Escherichia coli phosphoenolpyruvate carboxylase: Characterization and sedimentation behavior

ARCHIVES

OF

BIOCHEMISTRY

AND

Escherichia

BIOPHYSICS

128, 611-622 (1968)

cofi Phosphoenolpyruvate

Characterization

and THOMAS

Bio-MedieaE

Division,

Carboxylase:

Sedimentation E. SMITH

Lawrence Radiation Laboratory, Livermore, California 94550

Received

May

Behavior

29, 1968; accepted

July

University

oj Calijornia,

19, 1968

Phosphoenolpyruvate (PEP) carboxylase was partially purified from extracts of Escherichia coli. Sedimentation analyses of the enzyme in sucrose gradients indicate association-dissociation reactions involving monomeric units of 94,000 molecular weight. It has an SZO,, of 8.4 (corresponding to a dimer) in the absence of substrates, metal ions, metabolic regulators, or urea. The SZO,~ decreases to 5.8 (monomer) when 1 M urea alone is included in the centrifugation medium, It increases to 12.2 when Mg2+ is included or to 13.2 (tetramer) when aspartate, an allosteric inhibitor of PEP carboxylase activity, is included in the centrifugation medium. Sedimentation analyses of the enzyme in the presence of urea containing PEP, Mg*+, and acetyl-coenzyme A give an SZO,,~of 8.1, characteristic of the dimer. Assays for enzymatic activity in 1 M urea under essentially the same conditions show that the enzyme has 62 to 75% of maximum enzymatic activity, indicating that the dimers are enzymatically active. The enzyme is isoelectric at pH 4.92. The K, values for its substrates are: 0.17 mu for acetyl-coenzyme A, 0.44 mM for Mg 2+, 0.63 mM for PEP, and 1.75 mM for bicarbonate. It is inhibited reversibly by p-mercuribenzoate. Increasing concentrations of NaCl or KC1 up to 0.5 M cause a precipitous drop in enzymatic activity; however, neither 0.5 M NaCl nor 0.5 Y KC1 shows an appreciable effect on the &o,~. Although 1 M urea causes dissociation of the molecule and inhibits activity to some extent, it does not interfere with the regulation of catalytic activity by acetyl-coenzyme A.

To understand the catalytic and regulatory mechanism of an enzyme in a metabolic pathway, it is important to know the basic nature of the protein catalysis itself. Ideally, such studies should begin with a completely pure protein. This is not always practical nor necessary because of the proved usefulness of such analytical methods as density gradient centrifugation (1). In the work reported here, a partially purified extract of E. coli was studied in an attempt to learn more about the protein responsible for the anaplerotic carboxylat’ion (2) of phosphoenolpyruvate (PEP).’ One of the functions of this enzyme, phosphoenolpyruvate car-

boxylase (EC 4.1.1.31), is to maintain an operative TCA cycle by maintaining adequate levels of the substrate oxalacetie acid (2) for the first enzyme of that cycle, citrate condensing enzyme. It does so in response to the other substrate for citrate condensing enzyme, acetyl-coenzyme A. Acetyl-coenzyme A, however, functions only in a catalytic role for PEP carboxylase and apparently only to regulate catalytic activity (3). The main product of the carboxylase reaction may also be used in another metabolic pathway, i.e., the formation of aspartic acid for protein synthesis. Just as the PEP carboxylase can be “turned on” by acetyl-coenzyme A, it can be “turned off” by aspartic acid (4). In this respect, this protein fits the broad criterion of exhibiting heterotropic allosteric effects (5). These studies were initiated in an attempt

1 Abbreviations used: PEP, phosphoenolpyruvate; TCA, tricarboxylic acid; MTT, 3(4,5-dimethylthiazolyl-2-)2,3-diphenyltetrazolium bromide; DTT, dithiothreitol; PMB, p-mercuribenzoate; AcCoA, acetyl-coenzyme A. 611

SMITH to explain in terms of some molecular parameter(s) the differential effects of various reagents on the catalytic and regulatory properties of this protein. This report deals primarily with: (a) a general characterization of the enzyme (which is, in part, an extension of the work of C&novas and Kornberg (3)), (b) the effects of ionic strength and urea on regulatory and catalytic activity, (c) approximations of the molecular size by sedimentation in sucrose gradients, and (d) relating the third kind of observations to the first two. MATERIALS

AND METHODS

Purification of PEP carboxylase. PEP carboxylase was extracted from E. coli strain B. The E. co& obtained from Grain Processing Company, Muscatine, Iowa, were suspended in 0.01 M phosphate buffer at pH 7.8 containing 1F4 M EDTA, disrupted in a Waring blender with glass beads (6), and treated with streptomycin sulfate as described by Richardson et al. (6). The large volume of supernate usually resulting from this treatment was made 50yo saturated with ammonium sulfate. The precipitate containing the PEP carboxylase activity was dissolved in 0.01 M sodium potassium phosphate at pH 7.8 containing 10m4M EDTA. The protein concentration was adjusted to approximately 40 mg/ml and further purified as outlined in Table I. The procedure for each of the steps was routine and does not require detailed description. Column dimensions, dialysis times, etc. were as follows: All dialyses were for 6 hours against 3 changes of approximately 100 volumes of 0.01 M phosphate buffer at pH 7.8 containing 1c4 M EDTA. DEAE chromatography was carried out on a 3.8 X 50 cm column bed of Whatman DE-52 (Reeve Angel and Co.). Proteins were eluted from the column with a linear gradient of a phosphate-EDTA buffer, pH 7.8, between 0.01 and 0.3 M delivered over a 24 hour period at a flow rate of 0.8 ml/mm. Hydroxylapatite chromatography was carried out on a 1.9 X 15.5 cm bed of Bio-Rad HT suspension of hydroxylapatite. Protein was eIuted with a linear gradient of 0.01 to 0.3 M phosphate-EDTA buffer at pH 7.8 delivered over a 12 hour period at 0.8 ml/min. Gradients were produced and flow rate was maintained with a Phoenix Varipump model 4000. Ammonium sulfate fractions were made indiscriminately with either crystalline ammonium sulfate or saturated solutions. Assay of enzymatic activity. Enzymatic activity was measured by following the rate of oxidation of NADH spectrophotometrically at 340 rnp by

