β-Alanine synthase: Purification and allosteric properties

ARCHIVES

OF BIOCHEMISTRY

AND BIOPHYSICS

Vol. 293, No. 2, March, pp. 254-263,1992

@Alanine Synthase: Purification and Allosteric Properties’ Margaret M. Matthews, Wei Liao, Kalla L. Kvalnes-Krick, Department of Biochemistry and Biophysics, Chapel Hill, North Carolina 27599-7260

University

and Thomas W. Traut2

of North Carolina School of Medicine,

Received August 2, 1991, and in revised form October 22, 1991

&Alanine synthase has been purified greater than lOOO-fold to homogeneity from rat liver. The enzyme has a subunit molecular weight of 42,000 and a native size of hexamer. The enzyme undergoes ligand-induced changes in polymerization: association in response to the substrate, N-carbamoyl-&alanine, and the inhibitor, propionate; and dissociation in response to the product, @-alanine. The ability of the substrate to associate the pure native enzyme to a larger polymeric species was exploited in the final purification step. The purified enzyme had a pI of 6.7, a K,,, of 8 ELM, and a k&K,,, of 7.9 X lo4 M-l s-l. Positive cooperativity was observed toward the substrate N-carbamoyl-/3-alanine, with nH = 1.9. Such cooperativity occurred at substrate concentrations below 12 nM, so that this activation most likely occurs at a regulatory site, with a significantly stronger affinity for Ncarbamoyl-&alanine than that shown by the catalytic site. The enzyme was sensitive to denaturation, which could be minimized by avoiding heat steps during the purification and by the presence of reducing agents. Such denatured enzyme had little change in V,,,, , but had much to associate or higher K,,,, and had also lost the ability dissociate in response to effecters. After purification, enzyme stability was achieved by the addition of glycerol and detergent. o 1992 Academic PWS, IW.

ered biosynthetic, due to the various physiological roles that have been reported for @alanine in different organisms (1). In humans, inborn genetic errors in the preceding enzymes of this pathway that result in elevated or depressed @alanine levels are accompanied by severe neural dysfunction (2-5). These biological and clinical findings suggest the importance of controlled /3-alanine production and are consistent with the observed allosteric regulatory properties of @-alanine synthase (6). Our earlier studies showed that @-alanine synthase, partially purified from rat liver, was a dissociating enzyme that is readily converted from the native hexamer to an inactive trimer by the product @-alanine, or becomes a larger and more active polymer in response to the substrate NCPA3 (6). That enzyme had good affinity for NCflA, with a K,,, of 6.5 PM. When the enzyme from rat liver was completely purified by another laboratory, it gave no evidence of dissociation or association and had much poorer affinity for NCPA, with a K,,, of 170 PM (7). Since a heat step was used in the latter purification, some denaturation may have occurred to the enzyme. We have purified @-alanine synthase to homogeneity from rat liver, with a purification protocol that retains the allosteric character previously described for the partially purified enzyme. EXPERIMENTAL

fl-Alanine synthase (iV-carbamoyl-@-alanine amidohydrolase, EC 3.5.1.6; also called /3ureidopropionase) catalyzes the third and final step in the catabolism of uracil and thymine to produce NH3, COP,and the corresponding &unino acids, /3-alanine and 2-methyl+-alanine. In mammals, the enzyme is found primarily in the liver, where the bulk of pyrimidine catabolism occurs. Since this enzyme synthesizes fl-alanine, it may also be considi Supported by Grant 5-ROl-GM40626 from NIH. 2 To whom correspondence should be addressed. 254

PROCEDURES

Materials K[“C]NO was purchased from DuPont-New Carbamoyl-j3-alanine, &alanine, a-antitrypsin,

England Nuclear. Nphenylmethylsulfonyl

3 Abbreviations used: CHAPS, 3-[(3-cholamidopropyl)-dimethylammoniol]-1-propanesulfonate; CHAPSO, 3-[(I-cholamidopropyl)-dimethylammonioll-2-hydroxy-1-propanesulfonate; DTI’, dithiothreitol; NC@A, N-carbamoyl-@-alanine; PMSF, phenyhnethylsulfonyl fluoride; PTC, phenylthiocyanate; PTH, phenylthiohydantoin; QAR, diethyl(2hydroxypropyl) aminoethyl; S-20, supernatant at 20,OOOff SDS, sodium dodecyl sulfaw, TFA, trifluoroacetic acid; PAGE, polyacrylamide gel electrophoresis. 0003-9861/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

ALLOSTERIC

B-ALANINE

fluoride (PMSF), and CM-Sephadex were from Sigma. Aprotinin, leupeptin, dithiothreitol (DTT), and gel filtration standards were from Boehringer-Mannheim. QAE-Sepharose fast flow resins, plus the Superose 6 gel filtration column, were purchased from Pharmacia. The phenyl-P-5W column, gel electrophoresis standards, and the isoelectric focusing apparatus (Rotofor cell) were from Bio-Rad. Filters (0.45 and 1.2 pm) were purchased from Micro Separations, and microconcentrators were from Amicon. The hollow fiber dialysis unit was purchased from Spectrum. Freshly frozen rat livers (Sprague-Dawley) were obtained from Zivic-Miller (Allison Park, PA).

