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THE JOURNAL OF Bm~oorcar. CHEMISTRY Vol. 242, No. 6, Issue of March 25, PP. 1146-1154, 1967

Printed in U.S. A.

On the Mechanism of Activation of L-Threonine Deaminase from Clostridium tetanomorphum by Adenosine Diphosphate”

(Received for publication, July 20, 1966)

ATSUSHI NAKAZAWA$ AND OSAMU HAYAISHI

From the Department of Medical Chemistry, Kyoto University Faculty of Medicine, Kyoto, Japan

SUMMARY

In order to clarify the mechanism of activation of L-threonine deaminase by adenosine diphosphate, the enzyme was purified about 700.fold from sonic extracts of Closlridium tetanomorphum, and kinetic and ADP-binding studies were carried out. Plots of reaction rates against L-threonine concentrations gave a sigmoid curve in the absence of ADP, whereas a hyperbolic curve was obtained in the presence of low3 M ADP. Double reciprocal plots were parabolic in the former and linear in the latter case.

Different pH profiles of the apparent k=, and maximal velocity were observed with and without ADP. A linear Arrhenius plot was obtained in the absence of ADP, whereas a discontinuity in the slope was found in the presence of ADP. Values of the activation energy were calculated to be 13.9 and 11.4 kcal per mole over the temperature range of 4-37’ in the absence and presence of ADP, respectively.

The Michaelis constant for ADP as an activator was 2.3 X 10-K M at a threonine concentration of 10B3 M. No mutual effect of ADP and threonine on K,,, and K, was observed. Adenosine triphosphate offset activation of the enzyme by ADP, and increased the sigmoid nature of the rate-substrate concentration curve.

D-Threonine and semicarbazine inhibited the deaminase activity competitively toward L-threonine both in the presence and in the absence of ADP. p-Chloromercuribenzoate inhibited the enzyme competitively toward L-threonine in the absence of ADP, but noncompetitively in the presence of ADP.

The dissociation constant for ADP, estimated by equilibrium dialysis, was 3.0 x 10v6 M, which agreed reasonably well with the value obtained from reaction kinetics. D- Threonine did not interfere with the binding of ADP.

On the basis of kinetic analysis, two substrate sites are postulated, a catalytic site with a dissociation constant for substrate (KS) of 3.5 x low3 M, and an activating site with a K, of 3.3 x 1O-2 M.

* This investigation has been supported in part by Research Grants CA-04222 and AM-10333 from the National Institutes of Health, United States Public Health Service, and by grants from the Jane Coffin Childs Memorial Fund for Medical Research, the Squibb Institute for Medical Research, and the Scientific Research Fund of Ministry of Education of Japan. The data were taken from a dissertation submitted by Atsushi Nakazawa in April 1966 to the Graduate School of Kyoto University in partial fulfillment of the requirements for the degree of Doctor of Medical Science.

1 Recipient of the Sigma Chemical Company Postgraduate Fellowship.

During the course of a study on the metabolism of n-threonine by cell-free extracts of Clostridium btanomorphum, evidence was presented that adenosine diphosphate specifically stimulated the deamination of n-threonine to a-ketobutyrate by threonine deaminase (1). The effect of ADP, which was most marked at low concentrations of the substrate, was ascribed to a decrease in the apparent K,,, of the enzyme for the substrate rather than to direct participation of ADP in the reaction. Subsequent studies revealed that ADP was bound by the enzyme protein and protected it against inactivation by dilution or heat (2). In a teleological sense, ADP appeared to be a metabolic regulator of the anaerobic energy production in this microorganism during the catabolism of L-threonine (3).

Recently, similar phenomena have been reported from a number of laboratories and are usually referred to as “allosteric effect” (4), in which the enzyme activity in a key position of cellular metabolism is controlled by specific metabolites or nucleotides. Two kinetic features have generally been notice- able with the enzymes which are activated by allosteric effecters: first, a sigmoid curve of rate of reaction as a function of substrate concentration; second a change in the apparent K, for the substrate in the presence of the effector. The threonine deaminase also showed these two features, as will be described below. Some interpretations which have been reported to ex- plain these features were based on the marked difference between the oxygen saturation curve of hemoglobin and that of myo- globin. In all these interpretations, the decrease in K, values was considered as the increase in affinity of the enzyme for the substrate in a direct sense. A possibility arises, however, that the decrease in K,,, can also be achieved without changing the actual affiity for the substrate, especially for the enzymes that have kinetics of an unusual type.

In this paper, we describe the results of kinetic studies and binding experiments with ADP-14C in order to clarify the mechanism of stimulation of the threonine deaminase by ADP. The experimental evidence is consistent with the following interpretations: (a) threonine is bound to the enzyme protein at two sites, one “catalytic” and the other “activating”; (b) ADP may be bound at a site which is distinct from the two sites for threonine (“ADP site”); and (c) ADP functions in a manner similar to threonine at the activating site in activating the deaminase. Indeed, ADP may stimulate the enzyme reaction without increasing the affiity of the enzyme for the substrate in a direct sense.