TABLE I PURIFICATION OF E. coli PEP CARBOXYLASE -

Purificationstep

Crude extract” (NH&SO4 fractionation (35-5Oyo) Dialysis DEAE chromatography (NH&S04 precipitation (O-500/,) Dialysis Hydroxylapatite (NH&S04 precipitation (0-5Ooj,)

-

‘jpecific a.ctivity’ ‘Ii’ield

1?rotein bx)

(units/ w)

1617 344.45 522 336.06

0.21 0.64

__ 1 0.98

493 316.59 252 308.52

0.64 1.22

0.92 0.90

145 283.20

1.95

0.82

115 181.17 24 154.02 20 114.79

1.58 6.42 5.75

0.53 0.45 0.33

-

-

5 The crude extract was obtained from a O-50% saturated (NH.&SO1 fractionation of several preparations after streptomycin sulfate treatment. The (NH&S04 precipitates were dissolved in buffer and stored frozen until used as described. coupling the PEP carboxylase reaction with ma&c dehydrogenase (EC 1.1 .1.37). All rate measurements were made in a Bausch and Lomb Spectronic 505 equipped with a time-rate generator and a Sargent SR recorder as readout. The normal reaction mixture consisted of 6 my PEP, 10 mM KHCOa, 5 mM MgS04, 1.05 mM acetyl-coenzyme A, 10 pg malic dehydrogenase, 0.1 to 0.15 mm NADH, and 100 mM Tris-HCl at pH 8.5, in a total volume of 1 ml. Unless otherwise stated, PEP carboxylase (3 to 20 rg) was added last and a measure of the reaction rate was begun within 30 seconds. The instruments, in general, stabilized within the first few seconds, from which point reaction rates were linear with time and proportional to the amount of enzyme added. Exceptions are noted. Protein assay. Protein concentration was determined spectrophotometrically by 280 mp/260 nn~ absorbance (7) or calorimetrically by the method of Lowry et al. (8). Zonal centrifuSucrose gradient centrijugation. gations were done as described by Martin and Ames (1) in 5 to 20yo (w/v or w/w) linear sucrose gradients. Centrifugation was for 16 hours at 35,000 rpm. The “corrected” Sz0,wrates were calculated using the tables of McEwen (9). For these caIculations the average rotor speeds for the 16 hour centrifugations were calculated from the revolution counter of the centrifuge. The braking system of the centrifuge was employed during deceleration. Since the acceleration and decelera-

E. coli PHOSPHOENOLPYRUVATE tion times of the Spinco L2 centrifuge were negligible compared to the total centrifugat.ion times, they were not considered in the overall calculations. Solutions (100 ~1) containing from 0.5 to 1.5 mg of PEP carboxylase extract plus 0.12 mg (0.8 unit) of diaphorase were layered gently onto the gradients. Sedimentation rate was independent of concentration within the range indicated. Reduced-NAD:lipoamide

oxidoreductase

(diaph-

erase, EC 1.6.4.3) assay. Porcine heart lipoamide oxidoreductase (obtained from Calbiochem) was used in the gradients also as a reference marker for sedimentation coefficient and molecular weight determinations. It was assayed either colorimetrically by reduction of thiazole blue (MTT) at pH 9 with NADH as described previously (IO), or spectrophotometrically by reduction of K3Fe(CN)6 as described by Massey (11). Sephadex G-200 gel jZtration. Gel filtration studies were performed on approximately 40 cm3 bed volumes of Sephadex G-200 in 1.5 cm diameter columns. The Sephadex was swollen and t,he columns run in 0.01 M Tris-HCl buffer containing 1O-4M EDTA at pH 7.8. In some cases, 1 M urea was included in the buffer. The columns were run at room temperature. Electrofocusing. All electrofocusing experiments were done in pH gradients of either 3 to 10 or 3 to 6 at 5” as described by Vesterberg and Svensson (12). The column and ampholytes used in the electrofocusing experiments were purchased from LKB Instruments. All pH measurements were made at approximately 21” on a Sargent model DR pH meter. Determination of dissolved CO2 and storage of the incubation mixture. All solutions were made in boiled water and handled under Indicarb (a CO2 absorbent obtained from Fisher Scientific Company). However, despite this precaution some dissolved CO2 remained. This was determined TABLE PMB

II

INHIBITION OF PEP CARBOXYLASE PRESENCE OF SUBSTRATES

Condition of PMB inhibition

Control (no PMB) No substrates AcCoA, PEP, Mg AcCoA, PEP AcCoA, Mg AcCoA

Activit (munits 3

17.5 6.5 6.6 5.8 6.0 5.8

IN

THE

Inb$i;ion D

0 63 62 67 66 67

Except for the control, W4 M PMB was added to the cuvettes containing the indicated substrates.