Methods Enzyme assays. 5-14C-labeled NCPA was prepared by condensation of K[“C]NO (40 mCi/mol) with @-alanine (8) and purified by CMSephadex chromatography (6). For kinetic studies all enzyme assays were done at 37°C in 50 mM KPO, buffer (pH 7.0) plus 1 mM DTT, with NCPA at 250 pM. Reactions were incubated for varying times (total conversion of substrate was normally kept below 10%) and quenched with 4 M HClO*. Product formation was determined by trapping the reaction product, [“C]Ox, as [“C]carbonate on alkali-treated filters which were quantitated by liquid scintillation spectrometry (9). Assays were done in triplicate or quadruplicate, and average values + SD are reported. For the kinetic studies of Fig. 9 (Hill plot), the concentration of [5i4C]NC@A was varied from 5 to 100 nM (0.3-6 nCi) in a lo-ml reaction volume. This large reaction volume made possible the quantitation of sufficient cpm while working in the nanomolar range of substrate, yet keeping the amount of substrate consumed during the assay below 10%. Reactions were started by addition of 10 al of enzyme sample and quenched as above. Since the substrate NC@A produced positive cooperativity at very low concentrations, the enzyme af8nity for the substrate as an activator was defined by an activation constant, S,. This was determined from the Hill plot as the concentration of substrate at the midpoint of the curve with a slope of 1.9. To monitor enzyme purification, column chromatography fractions were each assayed in a 5O&total reaction volume. Reactions were started by addition of a lo-p1 enzyme aliquot. A modified Bradford assay using Coomassie blue G (10) was used to determine protein concentration in all samples, with bovine serum albumin as a standard. Buffers. All buffers were adjusted to pH 7.5 at 24°C and contained 1 mM DTT. Livers were homogenized in Buffer A, containing 20 mM KPO,, 0.25 M sucrose, 1 mM EDTA, plus the protease inhibitors PMSF (1 mM), leupeptin (0.5 mg/liter), antitrypsin (25 mg/liter), and aprotinin (70 units/liter). For anion exchange chromatography (QAE column) Buffer Bl, containing 20 mM KPO, plus 10% glycerol, and Buffer B2, containing 60 mM KPOl plus 10% glycerol, were used in step elutions; both buffers contained the protease inhibitors PMSF (1 mM) and leupeptin (0.5 mg/liter). Buffer C, containing 20 mM Tris, was used for the HPLC hydrophobic interaction chromatography (phenyl-P-5W column). Gel filtration chromatography on a Superose column was done using Buffer D, containing 20 mM Tris plus 50 mM NaCl. Buffers used in HPLC and FPLC columns were filtered through 0.45-pm filters and degassed before using. Except for the FPLC Superose column and the HPLC phenyl column, all buffers were chilled and used at 4°C. QAE-Sepharose fast flow resin (column: 5 X 5 cm) was equilibrated in Buffer Bl. (For reuse, this column was washed in Buffer B2 plus 0.5 M NaCl to remove tightly bound protein). Before application of sample, the Superose 6 gel filtration column was equilibrated with at least 60 ml of Buffer D, containing 2.5 mM NCfiA (during the purification). A phenyl-P-5W HPLC column (7.5 X 75 mm) was equilibrated in Buffer C plus 0.6 M ammonium sulfate. A 0.5-ml sample was loaded onto the column and run at a flow rate of 0.25 ml/min. The column was washed for 20 min in Buffer C containing 0.6 M ammonium sulfate, and the enzyme was eluted along a 15-ml (60-min) linear gradient from 0.6 to 0 M ammonium sulfate. The pooled sample from the QAE column was Isoelectric focusing. concentrated by precipitating the protein (about 150 mg in 275 ml) at

