32 48 87

128 57

101 157

75 144 255

17 (IQ)," 15d 143

37 79

187 19 34

6e

a Mean of the three hydrolyses is shown. Serine and tyrosine values were obtained by extrapolation to zero hydrolysis time. The 72-hour hydrolysates of leucine, isoleucine, and valine were used.

* Integral residues are the mean of the analysis shown and a second complete analysis on another enzyme preparation.

e Determined as cysteic acid after performic acid treatment. The value in parentheses was corrected for an 89.5% experimental recovery of cystine slandard (22). Duplicate analyses on two separate performic acid digests were performed.

rl Determined as S-carboxymethylcysteinc. Two aliqrlots of the same alkylated preparation were altalyzed. The carboxy- methylated enzyme gave a single schlieren peak when sedimcnted in 6 M guanidine hydrochloride.

e Estimated from the ultraviolet absorpt,ion spectrum in 0.1 M

NaOH (23).

the absence of mercaptoethanol was 15yo of that obtained under

standard assay conditions. Activation of the “stored” enzyme by mercaptoethanol was markedly dependent on thiol concen- tration, with 1.33 mM optimal (Fig. 7). Glutathione or dithio-

threitol could activate the enzyme in place of mercaptoethanol. With optimal concentrations of these agents (6.7 and 0.3 mM, respectively), t’he enzyme was 25% more active than with optimal mercaptoethanol. All three thiols, at concentrations above those optimal for activation, were inhibitory. Inhibition with mercaptoethanol was competitive with substrate, with a Ki of 25 InM determined at 33 and 133 mM mercaptoethanol. Thus, appropriate concentrations of glutathione, dithiothreitol, and mercaptoethanol could activate or inhibit histidine am- monia-lyase. Cysteine, which also inhibited the enzyme, differed in being unable to activate the stored preparations at

any concentration. Activation of Stored Enzyme by Mercaptoethanol-The condi-

tions for activation of stored histidine ammonia-lyase by mer- captoethanol have been further defined.

Activation depended on the time, temperature, and pII

of incubation. Under standard assay conditions (diethanola-

mine-Cl (pH 9.0), 1.33 mM mercaptoethanol, 25”) the enzyme was fully activated in 15 min; at O”, activation was only 60% complete after 25 min. In pH 7.2 potassium phosphate buffer at 25”, 0.028 M mercaptoethanol was required to activate the enzyme, with activation complete in 10 min. Urocanate formation by fully activated enzyme was always linear with time (Fig. 8A).

0 4.0 8.0 12.0 16.0 MERCAPTOETHANOL (mM1

-...I

FIG. 7. The effect of mercaptoethanol concentration on his- tidine ammonia-lyase activity. Duplicate 5-J aliquots of puri- fied enzyme were assayed as described under “Materials and Methods.” Mercaptoethanol was added in the final concentra- tions shown. MnC12 was omitted from the reaction mixture. Activity is expressed in units per ml of the original enzyme solu- tion.

L I 0 2 4 6 0 2 4 6 8 IO

TIME(minutes)

FIG. 8. The effect of substrate on the activation of histidine ammonia-lyase by mercaptoethanol. A, 25 ~1 of enzyme stored for 2 months after protamine sulfate treatment were assayed as described under “Materials and Methods” (- - -) or with mercap- toethanol and substrate added simultaneously (+-). MnClz was not added to the reactions. The abscissa is the time after L-histidine addition. B, 25 ~1 of enzyme eluted from DEAE-cel- lulose were assayed as in A, only without addition of mercapto- ethanol. At the arrow, mercaptoethanol was added to a final concentration of 1.67 mM. (The curve is discontinuous for the interval when the cell contents were being mixed.)

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Issue of February 10, 1969 M. M. Rechler 557

Substrate did not prevent activation of histidine ammonia- lyase by mercaptoethanol. When L-histidine was added to the assay mixture either before or with mercaptoethanol, enzyme activation still occurred. In these instances, with enzyme activation and the enzymatic reaction proceeding concurrently, an initial low rate of urocanate formation (or lag) was observed (Fig. 8, B and B).

Stored enzyme activated by mcrcaptoethanol remained fully active when the mercaptoethanol concentration was reduced to 0.06 mM by dilution or limited dialysis against 0.02 M potassium phosphate (pH 7.2). On the other hand, complete removal of the mercaptoethanol from activated enzyme by Sephadex G-25 chromatography caused almost a complete reversion to the nonactivated state within 12 hours.

When the enzyme was incubated and assayed in the absence of air, both activating and inhibiting effects of mercapto- ethanol could be shown at the appropriate concentrations.

Comparison of Activated and Untreated Enzyme-Mercapto- ethanol-activated and untreated purified enzyme showed several different properties.

Stored purified enzyme assayed without mercaptoethanol ex- hibited only 15% of the activity present under standard assay conditions.