613

CARBOXYLASE

spectrophotometrically by using the same reaction mixture and conditions for the PEP carboxylase but in the absence of any added bicarbonate. The reaction was allowed to go to completion under a layer of mineral oil, and the amount of COz was calculated from the change in absorbance at 340 rnp due to the oxidation of NADH. There is a l-to-l relationship between CO2 concentration and NADH oxidation. In some cases, the limit of NADH oxidation was determined by plotting the reciprocal of the absorbance at 340 rnp versus the reciprocal of the time. The Y intercept at infinite time for the terminal linear portion of the curve was taken as the limit of NADH oxidation. This procedure was reproducible. The reaction mixture stored under Indicarb in a semiclosed system of burets (all air entering the system passed through a column of Indicarb) maintained a constant low level of endogenous CO1 for at least one day. RESULTS I.

CHARACTERISTICS

E.

coli

AND

PEP

PROPERTIES

OF

CARBOXYLASE

Some of the characteristics and properties of partially purified E. coli PEP carboxylase have been described (3). No significant differences have been observed in the gross kinetic properties of the enzyme. For comparative purposes, however, overlapping studies are summarized in this section. K,,, for substrates and activators. The apparent Km for each substrate was determined from Lineweaver-Burk (13) plots at the optimum concentrations of each of the other substrates. The apparent Km for each substrate is 0.44 mM for magnesium, 0.17 mM for acetyl-coenzyme A, and 0.63 mM for PEP. These compare with 0.98 rnbf, 0.14 mM, and 0.64 mM for magnesium, acetyl-coenzyme A, and PEP, respectively, as determined by C&novas and Kornberg (3). The Km determined for bicarbonate in these experiments is 1.75 mM. p-Mercuribenxoate (PMB) inhibition. E. co& PEP carboxylase activity is inhibited from 65 to 80 % by 10e4 M PMB. This inhibition is independent of the presence of any or all substrates (Table II), and is almost completely and immediately reversed by low3 M DTT (Fig. 1). Complete restoration of activity is achieved with a 5 minute exposure of the inhibited enzyme to DTT even after a prior 10 minute incubation with

614

SMITH

PMB (dashed line, Fig. 1). C&novas and Kornberg (3) obtained similar results for the reversal of PMB inhibition by reduced glutathione. Stability. (a) As a function of pH: The pH for optimum activity is 8.5. Figure 2 shows the stability characteristics as a function of pH. The two upper curves show that for the partially purified enzyme there is relatively little change in stability as a function of pH between 5.5 and 8.75 during a half-hour storage at 15” in the presence or absence of DTT. There is little difference in long-term stability between pH 7.6 and pH 8.0. Thus, all preparations have been stored routinely at’ pH 7.8, and generally in a frozen state. (b) As a junction of temperature: At pH 8.5, which is optimum for activity (3), the enzyme is completely inactivated within 3 minutes at 60” (Fig. 3). Some protection against heat inactivation is given the enzyme by substrates at lower temperatures. With twice the normal concentrations of acetyl-coenzyme A, PEP, and magnesium, PEP carboxylase is one-half inactivated at 0.61

0.11

I

0

I I

1

I 2 MINUTES

I

, 3

\y 4

FIG. 1. Reversal of PMB inhibition of E. coli PEP carboxylase by DTT. PMB at W4 M was added at zero time. The reaction rate for a control incubation prior to the inhibition by PMB was 0.288 absorbance unit per minute. The changes in absorbance per minute after PMB addition and DTT (10-a M) addition are shown in parentheses. Dashed line; a 10 minute incubation with PMB followed by a 5 minute incubation with lO+ M DTT.

00 4

5

7

6

8

PH

FIG. 2. Stability of E. coli PEP carboxylase as a function of pH. p1-I measurements were made after dilution of the enzyme in 0.01 M phosphate over the indicated pH range. Activity in buffers containing 10-3 M DTT (A). Activity in buffers without DTT (a). Activity of an ammonium sulfate fraction after two weeks at room temperature (approximately 21’) in phosphate buffer without DTT (m). The shadow is an idealized estimate of average activity.

approximately 55’; with PEP and magnesium, at approximately 49’; with acetylcoenzyme A alone, at approximately 47.5”. In the absence of substrate, one-half inactivation occurs at pH 7.8 at 44’ and at pH 4.9 at 33”. In the presence of the substrates or the activator, temperatures below 40” cause some increased enzymatic activity. This activation was not seen upon heating at the normal storage condition of pH 7.8 nor at pH 4.9. At pH 4.9, heat inactivation occurs rather rapidly in acetate buffer.2 PEP carboxylase, however, does maintain most of its activity at this pH when stored suspended in 40% ammonium sulfate for 3 months at 2 to 4O. Isoelectric point. The isoelectric point of the E. coli PEP carboxylase was determined by electrofocusing in a salt-free pH gradient (12). One of the electrofocusing patterns obtained is shown in Figure 4. In two experiments using a pH gradient between 3 2 As shown below, PEP carboxylase is isoelectric at pH 4.9. This, however, is for the protein in a salt-free medium. Kinetic studies in Tris-aeetat.e buffers do not differ from those in Tris-Cl, but at the same time do not rule out some ionic interactions with a concomitant change in t,he isoelectric point.