SYNTHASE

255

70% ammonium sulfate. The precipitate was dissolved and dialyzed versus Buffer Bl overnight at 4’C. After the sample was diluted to 50 ml with Buffer Bl, containing 2% ampholytes (2.5 ml 40% Bio-Lyte ampholytes, pH range 5-7), the solution was put directly into the Rotofor isoelectric focusing cell, and focusing was carried out at 12 W constant power for 3.5 h. The apparatus was maintained at 0-2°C by recirculating a solution of 30% ethylene glycol maintained at 0°C. The Rotofor unit contains 20 separate cells, which were assayed for enzyme activity, and also for pH (to determine the enzyme’s PI’). Appropriate fractions were pooled, diluted to 50 ml with 10% glycerol + 1 mM DTT in distilled water, and maintained at 0°C. This sample was reloaded on the Rotofor (without additional ampholytes) and again subjected to a constant current of 12 W for 3 h. Fractions were again assayed, and those with peak enzyme activity were pooled and concentrated overnight using a Centricon-30. Gel filtration chromatography. For the purification, a Superose 6 FPLC gel filtration column (10 X 300 mm) was equilibrated with Buffer D (containing 2.5 mM NCOA), and 100 ~1 of enzyme sample was loaded; the column flow rate was 0.3 ml/min. For additional M, studies the column and enzyme samples were adjusted to Buffer D only, to Buffer D + 1 mM propionate, or to Buffer D + 15 mM @-alanine, and a lOO-~1 sample was then loaded onto the column. The elution position of the enzyme was commonly determined by assay for enzyme activity, though with pure @alanine synthase, its elution position was monitored by absorbance at 280 nm. The column was calibrated with the following protein M, standards: thyroglobulin (670,000), ferritin (445,000), catalase (240,000), aldolase (158,000), and chymotrypsinogen (25,000). A slab gel (0.75 mm X 11 SDS-polyauylumide gel electrophoresis. cm X 16 cm) containing a running gel (9% acrylamide and 0.24% NJ’methylene bisacrylamide) and 2-cm stacking gel (3% acrylamide and 0.1% N,W-methylene bisacrylamide) was prepared using a Bio-Rad casting apparatus. The gel was run in a Model SE-600 vertical slab gel unit (Hoefer Scientific Instruments) by the Laemmli procedure (11). Protein samples in the range 10 ng-10 pg, as well as Bio-Rad electrophoresis protein standards, were applied to sample wells. The gel was run at 15 mA throughout the stacking portion after which the current was increased to 25 mA. A procedure for staining proteins with Coomassie brilliant blue G-250 was used as described (12). Cysteines in &alanine synthase were modAmino acid composition. ified with I-vinylpyridine to yield pyridylethylcysteine as previously described (13). The modified fi-alanine synthase protein was purified from two other minor protein contaminants and unreacted vinylpyridine by chromatography on an aquapore-butyl (C, HPLC) column (2.1 X 30 cm; Applied Biosystems) with a 45-min linear gradient of 0.1% TFA to 0.06% TFA/70% acetonitrile, at a flow rate of 0.1 ml/min. After acid hydrolysis of ,5’-alanine synthase, two aliquots were subjected to PTCamino acid derivatization and HPLC analysis on a Model 420 derivatizer and Model 120A PTH analyzer (Applied Biosystems). These services were provided by the Protein Chemistry Lab (UNC-NIEHS at Chapel Hill).

RESULTS

Heating the Enzyme A previous study showed that heating the crude S-20 liver sample to 50°C was effective as a batch step for precipitating some of the contaminating proteins (7). Concerned about the effects of heating on /3-alanine synthase itself, we performed the experiments shown in Fig. 1, using a freshly prepared crude S-20 enzyme preparation. Enzyme samples containing 10 pg protein were heated in a final volume of 1.0 ml in 1.5-ml tubes. After 10 min of heating, tubes were placed on ice for 5 min; 10 ~1 of cooled enzyme was added to 500 ~1 of assay solution containing

256

MATTHEWS

-0.08

-0.04

0.00

l/[NCBA] FIG. 1. Effect of zyme (S-20 fraction: at 50°C for 10 min. enzyme; (0) heated

0.04

0.08

0.12

(PM) -1

heat treatment on fl-alanine synthase activity. En10 gg per assay; sp act 1 nmol/min/mg) was heated (m) Control, unheated enzyme; (0) control, heated with 1 mM propionate.

substrate concentrations shown in the figure, to measure initial rate enzyme activity. Compared to the unheated control, enzyme that was heated in buffer alone lost 50% of its activity (Fig. 1). Since Tamaki et al. (7) used propionate during their heat step, this ligand was also tested: enzyme heated in the presence of 1 mM propionate retained 95% of the activity. Although propionate stabilized enzyme activity during heating, there was still a significant change as measured by VJK,,,, with values of 10.1 (control, unheated), 4.8 (control, heated), and 3.1 (propionate, heated) pmol/min/~M. The unheated enzyme and the enzyme heated without ligand still had fairly similar Km values (16 and 19 PM, respectively)! By comparison, enzyme heated with propionate now had a K,,, of 63 PM. Since propionate, an inhibitor, was present at a final concentration of 20 PM in the assay, an increase in K,,,, due to the inhibitor, to a Kwp of 19 PM is predicted. Therefore, the observed K,,, with propionate is considerably larger, suggesting a change in the affinity of the enzyme caused by heating, even in the presence of propionate, a ligand which stabilizes enzyme activity. The S-20 enzyme preparation binds propionate effectively (Ki = 90 PM) and shows typical allosteric behavior toward propionate. In the presence of 1 mM propionate (Fig. 2B) the enzyme has an M, of 430 kDa, almost double the native size (Fig. 2A). Purification

were performed at 4”C, and the last two chromatography steps were done at room temperature (24°C). The procedure outlined is for about 100 g of liver, which was homogenized and centrifuged. The supernatant (S-20 fraction) was adjusted to 37% of saturation with ammonium sulfate, stirred gently for 30 min, and centrifuged for 1 h at 105,OOOg. This step accomplished both the first salt precipitation and the removal of subcellular particles to yield a cytosolic suspension. The supernatant was brought to 47% of saturation with ammonium sulfate and stirred for 30 min, and solids were pelleted at 20,OOOgfor 20 min. The supernatant was discarded; the pellet was resuspended in Buffer Bl and dialyzed overnight at 4°C (4X 1 liter buffer). The protein sample was then diluted 1:l with Buffer Bl for the QAE column and centrifuged at 12,OOOg,before application to the QAE-Sepharose column. After the sample was loaded, the column was washed with Buffer Bl (flow rate = 4 ml/min) until protein absorbance (280 nm) reached a constant lower value. Elution of the enzyme was accomplished with Buffer B2. Fractions of 9 ml were collected throughout the run and assayed for /3alanine synthase activity (Fig. 3). The enzyme activity eluted primarily with the 60 mM KP04 step. Fractions containing enzyme were pooled (95% of total enzyme activity), adjusted to 70% of saturation with ammonium sulfate, and stirred for 30 min at 4”C, and the enzyme precipitate was pelleted by centrifugation for 20 min at 20,OOOg. The pellet was resuspended in Buffer Bl and dialyzed overnight (4X 1 liter).