Both untreated and mercaptoethanol-activated enzyme showed simple Michaelis kinetics (Fig. 9). Five experiments with mercaptoethanol-activated enzyme indicated a mean K, for L-histidine of 3.8 mM. The mean K, for L-histidine ob- tained in three experiments with untreated enzyme was 16.4 mM. As seen from the data of Fig. 9, the V,, of the two en- zyme states also differed: 1400 units for 60 ~1 of untreated enzyme and 650 units for 10 ~1 of mercaptoethanol-activated enzyme.

Histidine ~Lnlmoilia-lyaae from liver or f’seudornonas was almost complet~cly inhibited by 0.01 IllM EDTA (8, 9, 26). The present studies confirmed the sensitivity of purified, mercaptoethanol- activated enzyme to disodium EDTAg (Table IV). At EDTA concent,rations ranging from 0.1 mM to 0.8 nt, inhibition did not exceed 92 Oi.‘O In contrast, 10 mM (Na)n-EDTA failed to inhibit ljurified enzyme which had not been activat’ed with mercagto- ethanol (Table IV). The activity of EDTA-treated, mercapto- ethanol-activated enzyme was lower than that of stored enzyme not activated with mercaptoethanol.

CMB” did not inhibit, and slightly enhanced, the activity of histidine ammonia-lyase untreated with mercaptocthanol. Mcrcaptoethanol-activated enzyme was inhibited up to 75% by

comparable concentrations of CMB (Table IV). Enzyme freshly released from intact cells did not require

mercaptoethanol for full activity, but otherwise closely re- sembled mercaptoethanol-activated stored enzyme. It had a low K, for L-histidine (1.7 mM), was inhibited 90% by 1 mM (Na)z- EDTA, and was inhibited 35% by 0.1 mM CMB. Mercapto- ethanol-activation of stored histidine ammonia-lyase seemed to reinstate the original active form of the enzyme, but the funda- mental difference between untreated stored enzyme and the activated state of the protein remains obscure. They could not be differentiated by sedimentation in the ultracentrifuge or

9 In analagous experiments, 1 mM (Ca)z-EDTA or (Mg)z-EDTA failed to inhibit the mercaptoethanol-activated enzyme.

1” Originally observed by Dr. Elijah Adams (personal com- mlmication).

11 The abbreviatiou used is: CMB, p-chloromercuribenzoate.

-400 0 400 800 1200 1600

& (M-'1

FIG. 9. Lineweaver-Burk plots (25) of untreated and mercap- toethanol-activated histidine ammonia-lyase. Mercaptoetha- nol-activated (O ): duplicate 10.~1 aliquots of purified enzyme were assayed as described under “Materials and Methods,” ex- cept for the omission of MnC12. Untreated (0): 60-4 aliquots of the same enzyme solution were assayed without the addition of mercaptoethanol or MnCl2. S = the final concentration (molar) of L-histidine; 2, = units of histidine ammonia-lyase.

Activity

___-~_

Without mercaptoethanol

Xercaptoethanol- activated

TABLE IV

Inhibition of hislidine ammonia-lyase by EDYA and CMB

Aliquots, 3 ~1, of purified hi&dine ammonia-lyase were incu- bated and assayed as previously described, except for the omission of mercaptoethanol (where indicated) and MnClz. Jlifferent enzyme preparations were Ilsed for the J2DTA and CMB experi- ments. Inhibitor was added 15 min before r,-histidinc, except where otherwise noted. All assays were performed in duplicate or triplicate.

Inhibitor concentration

WLM

(Na)s-EDTA 0 0.1 1.0

10.0 CMB

0 0.01 0.10 0.20 0.40 0.50 0.60 0.80

30

30, 29a 28, 2S8

16 18 23

26

25

208 18, 21' 18, 19”

129b

30" 57"

74b

a EDTA was added 30 set before substrate. * Aliquots, 3 ~1, of enzyme were activated in 1.33 mM mercapto-

ethanol (pH 9.0) in a total volume of 150 ~1. The activated enzyme was diluted into 2.75 ml of 0.133 M diethanolamine-Cl (pH 9.0) containing CMB. Substrate was added after 10 mins. The [CMB] in the final reaction mixture exceeded the [mercapto- ethanol] carried over from the initial incrlbation by 3- to B-fold.

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555 Hi&line Anamonia-lyase Vol. 244, No. 3

analytical disc electrophoresis at pH 9.5. There are 15 to 19 moles of half-cystine residues per mole of native enzyme (Table III). Of these, fewer than 2 moles of cysteine, in either un-

treated or mercaptoethanol-activated enzyme, reacted with dithionitrobenzoate in 6 M urea (27, 28).

EJect of Sodium Borohydride

Carbonyl reagents, including sodium borohydride, have been reported to inhibit histidine ammonia-lyase (7). After the enzyme was reacted with NaB3H+ tritium remained bound to the protein (7). Similarly, when potato tuber phenylalanine ammonia-lyase was treated with NaB3H4, tritium incorporation paralleled the loss of enzyme activity; cinnamate or phenyl- alanine protected against this inactivation (29). NaB3H4 is known to inhibit n-proline reductase and, in the process, to reduce an enzyme-bound pyruvate molecule to lactate (30).