E. coli PHOSPHOENOLPYRUVATE

CARBOXYLASE

615

decreases as a first-order function of KC1 concentration for the enzyme assayed in the absence of acetyl-coenzyme A (solid squares). For the same enzyme assayed in the presence of acetyl-coenzyme A (solid circles), the same plot gives a sigmoid curve (n = 2.3 from the Hill equation). Shown on the same graph for comparative purposes (open circles) are data for a PEP carboxylase isolat’ed from potatoes (10). The potato enzyme is not an allostericall~ regulated protein and it reacts kinetically m KC1 in a manner similar to that of the E. coli PEP carboxylase when acetyl-coenzyme A is omitted. These effects of KC1 on E. coli PEP carboxylase are not specific effects of K+ on the enzyme since essentially the same results are obtained with l\‘a+ (Table III). Of particular interest is the degree of t Oc acetyl-coenzyme A stimulation as a function FIG. 3. Stability of E. coli PEP carboxylase as of NaCl concentrations. Part I of the table a function of temperature. Aliquots of enzyme shows Ohat the degree of stimulation of corresponding to 5.6 units in 0.5 ml volume were activity by acetylcoenzyme A is relatively heated at the indicated temperatures for 3 min. constant and averages 16- to 17-fold when (A) Enzyme with PEP (12 mM), Mg (10 mM), and acetyl-coenzyme A (2 mM) in 0.1 M Tris-HCI buffer the salt is diluted to a constant concentraat pH 8.5. (B) Enzyme with PEP and Mg in the tion before the assay. On the other hand, same concentrations and buffer as A. (C) Enzyme when assayed in the presence of salt, the with only acetyl-coenzyme A. (D) Enzyme alone degree of stimulation increases markedly as in 0.1 M NaKHP04 buffer, pH 7.8. (E) Enzyme the salt concentration increases. This inalone in 0.1 M sodium acetate buffer, pH 4.9, concreased acetyl-coenzyme A stimulation is taining 1c4 M EDTA and 10-a M DTT. apparent notwithstanding the overall decrease in measurable activity. and 10, the peak of enzymatic activity was Urea e$ects. Preincubation of E. coli PEP at pH 4.91 and 4.97. In two experiments carboxylase in urea at concentrations up to using a pH gradient between 3 and 6, the and including 1 M has no appreciable effect peak of enzyme activity was at pH 4.89 on the stimulation of activity by acetyland 4.92. Thus, the isoelectric point of PEP carboxylase appears to be 4.92 s a coenzyme A (part I of Table IV). As the concentration of urea was increased, however, standard deviation of 0.04. there was some inactivation of the enzyme. Similarly, the same effects were observed II. EFFECTS OF IONIC STRENGTH AND UREA if the enzyme was added directly to incubaON ENZYMATIC ACTIVITY tion mixtures containing urea, i.e., the KC1 and NaCl efects. The activity of E. degree of acetyl-coenzyme A stimulation coli PEP carboxylase decreases precipitously remained essentially unchanged (part II of when assayed in the presence of increasing Table IV). This is in contrast to the effects concentrations of KC1 (Fig. 5). Effective of increasing concentrations of NaCl or removal of the KC1 by dilution completely restores enzymatic activity. In fact, the KC1 on PEP carboxylase activity. As shown recovered activity is greater in some in- in Table IV, however, 2 M urea did appear to stances than before the KC1 treatment. A affect t’he enzymatic act’ivity differently replot of the two lower curves of Figure 5 as depending on the presence of acetyl-cothe logarithm of activity versus KC1 con- enzyme A, i.e., a 4- to &fold greater stimulacentration (Fig. 6) shows that the activity tion of activity by acetyl-coenzyme A than

616

SMITH II

I

I

I

I

I

I

I

I

I

I

I

440 I

TUBE NUMBER FIG. 4. Isoelectric point of E. coli PEP carboxylase. Electrofocusing was carried out in

a pH 3-10 gradient at 5” and 300V for 40 hr; 50~1of the hydroxylapatite fraction containing 0.8 mg protein was added to about the middle of the column; 2 ml fractions were collected. pH was measured at room temperature, 21”. expected. This, unlike the salt effect, is not real, but an artifact of the measurements and perhaps, also, of some contaminant in the urea solutions. Especially at this concentration of urea, rate measurements were not linear with time. There was a progressively greater increased inhibition of activity with time making it difficult to measure initial rate directly. It can be shown, however, that even in 2 M urea there was no interference with acetyl-coenzyme A stimulation of enzymatic activity. When, at various points of time on the nonlinear rate curve, changes in rate were calculated and the logarithm of these rates plotted as shown in Figure 7, straight lines were obtained indicative of a first-order (perhaps pseudo first-order) decrease in reaction rate. From these curves approximate first-order rate constants, Ic’, for this apparent inactivation were found to be 0.147 min-1 and 0.625 min-l in the presence and absence of acetyl-coenzyme A,

respectively. The respective tl/z values for this reaction are 4.72 minutes in the presence of acetyl-coenzyme A and 1.11 minutes in the absence of acetyl-coenzyme A. When the inactivation half-times were considered and reaction rates calculated at times when comparable amounts of enzyme were present, the ratio of activity in the presence of acetyl-coenzyme A to activity in its absence was 81, consistent with the other data of Table IV. Thus, when substrates are present, there is no preferential inactivation of regulatory versus catalytic sites of E. coli PEP carboxylase by urea up to and including 2 M. The above k’ data are not absolute values but are empirical values for this set of data only. When, for example, Mann “ultra pure” grade of urea was used instead of its regular grade, no change in rate was observed with time except at 2 M urea-and then only in the absence of acetyl-coenzyme A. The apparent inactivation h+ for that