O.OOI

IB

fraction

always gave a slightly higher of the enzyme.

Km value (about 16 PM) than other preparations

m

Cl.06 -I

h

F E ‘=o 0.04 E 5 > 0.02 .-

To avoid the effects of heating, we developed a new scheme for the purification of @-alanine synthase from rat liver, as summarized in Table 1. The first four steps 4 An S-20 crude homogenate

ET AL.

0.00

0

i:‘1 10

11

elution

12

13

volume

14

15

16

(mls)

FIG. 2. Association of @alanine synthase in response to propionate. Chromatography was done in control buffer (A) or in the presence of 1 mM propionate (B). Arrows in (A) indicate the elution position of A4, standards: ferritin (445 kDa) and catalase (240 kDa).

ALLOSTERIC

P-ALANINE TABLE

Purification

Total protein (md

Volume (ml)

Fraction s-20 AS 37-47% QAE-Sepharose Rotofor 1 Rotofor 2 Phenyl-Sepharose Superose FPLC

of P-Alanine

407 100 275 14 28 26 2.1

HPLC

16,280 2,120 148 36.4 14.0 3.8 0.84

The enzyme sample was added to a solution of ampholytes and subjected to isoelectric focusing (Fig. 4) as described under Methods. After the two isoelectric focusing runs, the enzyme was concentrated using a Centriconmicroconcentrator. To avoid protein precipitation during this step, the pooled sample (pH near 6.7) was adjusted to pH 7.5. The sample was then adjusted to 0.6 M ammonium sulfate and applied to the phenyl-Sepharose HPLC column (Fig. 5). The first two enzyme activity peaks were pooled and concentrated. The final chromatography step utilized a Superose 6 FPLC gel filtration column equilibrated with Buffer D; 2.5 mM NCPA was included in the column buffer to promote association of @-alanine synthase to a larger polymeric species (6), thereby facilitating its separation from smaller contaminating proteins. The column elution profile and SDSPAGE gel of the corresponding column fractions are shown in Fig. 6. This column produced two major uv absorbance peaks, with most of the fi-alanine synthase activity eluting with the first protein peak. The correspond-

12-0

257

SYNTHASE

I

Synthase

from

Rat Liver Purification x-fold

Recovery (%)

Specific activity (nmol/min/mg) 0.80 2.8 25.3 98.9 228 721 877

100 45 29 28 25 21 5.7

1 3.5 32 124 285 901 1096

ing SDS-PAGE gel shows a single protein band which increases in intensity coincident with /3-alanine synthase activity. The subunit M, of ,&alanine synthase was determined from a standard curve to be 42,000 (repeated more than three times each, with two different sets of M, standards). The gel shows that the first protein peak was largely pure fl-alanine synthase; however, a small amount of @-alanine synthase activity (with correspondingly decreased amounts of the M, 42,000 band) continued to elute under the second major protein peak. The gel shows the M, 42,000 band to be the major protein in the sample loaded on the Superose column (Fig. 6A, lane 1). For the procedure outlined in Table I, a significant loss in recovered activity occurred at the original ammonium sulfate step, and again later with the final chromatographic step where glycerol had to be omitted. Very little enzyme activity was lost on the two successive isoelectric focusing steps. The pure enzyme in Table I had a specific activity of 877 nmol/min/mg protein; this represents a moderate rate of catalytic activity, with a kcat of 0.63 s-l.

8

1.5

1.2 7

i 0.9

0.6 6 0.3

0

300

600

elution

volume

900

1200

(mls)

FIG. 3. Anion-exchange chromatography. The enzyme sample was applied to the QAE-Sepharose column and was eluted with 60 mu KPO, buffer.

0

5

10

15

20

Cell Number FIG. 4. Isoelectric focusing of @alanine synthase. Enzyme from the QAE column (Fig. 3) was focused in successive runs on the Rotofor. This figure represents the second run.

258

MATTHEWS

ET AL.

fairly consistently observed at most stages of the purification. This leads to a specificity constant, kJK,,, , of 7.9 x lo4 M-’

t E .E 2

R a

E > -0 0

10

20

elution volume (mls) FIG. 6. Hydrophobic interaction chromatography. The concentrated sample from the isoelectric focusing step was applied to a phenyl-sepharose HPLC column and eluted with a decreasing gradient of ammonium sulfate (0.6-O M), producing three peaks of enzyme activity.