Purified preparations of histidine ammonia-lyase were inhibited completely by 10 mu NaBH4 in 0.15 M diethanolamine-Cl (pH 9.0, 10 min, 25”), and were inactivated 80% by 1 mM reagent. Ten millimolar n-histidine, added 1 min before NaBH4, did not protect against either concentration of borohydride. When purified enzyme (1.5 mg) was treated with 10 mM NaB3H4 (250 mC per mmole) in diethanolamine-Cl (pH 9.0), separated from excess reagents on a Sephadex G-IO column, 40 x 1 cm, equilibrated with the same buffer, and dialyzed for 18 days against frequent changes of 0.05 RI NHdHCOs, tritium remained bound to the protein (1.8 moles of 3H per 210,000 g).i2 Reaction of the enzyme with NaB3H4 did not alter the protein absorption spectrum at neutral pH. Preliminary attempts have been made to identify the tritiated moiety. Tritium-labeled enzyme was hydrolyzed with 3 ml of 6 N HCl for 4 hours at 105” in evacuated, sealed tubes; tritiated material in the hydrolysater3 did not cochromatograph with lactate or glycolatc in ether- acetic acid-water (13 : 3 : 1).

DISCUSSION

Histidine ammonia-lyase was purified from a pseudomonad and appeared homogeneous by the following criteria: (a) the enzyme sedimented as a single species in the ultracentrifuge; (b) enzyme distribution during diffusion was gaussian; (c) log C against R2 plots of sedimentation equilibrium data were linear; (d) analytical disc gels at pH 9.5, 8.9, and 7.8 revealed a single protein species, with coincident enzymatic activity recoverable from the first two gels. The specific activity of the homogeneous enzyme was, however, up to 5 times lower than that reported by other investigators (4, 5, 7). Comparison with Peterkofsky’s purification, which has been reported in detail (4), indicated that the difference in specific activity was already manifest in the crude extracts. There is no evidence that mutation of the organism, or modified assay, growth, or extraction conditions are responsible for this variation.

In the above purification, enzyme specific activity did not increase after preparative gel electrophoresis and Sephadex G-150 chromatography despite the removal of two to four inactive

I2 The calculation of stoichiometry assumes no isotope dis- crimination.

13 Only35% of the original tritium radioactivity was recovered after acid hvdrolvsis and lvolshilization. It is unclear whether

I -

this represents &tual loss of tritiated material or increased quenching. (In one experiment, the products of acid hydrolysis were incompletely soluble.)

proteins (Fig. 1; Table I). There is no evidence that a portion of the enzyme was inactivated during electrophoresis: purified enzyme has not been resolved into an active and inactive species. There is no indication that an essential cofactor or metal was lost or modified: purified enzyme remained inhibitable by di- sodium EDTA; Mn++, the only metal ion among a large group tested which stimulated enzyme activity, did so by only 30%; there was no spectral or chemical evidence for the presence of B12 coenzyme or pyridoxal phosphate. Although the sugges- tion of a nonpyridoxal carbonyl cofactor (7) is an attractive one, it remains to be established for histidine ammonia-lyase.

Previous estimates of the molecular weight of Pseudomonas and B. subtilis histidine ammonia-lyase by sucrose gradient centrifugation ranged from 198,000 to 220,000 (5, 31, 32). In the present study, the molecular weight of the homogeneous enzyme was found to be 211,000 by sedimentation equilibrium and 214,000 by sedimentation velocity and diffusion. These results have been obtained, however, under conditions different from those of the usual enzyme assay. When the ultracentrifuge studies were performed at assay pH, temperature, and mercapto- ethanol and protein concentrations, identical results were ob- tained. This suggests that the enzymatically active species of histidine ammonia-lyase has a molecular weight of 210,000.

Native enzyme was dissociated into subunits of molecular weight 35,000 by 6 M guanidine hydrochloride-O.1 M mercapto- ethanol. Only one protein band was observed when disc electrophoresis was performed on the dissociated enzyme at pH 9.5 in 6 M urea. In preliminary experiments, trypsin digestion of carboxymethylated enzyme in 2 M urea yielded 20 definite and 10 questionable peptides, with no residual undigested material. This suggests a considerable degree of identity among the six hist.idine ammonia-lyase subunits.

Acknowledgments-My warmest thanks are due Dr. Herbert Tabor, in whose laboratory this work was conducted, for his continual guidance and encouragement. I am most grateful to Dr. William R. Carroll and Mr. Ellis Mitchell for assistance with the ultracentrifuge studies, to Dr. John C. Keresztesy and Mr. David L. Rogerson for growing large batches of bacteria, to Mr. George Poy for performing the amino acid analyses, and to Dr. Leonard Kohn for many stimulating discussions.

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Matthew M. Rechler)Pseudomonas

The Purification and Characterization of l-Histidine Ammonia-lyase (

1969, 244:551-559.J. Biol. Chem. 

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