E. coli PHOSPHOENOLPYRUVATE

single experiment was 0.104 min-l. The fact that this inactivation decreases in severity as the purity of the urea increases indicates that some impurity in the urea progressively inhibits enzymatic activity (more in the absence of acetyl-coenzyme A t’han in its presence) and that the apparent increased stimulation (last line of Table IV) is not directly related to urea effects per se on PEP carboxylase. The absence of differential effects of urea on the catalytic and regulatory functions of the enzyme is not limited to acute experiments such as t’hose just described. Prolonged exposure to 1 M urea gave essentially the same results. PEP carboxylase incubated in 1 M urea/l0 % sucrose solutions at pH 7.8

Y %

CARBOXYLASE

617

I.0 0.8

\

l

::

-I

0.6

\

[K Cl] (MOLES/LITER)

FIG. 6. Logarithmic replot of two lower curves of Figure 5. Activity in absence of acetyl-coenzyme A, squares. Activity in presence of acetylcoenzyme A, closed circles. Activity of potato PEP carboxylase, opened circles.

[K Cl]

(MOLES/LITER)

FIG. 5. Effect of ionic strength on E. coli PEP carboxylase activity. Aliquots (15 ~1 containing 75 munits of activity) were diluted with 15 pl of KC1 at twice the indicated concentrations of KCI. After 30 min at 20”, 10 ~1 of each tube was assayed in the presence (upper solid line) and absence (upper dashed line) of acetyl-coenzyme A. The final KC1 concentration was adjusted to 0.01 M. The two lower curves demonstrate the relative activity of 5 ~1 of the original enzyme assayed directly in the presence of the indicated concentrations of KC1 in the presence (solid line) and absence (dashed line) of acetyl-coenzyme A.

for 18 or 19 hours still retained activity (Table V) that was stimulated 20-fold or better by acetyl-coenzyme A when assayed either in the presence or the absence of 1 M urea. The relative activity in urea, however, was only about two-thirds that observed when urea was omitted from the incubation mixture. Substrate saturation curves indicated that about twice as much Mg2+ and acetyl-coenzyme A are needed to saturate the urea pretreated enzyme when it is assayed in the presence of 1 M urea. Double reciprocal plots of rate versus PEP concentration were made after measuring initial rates in the presence of 2 mM acetyl-coenzyme A and 10 mM Mg”+. K, values for PEP in 1 M urea were found to be 2.65 mu and 1.78 mM in two different experiments. The corresponding K, values for t,he same enzyme preparation assayed in the absence of urea were 0.94 mu and 0.85 mu, respectively. The ratios of V,,, (mclmoles/min) for the analyses in the presence of urea to that in the absence of urea were 73.4/98.1 for one experiment and 67.6/97.8 for the other, indicating that the enzyme species present during assay in 1 1\1urea catalyzes the reaction at a somewhat slower rate.

618 III.

SMITH

SEDIMENTATION COEFFICIENT OF PEP CARBOXYLASE IN THE PRESENCE OF AN ALLOSTERIC INHIBITOR, SUBSTRATES, SALT, AND UREA

Sedimentation analyses. The data of Tables III-V show that KCl, NaCI, and urea affected either the catalytic or the regulatory and catalytic properties of the E. coli PEP carboxylase. The question that this section will attempt to investigate is whether these effects could be explained on the basis of association-dissociation reactions of the protein. Representative data for this series of experiments are shown in Table VI and Figure 8. In all cases, 8~0,~ values were calculated from the tables published by McEwen (9). Similar calculations were made for the reference protein, diaphorase, used in all of these experiments. It may be observed that the S20,Wfor this protein did TABLE

III

EFFECT OF NaCl CONCENTRATIONS ON ACETYLCOENZYME A STIMULATION OF E. coli PEP CARBOXYLASE Activity

Prph$XI NaCl

(M)

-AcCoA

(munits) SAcCoA

Ratio of activity t$gy

I 0 0.10 0.25 0.50 0.75 1.00

11.43 16.40 12.41 12.43 10.29 9.26 Activity

Assay c~ncn of N&l (M)

-AcCoA

146.86 243.25 209 .49 192.93 182.72 200.16 (munits) +AcCoA

12.8 14.8 16.9 15.5 17.8 21.6 Ratio of activit +-“A”c”c”

II 0 0.05

46.62 28.22

0.10

14.86

0.25 0.50

4.28 0.06

757.65 770.50 740.35 369.75 64.30

16.3 27.3 49.8 86.4 1071.7

Part I. Approximately 0.8 unit of enzyme was incubated for 30 min at 20” in the NaCl concentrations indicated. Aliquots (10 ~1) were removed to an incubation mixture for assay. The final NaCl concentration in each assay mixture was 0.01 M. Part II. Aliquots of enzyme were added to incubation mixtures containing the NaCl concentrations indicated.