Both detergents and reducing thiols must be present to stabilize the purified enzyme in solution. The enzyme is hydrophobic and adsorbs easily to surfaces. A tube that had stored concentrated enzyme was emptied and washed; a subsequent second wash with 3% SDS resulted in solubilizing some fi-alanine synthase as shown in lane 13 (Fig. 6A). Enzyme activity was barely detectable after storage at 4°C for 26 days in buffer alone (Fig. 7). Addition of the detergent CHAPS0 at 3% resulted in improved recovery for 10 days, with a third of the activity still measurable after 26 days. Also shown is the importance of fresh reducing thiols; for both experiments in Fig. 7, at the early times there were increases in enzyme activity after the addition of fresh DTT to stored enzyme. In fact detergent produced an apparent increase in activity (relative to the control) in the first few days, probably because the detergent helped resolubilize enzyme that had come out of solution. This effect by detergent is similar to the resolubilizing effect of SDS shown in lane 13 of Fig. 6A, except that detergent did not denature the enzyme. The detergent CHAPS gave results almost as good as those of CHAPSO, whereas digitonin was not effective.

s-l.

Since our earlier studies (6) suggested that the substrate activates the enzyme, the relationship between P-alanine synthase activity and substrate concentration was examined at very low levels of N-carbamoyl-P-alanine. The Hill plot (Fig. 9) demonstrates complex kinetics for the enzyme in the substrate concentration range 5-100 nM. Below 12 nM substrate, the slope of the Hill plot (nn = 1.9) indicates positive cooperativity for the enzyme. Above 12 nM substrate (nH = l.O), the enzyme follows MichaelisMenten kinetics, comparable to what is seen in Fig. 8. This biphasic Hill plot indicates that a transition affecting the kinetics of the enzyme became complete at approximately 12 nM NCPA, a substrate concentration which is almost 3 orders of magnitude below the Michaelis constant for the enzyme (Km = 8 f 2 PM).

11

12

13

14

elution volume Kinetics With P-alanine synthase from a partially purified rat liver preparation, we measured a K,,, of 6.5 PM (6), whereas Tamaki et al. (7) have measured a Km of 170 PM for the pure enzyme. To reconcile the possibility that enzyme purity contributed to these differences, we examined the kinetics of pure rat liver P-alanine synthase obtained by our own purification protocol. Figure 8 shows a Lineweaver-Burk plot of velocity versus substrate concentration for pure P-alanine synthase. At this range of substrate concentration the enzyme exhibited Michaelis-Menten kinetics with K,,, = 9.6 PM (Fig. 8), a K,,, value that we

15

16

17

(mls)

FIG. 6. Gel filtration chromatography in the presence of N@A. (A) Samples along the Superose 6 gel filtration chromatography profile (shown in B) were analyzed by SDS-PAGE. Molecular weight markers (for the far left and right lanes) contained 2 ag each of myosin (ZOO,OOO), P-galactosidase (116,00), phosphorylase b (97,400), bovine serum albumin (66,200), and ovalbumin (45,000). Lane 1 contained the enzyme sample loaded on the column; lo-p1 aliquota from fractions of the column elution profile were applied to lanes 2 through 11 (at 12.6 to 15.3 ml of the elution); lane 12 contained the concentrate of pooled enzyme from 12.6 to 14.1 ml, and lane 13, the SDS wash from the tube for lane 12. (B) A lOO+l sample of the phenyl-P-5W enzyme peak was concentrated and chromatographed on a Superose 6 gel filtration column; both the sample and the column buffer contained 2.5 mM NCj3A. @Alanine synthase activity (4) and corresponding protein absorbance (-) at 260 nm are shown.

ALLOSTERIC

0

5

10

15

20

25

@ALANINE

SYNTHASE

30 1

Time (days) FIG. ‘7. Storage stability of fl-alanine synthase. Pure enzyme was stored at 4°C in buffer alone (m) or in the presence of detergent at 3% (A). Both solutions originally contained 1 mM DTT, and an additional 1 mM DTT was added at the times indicated by arrows.

Enzyme Size By denaturing electrophoresis the subunit has a size of 42,000 + 2000. The amino acid composition (Table II) is consistent with a protein containing 398 amino acids. Only two Met residues were detected, which must both be internal since efforts at sequencing showed the protein to have a blocked N-terminus, while cyanogen bromide digestion always produced three peptides. The purified enzyme still showed an allosteric response to ligands as seen by change in the native size. In the absence of ligands, the native size of the purified enzyme was 240,000 (Fig. 10); in the presence of 2.5 mM NC/IA, the enzyme had a size of about 410,000, while 15 mM @alanine caused dissociation to an M, of 160,000 (Fig. 10). The concentration of the enzyme is a potential variable in judging its oligomer size. Experiments that tested the

10

i

-0.1

0.0

0.1

0.2

(yM) -’

FIG. 8. Lineweaver-Burk plot of purified function of N-carbamoyl-@-alanine.

fl-alanine

FIG. 9.