TABLE EFFECT

OF

COENZYME

UREA

A

IV

CONCENTRATIONS STIMULATION

ON OF

ACETYL-

E. coli PEP

CARBOXYLASE Preincubation concn of urea CM)

Activity -AC&A

(munits) +AcCoA

Ratio of activity f_“A”,“c”,“A/

I 0 0.05 0.10 0.25 0.50 1.00

19.90 11.41 14.41 13.41 12.59 1.38 Activity

Assay c~ncn of urea (M)

-AC&A

297.43 244.05 261.25 244.05 182.96 20.98 (munits) +AcCoA

14.9 21.4 18.1 18.2 14.5 15.2 Ratio of activity +-“A”,“c”,“A

II 0 0.1 0.2 0.5 1.0 2.0

39.39 33.28 33.12 27.97 23.47 2.32

724.90 686.50 631.05 601.30 485.55 216.25

18.4 20.6 19.1 21.5 20.7 93.2

These experiments were done in the same manner as those of Table III. No attempt was made to adjust the final urea concentration in the assay mixture of Part I since concentrations of urea up to the 0.01 M maximum (1OJ to 1 ml) had no effect on activity.

not vary systematically as the various constituents were added to the centrifugation medium. For this particular series of experiments a total of 66 gradients were analyzed in such a manner as to allow S20,W estimations for the diaphorase. The average X20,W, standard deviation, standard error, and coefficient of variance for this protein were 5.7, 0.45, 0.06, and 0.08, respectively. Reported S20,Wvalues for diaphorase (or lipoyl dehydrogenase) determined by other analytical methods range from 5.3 to 5.7 (14), all with an assumed partial specific volume of 0.73. In these experiments, molecular weight was estimated from the ratio of the calculated 820.~ using the procedure of Martin and Ames (1). The S20,Wused for the diaphorase was either the average given above or the one determined in the partic. ular experiment in question. To simplify

E. coli PHOSPHOENOLPYRUVATE

calculations with the McEwen (9) tables an assumed protein density of 1.4 was used. The data presented in Table VI show that E. coli PEP carboxylase can exist in approximately three different forms having L%,~ values of about 5.8, S.4, and 12.2. A composite diagram of some of the activity patterns observed for these three entities is shown in Fig. S. Peak B of Fig. S is typical of the sedimentation pat’tern observed with PEP carboxylase in the absence of substrates, divalent, metal ion activators, and allosteric regulators. Numerical dat,a for these experiments are present’ed in line 1 of Table VI. The addition of Mg2+ to t’he centrifugation medium causes PEP carboxylase to sediment with an X20,n,of 12.2 (line 4, Table VI). This value is not changed by the inclusion of PEP alone nor PEP and acetyl-coenzyme A to t,he centrifugation medium (lines 3 and 2, Table VI). Peak C of Figure 8 is typical of the sedimentation pattern observed under these conditions. In the presence of 1 JI urea alone or together with PEP and iUg2+, PEP carboxylase sediments in the sucrose gradients with an Szo,n.of 5.8 (lines 7 and 8 of Table VI and peak A of Fig. S). However, if acetyl-coenzyme A is included in the centrifugation MINUTES l.B-

,

16-

-.-. .

0.2 ,

,

.

04 , . . . .

06

/ .

l

0.8 ,

I .

.-.

.

.

.

! .

.

1.0 , I

1.2 , I

.::.-

.

1.4 -

14-

'...

O-

-. -.

------+-CCttH

-0.2I 0

I I

I

I 2

I

I 3

I 4

I

I 5

I

I 6

I 7

MINUTES

FIG.

7. Logarithmic

plots

of reaction

rates

in

presence of 2 ?IIurea as a function of time. These data were calculated of Table IV. Upper figure: reaction rate coenzyme A. Lower the figure: reaction coenzyme A.

from the original rate curves curve and scale at top of the in presence of 1 mM acetylcurve and scale at bottom of rate in absence of acetyl-

619

CARBOXYLASE TABLE

RELATIVE

ACTIVITY

Lctivity

T

Enzyme

V

OF E. coli PEP CARBOXVLASE IN UREA

Additions to mixture

4 6

pretreatment amay

I 1 M urea 1 y urea Rat,io of activity + /urea

(munits) Ratio of activity fAcCoA/ -AcCoA

+AcCoA

None 1 M urea

II 1 M urea 1 M urea Ratio of activity +/urea

None 1 M urea

-

8 ) 358 1 44.8 0.62 0.62

-I

I

I

Aliquots of PEP carboxylase were incubated for 19 hr at 5” in 1 M urea-lO%i, sucrose (w/w) dissolved in 0.01 M Tris-HCI pH 7.8 containing 10-4 M EDTA. Fractions of the enzyme were assayed as described above in the presence and absence of 1 M urea.

medium containing the urea along with PEP and Mg”f, the sedimentation rate for the enzyme is more characteristic of the controls (compare line 9 with line 1) than of those in urea alone (line 7) or urea with PEP and Mg2+ (line S), indicating that acetyl-coenzyme A can prevent the urea induced changes in the sedimentation pattern. Acetyl-coenzyme A alone, however, appears to reduce the rate of sedimentation slightly (line 5, Table VI), but the significance of the difference between that value and the controls is questionable. Aspartate, similar to Mg2+, causes PEP carboxylase to sediment with an Szo,W of 13.2 (line 10, Table VI). The activity profile of these gradients were more like peak C of Figure S, but the peak was more symmetrical, indicative of less heterogeneity than in the case with Mg2+. As with acetyl-coenzyme A, PEP, and Mg2+ (line 9), aspartate also prevents t’he shift in the sedimentation pattern to 5.8s (line 11) as is observed in urea alone.

620

SMITH TABLE

SEDIMENTATION

VI

COEFFICIENTS CARBOXYLASE

Additions to centrifugation mediums

I

FOR

Estimated Szo,w Diaphoraseb

1. None (16) 2. AcCoA, PEP, Mg2+ (4) 3. PEP, Mg2+ (3) 4. Mg2+ (2) 5. AcCoA (2) 6. KC1 (8) 7. Urea (6) 8. Urea, PEP, Mg2+ (6) 9. Urea, PEP, Mg2+, AcCoA (4) 10. Aspartate (2) 11. Aspartate, urea (3)

E. coli PEP

6.0 f 5.3 f

0.43~ 8.4 f 0.75 0.25 12.1 f 0.60

5.9 f 0.13 5.4d 5.9 5.7 f 0.18 5.8 f 0.36 5.2 f 0.61 5.6 f

PEP csrboxylase

0.09

5.9 5.5 f 0.03

12.2 f 0.17 12.2 7.4 8.6 f 0.70 5.8 f 0.38 5.8 f 0.70 8.1 f

0.54

13.2 7.3 f 0.09

Sedimentation experiments were performed as described in “Methods.” The sedimentation rates were calculated using the tables of McEwen (9). a Numbers in parentheses represent the number of gradients an xlyzed. 6 Diaphorase *efers to lipoyl dehydrogenase. c All calculatkons are given f the standard deviations except (d) those in which less than three gradients were analyzed; the average is reported. d The concentrations of the additions were: 1 mM AcCoA, 6 mM PEP, 5 mM MgSOa, 0.5 M KCl, 1 M urea, and 15 mM aspartate. DISCUSSION

Estimation of molecular parameters by the procedure of Martin and Ames (1) has the obvious advantage of not requiring absolutely pure materials. What is needed, however, is a specific assay for the unknown material and another well-characterized protein whose molecular weight, sedimentation coefficient, and partial specific volume are known. If it can be assumed that the partial specific volume and shape of the unknown are equivalent to the reference, then meaningful estimates of the molecular weight and sedimentation properties of the unknown can be obtained by comparing the relative movements of the two materials through the gradients.

011 I Ill III III1 I I 0.8 1.0 1.2 14 16 1.8 20 2.2 2.4 2.6 2:s 30 32 34 3.6 38 CENTIMETERS

FROM MENISCUS

FIG. 8. Composite diagram of sucrose density gradients of E. coli PEP carboxylase. Enzymatic activity is expressed as a change in absorbance at 340 mw/min. (A) Activity pattern with 50 ~1 aliquots after centrifugation in 5-2Ooj, (w/w) sucrose gradients containing 1 M urea. (B) Activity diagram with 5 ~1 aliquots and no additions to the centrifugation medium. (C) Activity diagram with 5 rl aliquots after centrifugation in 5-20y0 (w/w) sucrose gradient,s containing 5 mM MgSOd.

More recently, McEwen (9) calculated and compiled tables that will allow more easily an estimation of the corrected S20,w for proteins as determined by the gradient centrifugation technique. These tables take into account the changing viscosity and density of the sucrose gradients, but require some independent knowledge of the density (or partial specific volume) of t’he unknown protein. In the studies reported here, the tables of McEwen (9) have been used to estimate sedimentation coefficients for PEP carboxylase and lipoyl dehydrogenase after sedimentation in sucrose gradients. Assumptions consistent with the techniques were taken into account in all calculations. It was observed that PEP carboxylase can exist in essentially three different forms having S20,w values of 5.8, 8.4, and 12.2-13.2, depending on the additional constituents in the sucrose gradients. In all of the experiments performed, the reference protein, lipoyl dehydrogenase, had an average S20.wof 5.7 f 0.45. The ratio of the sedimentation coeficients (S) of a standard and unknown may be related to the ratio of their respective molecular weights (mw) by the equation (1) : x1/s* = (mwl/rTawp

E. eoli PHOSPNOENOLPYRUVATE Thus, for a twofold change in molecular weight of a protein in which the density and shape (spherical) remain the same, SJS, = 1.588. Therefore, if PEP carboxylase associates or dissociates under t,he various treatments, and if the controls (S20,, = 8.4) are taken as the standard case, its sedimentation coefficient lvould change t,o 5.3 upon dissociation and to 13.3 upon association. Values for the sedimentation coefficient of PEP carboxylasevery close to these are obtained when the enzyme is centrifuged in gradients containing 1 M urea (S?O,, = 5.8) and 15 mM aspartate (SgO,, = 13.2). Therefore, consistent with these data, is the assumption t,hat S.4S PEP carboxylase dissociates into two smaller entities in the presence of urea and that t,wo of the S.4S entities associate to a larger one in the presence of aspartate and 1\Ig2+. Since there is no evidence that the 5.8s material dissociates further, it will be referred to as monomer, the 8.4s as dimer, a,nd the 12.2-13.2s as tetramer. Consistent with the same argument and also with the same limitations, the e&mated molecular weights for these three entities calculated by the procedure of Martin and Ames (1) from the sedimentation coefficients determined in these experiments for lipoyl dehydrogenase (5.7) and the PEP carboxylase controls (8.4) are: 94,000 for the 5.3S, 188,000 for the 5.45, and 376,000 for the 13.3s species. The molecular weight of lipoyl dehvdrogenase (105,000) used in these calculations is an average of t,he data reviewed by Massey (14). In the absence of substrates, activators, metal ions, or urea, t,he predominant enzymatic species is the dimer. In t.he presence of Mg2+, it associates to the bet.ra,mer. Neit,her PEP nor aeetyl-coenzyme A alone nor the two together can promote this association in the absence of Mg2+. The 12.2 observed as the sedimentation coefficient for t,he Mg2+-PEP carboxylase complex corresponds to a weight average moIecular weight of 329,000. If predominantly an association-dissociation equilibrium exists between onty dimers and tetramers, about 75 % would be present as the t,etramer. Considering the high probability t,hat Mg2+ or other divalent met.al ions that may activate