O(M)

Hill plot for @alanine

TABLE Amino

Acid

Composition

II of @Alanine

Amino acid

22.1 40.3 17.7

11.1 14.7 37.0 8.5 53.9 25.2 38.5 20.0 11.8

19.7 33.0 2.4 16.9 25.4

398

as a n Determined

Synthase

mol/mol enzyme

Total synthase

synthase activity.

effect of protein concentration on such measurements are shown in Fig. 11. Except at the very high enzyme concentrations that are desired during the final purification step (Figs. 6, lo), the observed &f, for @-alanine synthase was quite steady at about 240 kDa in buffer alone, or at about 320 kDa in the presence of 2.5 mM NCBA. Thus, in the enzyme concentration range that we routinely employ, this is not itself a variable. Nonetheless, the pure enzyme is not always responsive to ligands that promote association or dissociation. An example of such a pattern is illustrated in Fig. 12, where the middle peak in the AZ80 profile represents fl-alanine synthase. These experiments had originally been done

Asp + Asn Glu + Gln Arg His LYS Ala Cys” ‘JY Pro Ser Thr Tyr Ile Leu Met Phe Val

I

I

l/[NCBA]

100

[NCRA;

as pyridyl-ethyl

derivative.

260

MATTHEWS

ET AL. contra

+ NCDP

11

12

13

14

elution volume

15

16

(mls)

FIG. 10. M, shift of pure j3-alanine synthase in the presence of NC@A or &alanine. Protein M. standards are shown (0). @-Alanine synthase elution: control, no ligand (0); in the presence of 2.5 mM NCBA (U); in the presence of 15 mM @-alanine (A).

using the enzyme alone, and were then repeated in the presence of internal M, standards to emphasize the reproducibility of the chromatography system, and the lack of response to normal allosteric effecters. This unusual chromatographic behavior led to a reexamination of the enzyme’s kinetics. Figure 13 shows biphasic kinetics, with most of the enzyme activity now associated with a very high K,,, of about 150 PM (suggestive of denaturation, as in Fig. l), while about 15% of the total enzyme activity still shows a low K,,, of 16 FM. DISCUSSION

Purification

/I-Alanine synthase was purified from rat liver to apparent homogeneity (Table I, Fig. 6). N-Carbamoyl-Palanine was added to the final column to increase the

O-IA,, 8 10

,, , , , , , 12

14

16

elution volume

16

20

(mls)

FIG. 12. Gel filtration chromatography of denatured @-alanine synthase. Samples of pure enzyme were chromatographed in buffer (A), in the presence of 2.5 mM NC@A (B), or in the presence of 15 mM @-alanine (C). Samples included internal M, standards: ferritin (first peak) and chymotrypsinogen (third peak).

native molecular size of the enzyme and thus enhance the separation of fi-alanine synthase from smaller protein contaminants. With the benefit of an appropriate affinity chromatography ligand, the exploitation of this allosteric behavior proved to be a valuable strategy for specific isolation of the enzyme. From about 100 g of rat liver, the final yield of pure protein was about 800 pg. The presence of glycerol, where possible, was very helpful during the purification, and the presence of detergents helped sta-

c

3.0

3

.

E

E 1

slmmo 0 20

40

60

Protein

80

2.0

.

E

.

2

1.0

-IL

0.0 -0.10

J c

100

(pg)

FIG. 11. Effect of enzyme concentration on native M,, as determined by gel filtration chromatography, in standard buffer (0) or in the presence of 2.5 mM NCj3A (0).

0.00

l/[NCXA] FIG.

13.

Lineweaver-Burk

0.10

0.20

(PM) -’

plot of denatured fi-alanine

synthase.

ALLOSTERIC

B-ALANINE

bilize the pure enzyme. The final recovery was normally in the range 3-6% of the activity in the starting homogenate. The overall purification fold was normally about 1100, signifying that in the liver @-alanine synthase is about 0.1% of the cytoplasmic protein, and somewhat above average in abundance. Some of the properties of the pure enzyme are summarized in Table III. From over 10 measurements, with different sets of reference proteins, the subunit M, was 42,000. This suggests that the native polymeric form of the enzyme (M, 240,000,5 Figs. 10,ll) is a hexamer. The amino acid composition data show the enzyme, relative to average proteins (14), to have significantly lower amounts of Asx, Met, and Lys. /3-Alanine synthase also has a significantly higher proportion of glycine, which constitutes 13.5% of amino acid residues. Overall, the enzyme has an average composition of hydrophobic amino acids, but has a much lower amount of charged residues; this latter feature probably contributes significantly to the observed hydrophobic properties of the protein. The native molecular size of freshly prepared crude enzyme was significantly increased in the presence of 1 mM propionate (Fig. 2). Enzyme prepared by Tamaki et al., using a heat step, showed no change in M, in response to propionate, or to any other ligands (7). The native molecular size of the pure enzyme was likewise increased in the presence of 2.5 mM NC@A to an M, value of about 320,000 at most enzyme concentrations, or to 430,000 at >400 pg protein (Fig. 11). When the pure enzyme was chromatographed in the presence of 15 mM P-alanine, its elution position was retarded to give an M, value of 160,000. Depending on the actual concentration of the effector, the enzyme can have an observed M, that is not an integral value of the subunit M,, but simply reflects a statistical average for interconverting polymeric assemblies (e.g., hexamers and trimers) during the time course of chromatography. By adjusting the concentration of ligand during molecular weight studies, the enzyme can be titrated from a hexameric form to trimer with @-alanine, or from hexamer to dodecamer with NCj3A (6). In the present case, each effector was used only at a single concentration to verify that this allosteric response had not been lost during purification. The elution of /3-alanine synthase on the phenyl-P-5W HIC column (Fig. 5) produced multiple peaks of @-alanine synthase activity. Grinberg et al. (15) have characterized similar behavior for ,&lactoglobulin, a protein which is known to undergo self-association, using HPLC hydrophobic interaction chromatography. The multiple P-lactoglobulin peaks were shown to represent rapidly equilibrating molecular weight species which are discretely ’ Sedimentation studies had given an average M, of 235,000 for native enzyme (61, consistent with the current average A4, of 240,000 observed by gel filtration chromatography. The expected M, for a hexamer of 42,000 Da subunits is 252,000.