CARBOXYLASE

621

the enzyme are never depleted in the cell, it may be suggested that the enzyme exists in the cell as the tetramer and that association-dissociation normally is not involved in regulation of activity. In addition, aspartate, which has effects on enzymatic aetivit.y in the opposite direction t,o those of Mg*+, shifts the association-dissociation equilibrium toward the tetramer, and it does so more completely. Although these data will not allow any definitive st.atement concerning conformation changes, it is highly probabIe that aspartate changes the conformation in the tetramer to one that prevents facile molecular dissociation. Moreover, it is apparent that, in this configuration, the enzyme is more stable to heat. Izui et al. (4) reported that essentialIy compIete protection of enzymatic activity was observed after heating PEP carboxylase for 5 minutes at 55” in the presence of 15 rnht aspartate. This same concentration of aspartate produces tetramers. Under other conditions in which tetramers are expected, i.e., combinations of substrates containing Mg2+, greater heat stability is seen also, but not as much as with aspartate (4). Sodium and potassium chloride probably affect the dimer in a manner similar to t.he aspartat.e effect on the tetramer. Even though the tetramer is the predominant enzyme species in t.he presence of substrates containing Mg2f or aspartate, and the monomer is the predominant species in the presence of usea, the combination of these substrates with urea or aspartate Kith urea results in a sedimentation coefficient more characteristic of the dimer than of the other two molecular ent.ities. In t.he absence of urea, Mg2+ is the most important constituent for producing tetramers; in t.he presence of urea, Mg*+ and PEP have no effect, on the sedimentation of PEP carboxylase unless acetyl-coenzyme A is added. Then the enzyme sediment predominantly as the dimer. It may be recalled (Table V and concomitant discussion in the text) that PEP carboxylase has activit,y in 1 M urea both with and without, acetyl-coenzyme A. Since in the absence of acetyl-coenzyme A the enzyme sediments as a monomer, it

622

SMITH

seems appropriate to assume that the monomers have activity. But an average molecular weight calculated from the observed sedimentation coefficient indicates a size of 108,000. And, if the real molecular size of the monomer should be 94,000, about 15 % of the material present could be dimers and they could be responsible for the activity. The standard deviations in the S20,w calculations, however, are too large to allow an estimation of t’he molecular weights with the precision necessary to unequivocally distinguish between 94,000 and 108,000. Both the data of Table V and the kinetics presented in that section of the text show that PEP carboxylase has enzymatic activity after exposure to 1 IN urea under conditions, both of sucrose concentration and time, similar to those used for sedimentation studies. In urea the activity is 62 to 75% of that in the absence of urea. If the predominant effect of acetyl-coenzyme A is to shift the urea-induced dissociation-association between dimers and monomers to favor dimers, then about 89% of the enzyme present in urea, PEP, Mg2+, and acetylcoenzyme A is the dimer, which could be solely responsible for the activity. If this shift is directly to a mixture composed of predominantly monomers and tetramers, only about 30% tetramer would be needed to give an apparent S20,w of 8.1 (average molecular weight of 178,000), as observed in Table VI. Since this would not be enough tetramer to account for the observed activity, it is more reasonable to assume that the dimers are active, and, as indicated in

Table V, respond to control by acetyl-coenzyme A. ACKNOWLEDGMENTS The author wishes to thank Manuel Perry for expert technical assistance in the performance of this research. This work was performed under the auspices of the United States Atomic Energy Commission. REFERENCES R. G., AND AMES, B. N., J. Biol. Chem. 236, 1372 (1961). KORNBERG, H. L., Angew. Chem , Intern. Ed. 4, 558 (1965). CBNOVAS, J. L., AND KORNBERG, H. L., Proc. Roy. Sot. 166, 189 (1966). IZUI, K., I~ATANI, A., NISHIKIDO, T., KATSUKI, H., AND TANAKA, S., Biochim. Biophys. Acta 139, 188 (1967). 5. MONOD, J., WYMAN, J., AND CHANGEUX, J. P., J. Mol. Biol. 12, 88 (1965). 6. RICHARDSON, C. C., SCHILDKRAUT, C. L., APOSHIAN, H. V., AND KORNBERG, A., J. MARTIN,

Biol.

Chem. 239, 22 (1964).

7. LAYNE, E., Methods Enzymol. 3, 447 (1957). 8. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 9. MCEWEN, C. R., Anal. Biochem. 20,114 (1967). 10. SMITH, T. E., Arch. Biochem. Biophys. 126, 178 (1968). 11. MASSEY, V., Methods Enzymol. 9, 272 (1966). 12. VESTERRERG, O., AND SVENSSON, H., Acta Chem. Stand.

20, 820 (1966).

H., AND BURR, D., J. Am. Chem. Sot. 66, 658 (1934). 14. MASSEY, V., in “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrhack, eds.), Vol. 7, p. 275. Academic Press, New York (1963). 13. LINEWEAVER,