261

SYNTHASE TABLE Physical

Properties

Subunit Mra Native M/ Native polymer size Polymerization effects Molar extinction Isoelectric point

coefficient

III

of ,&Alanine

Synthase

42,000 + 2000 240,000 + 5400 Hexamer +NC@A: M, > 400,000 + B-alanine: M, < 180,000 tZBO= 75,240 M-’ cm-’ pl = 6.7 f 0.15

’ At least 10 determinations.

adsorbed by the column matrix and reach definite endpoints which depend on sample concentration (15). Since P-alanine synthase shows at least two peaks by HIC chromatography, it is possible that these peaks are due to the presence of more than one molecular weight species which can exist in an equilibrium mixture in solution. Trailing enzyme activity observed for the gel filtration chromatography of the pure enzyme peak (Fig. 6) may also be due to the presence of more than one polymeric form of the enzyme. Molecular weight species have been shown to separate by various chromatographic applications, including gel filtration chromatography, even in the case of rapidly equilibrating polymeric systems (16). Thus, there is evidence for only a single protein species by SDS electrophoresis and by ionic charge (Figs. 3,4,6A), although multiple activity forms may occur due to the allosteric transitions of this dissociating enzyme between different polymeric species (Figs. 5, 6B). Much attention was given to the problem of enzyme recovery. @-Alanine synthase activity is stabilized by using buffer solutions with 310% glycerol, as has also been shown before (7). The ability of glycerol to alter the hydrogen-bonding and hydrophobic interaction character of proteins in solution (17, 18) prevented its use during hydrophobic interaction chromatography steps as it significantly reduced the binding efficiency of /3-alanine synthase to such resins. Kinetics Different K,,, values have been reported for the rat liver enzyme by Tamaki et al. (7) and our laboratory (6). A significant increase in K,,, was observed when the enzyme was treated according to Tamaki et al., by briefly heating at 50°C in the presence of propionate (Fig. l), resulting in a Km of 63 FM. One of the earliest characterizations of an allosteric enzyme (aspartate carbamoyltransferase of Escherichia cd) demonstrated how a heat step can denature the allosteric site of an enzyme without causing any loss of catalytic activity; after aspartate carbamoyltransferase was heated for 4 min at 6O”C, the standard inhibitor CTP no longer had any effect, while enzyme activity was actually increased (19). For /3-alanine syn-

262

MATTHEWS TABLE

Comparison

of the Properties

ET AL. IV

of Rat Liver @-Alanine Synthase Purified by a Previous Method (7) vs the Present Method Current results”

Ref. (7)

Affinity for ligands (at pH 7.0) Km: NCfiA Ki: fl-alanine Ki: propionate Sati*: NC@A L/Km Physical properties Native I’& Subunit M, Polymer size NCflA effect on M, E. (kcal/mol) ‘, above 19’C below 19°C

8+2nM 1.08 mM

7.9 X 10’ M-’ s-’

170 @M No inhibition (at 1 mM) 300 PM 170 PM 6.6 X lo3 M-’ S-’

240,000 f 19,400 42,000 + 2,000 Hexamer Association (at > 100 PM) 12.1 12.1

325,000 54,000 Hexamer None (at 1 mM) 10.3 23.8

90 /LM 9nM

’ Some data are from Matthews and Traut (6). * Substrate concentration that produces 50% activation. ’ From Arrhenius plot.

thase also, we suggest that the proposed regulatory site is more sensitive to heat denaturation. Thus heating did not cause any decrease in V,, when the enzyme was stabilized by the competitive inhibitor propionate (Fig. 1; Ref. (7)), but such heated enzyme showed a lower affinity for substrate (Fig. 1; Refs. (7,20)). Heating in the presence of propionate appears to produce a conformational form of the enzyme which is more vulnerable to the denaturation of the postulated regulatory site of the protein. This suggested that coupling between the catalytic site and a regulatory site for the substrate is consistent with the results, showing that propionate, although a competitive inhibitor, at low concentrations can produce some activation of the enzyme (21).6 This suggests that the catalytic site is relatively stable to heating since maximum activity is retained, but after heating the amount of substrate required to achieve V,,, is much higher because binding at the allosteric site, required to promote the transition to the active conformation, has become significantly poorer. This is consistent with our observation of the T + R transition near 9 nM NC@A (Fig. 9), while with enzyme purified by a heating step such a homotropic conformational transition occurred near 170 PM NC/IA (7). The intracellular concentration of iV-carbamoyl-/3-alanine has been estimated to be about 10 FM from steady-state studies (8) and by in uiuo measurements.7 Given this value, P-alanine synthase would be expected to function in the fully active, Michaelis-Menten form in duo, if it had a good affinity for this substrate (K,,, = 8 PM), as shown by our purified enzyme. By comparison, /?I-alanine synthase 6 Such an activating effect by low concentrations of propionate supported by our results showing that propionate favors association @-alanine synthase to the most active dodecameric form (Fig. 2). ’ K. Schnackerx (personal communication).

is of

purified by methods involving a heat step may have become denatured, since such enzyme preparations have been reported with Km’s of 170 PM when purified from rat liver (7), or of 500 PM when purified from calf liver (20). By comparison to the mammalian enzyme, @-alanine synthase from Euglena gradis also forms large polymers, with an M, of 1,500,OOO (22), and has reasonable affinity for NCj3A with a K,,, of 38 PM (23). We did obtain some samples of pure enzyme with a high Km of about 150 PM (Fig. 13). This enzyme preparation had also lost the normal allosteric response to effector ligands (Fig. 12). The activity (V,,) was not significantly changed, although it took much higher [NCPA] to approach V-. Even though the afhnity of the catalytic site for substrate decreased, the concentration of NCBA during gel filtration chromatography, 2.5 mM, was enough to have the catalytic site near saturation. Since no allosteric response was observed, as measured by change in M,, we interpret such data as evidence for denaturation, at a regulatory site different from the catalytic site. The simplest model for the enzyme that accommodates the present results, plus our earlier data (6), as well as studies cited (7, 20), requires that the enzyme have an allosteric regulatory site. Binding of the substrate at the regulatory site is obligatory to promote the active conformation (T + R). The conformational response of the enzyme has now been observed by three types of experiments: (i) in physical studies, the native M, of the enzyme increased in response to NCPA (Figs. 10,ll; Ref. (6)); (ii) in rapid time studies, there was a measurable lag time of 2 s after the addition of NCPA before enzymatic activity was observed (6); (iii) in kinetic studies, positive cooperativity toward the substrate occurred below 12 nM NCPA (Fig. 9). The last observation shows that the T --* R transition is mediated at a binding site with much higher

ALLOSTERIC

fl-ALANINE

affinity for NC@A (S,, = 9 nM) than was observed at the catalytic site (K, = 8 PM). The substrate therefore appears to be an automatic activator of the enzyme. Comparable substrate activation results have been observed with the DNA restriction enzyme Nuel(24).

Comparison of /3-Alanine Synthase from Rat Liver Purified by Different Protocols @Alanine synthase had previously been purified from rat liver by a different method than that used here (7), involving a heat step that may have produced some denaturation. The observed results for the two preparations differ significantly for many characteristics of the enzyme and are summarized in Table IV. Our subunit and native M, sizes are considerably smaller; in each case our values have been reproducibly obtained in many determinations. The affinity for ligands of our enzyme is much better, lower: the since both K,,, and Ki values are significantly K,,, for our enzyme is more than 20-fold lower, while positive cooperativity in response to NC@A (S,,) was 4 orders of magnitude more sensitive. Also, the enzyme purification previously reported produced ,&alanine synthase that was somewhat cold sensitive, as shown by a biphasic Arrhenius plot (7), where E, doubled below 19°C. The enzyme described by us has the same activation energy above and below 19’C (Table IV).

SYNTHASE

263

5. Braakhekke, J. P., Renier, W. O., Gabreels, F. J. M., DeAbreau, R. A., Bakkeren, J. A. J. M., and Sengers, R. C. A. (1987) J. Neural. Sci. 78, 71-77. 6. Matthews, M. M., and Traut, T. W. (1987) J. Biol. Chem. 262, 7232-7237. 7. Tamaki, N., Mizotani, N., Kikugawa, M., Fujimoto, S., and Mizota, C. (1987) Eur. J. Biochem. 169, 21-26. 8. Traut, T. W., and Loechel, S. (1984) Biochemistry 23,2533-2539. 9. Jones, M. E., Kavipurapu, P. R., and Traut, T. W. (1978) in Methods in Enzymology (Hoffee, P. A., and Jones, M. E., Eds.), Vol. 51, pp. 155-167, Academic Press, San Diego. 10. Read, S. M., and Northcote, D. H. (1981) Anal. Biochem. 116,5364. 11. Laemmli, V. K. (1970) Nature 227,680-685. 12. Neuhoff, V., Arold, N., Taube, D., and Ehrhardt, W. (1988) Ekctrophoresis 9, 255-262. 13. Yuen, S. W., Otteson, K. M., Colbum, J. C., Moore, W. T., Schlabach, T. D., DuPont, D. R., and Mattaliano, R. J. (1989) Protein Sequence User Bulletin, No. 38, Applied Biosystems, Inc. 14. Creighton, T. E. (1983) Proteins, p. 7, Freeman, New York. 15. Grinberg, N., Blanco, R., Yarmush, D. M., and Karger, B. I. (1989) Anal. Chem. 61,514~520. 16. Frieden, C. (1971) Annu. Reu. Biochem. 40,653-694.

1. Griffith, 0. W. (1986) Annu. Rev. Biochem. 66, 855-878. 2. Striver, C. B., Pueschel, S., and Davies, E. (1966) N. Eng. J. Med. 274,636.

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REFERENCES