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Issue of March 25, 1967 A. Nakazawa and 0. Hayaishi 1147

EXPERIMENTAL PROCEDURE

Materials-L- and n-Threonine were obtained from Tanabe Amino Acid Research Foundation. ADP was a gift of Takeda Chemical Industries, and ATP was a product of Sigma. Sodium cY-ketobutyrate was purchased from Mann; pyridoxal-P, from Calbiochem; and Sephadex G-25 and DEAE-Sephadex A-50, from Pharmacia. ADP-8-W (25.8 mC per mmole) was obtained from Schwarz and was purified by column chromatography on Dowex 1 (Cl-). All other chemicals were of analytical grade.

Growth of Organism-C. tetanomorphum (ATCC 3606) was grown for 15 hours at 37” in 5-liter Erlenmeyer flasks in 4 liters of glutamate medium (5) containing 0.5% yeast extract, 1.5% polypeptone, 0.5 $& sodium glutamate, 0.005 y. thioglycolic acid, and 0.25% K&CPOd. Cells were harvested with a Sharples centrifuge at room temperature and stored at -15”.

Assays-The activity of threonine deaminase was determined spectrophotometrically by measuring the formation of ol-keto butyrate as its 2,4-dinitrophenylhydrazone. The standard assay system contained 100 pmoles of n-threonine, 200 pmoles of Tris-HCl (pH 8.4), 2 c(g of pyridoxal-P, and the enzyme in a total volume of 1.0 ml. The reaction mixture was incubated at 37” for 10 min, and the reaction was terminated by the addition of 1.0 ml of 1 N HCl. The keto acid formed was determined as the 2,4-dinitrophenylhydrazone at 415 rnp by a modification of the method of Katsuki et al. (6) with a Shimadzu Bausch and Lomb Spectronic 20 spectrophotometer. One unit of enzyme was defined as that amount producing 1 pmole of a-ketobutyrate per min under the standard conditions. Specific activity was expressed as units per mg of protein. Protein was determined from the absorption at 280 and 260 rnp (7) with a Shimadzu DU spectrophotometer. Radioactivity was measured with a Pack- ard Tri-Carb liquid scintillation spectrometer, with the naphth- alene-dioxane system as a scintillator (8).

Binding Studies-For the determination of an average dissociation constant of ADP, equilibrium dialysis was carried out as follows. Visking dialysis tubing was soaked for several days in distilled water, which was changed periodically. Dialysis cells were made by milling rectangular grooves (1 X 4 X 0.5 cm) in polyvinyl chloride plates (5 x 6 x 1 cm) and pressing cellophane membrane between pairs of such plates. A thin film of silicone grease around the edge of the cellophane prevented capillary loss of solvent. Each pair of cells was fastened with bolts and nuts to hold the two halves of the container tightly together. To one compartment of each cell was added 0.30 ml of the enzyme solution (79 units, 2.8 mg of protein) in 0.03 M

potassium phosphate, pH 7.0, and, to the other, varying amounts of ADP-14C in 0.6 M Tris-HCl and 0.6 M NaCl, pH 8.4. The container was closed with adhesive vinyl tape to prevent evap- oration and was shaken in a shaking incubator at 25”. A glass bead (0.4 cm in diameter) was placed in each compartment to attain sufficient mixing. Practically complete equilibration usually took place in 4 hours. After 7 hours of dialysis, O.lO-ml aliquots were taken from each compartment to determine their radioactivity. The presence of protein and salt in samples did not interfere with the counting efficiency. During the 7-hour dialysis, the enzyme activity decreased by 12%, while 97% of the added ADP was recovered after the’ dialysis. The concentration of bound ADP (EADP) was obtained by subtracting the concentration of unbound ADP (A) from the concentration of ADP on the enzyme side of the membrane.

An average dissociation constant (K) of ADP can be derived graphically from the following equation.

(Et) K 1 (EADP)

= ;.--+l 0) n

where n represents the number of binding sites per molecule of enzyme protein, and (Et) is the total concentration of the enzyme (9). When the reciprocal of the concentration of bound ADP is plotted against the reciprocal of the concentration of unbound ADP at a constant enzyme concentration, a straight line is obtained, which intersects the abscissa at a point giving -l/K, and the ordinate at a point which gives l/n.(EJ.

To study the effect of various compounds on the binding of ADP, a technique similar to that described previously (2) was used. An incubation mixture, which consisted of 0.20 ml of enzyme solution (32 to 65 units, 1.9 to 5.0 mg of protein), 0.10 ml of ADPJ4C solution (5.6 mpmoles, 82,100 cpm), and 0.03 ml of 1 M Tris-HCl, pH 8.4, was incubated for 5 min at 37”. Then an aliquot of 0.20 ml was placed on a column of Sephadex G-25 (0.5 cm2 x 8 cm) equilibrated with 0.1 M potassium phosphate, pH 7.0, and the protein was eluted with the above buffer solution at room temperature. After the first 1.4 ml, 4drop fractions (about 0.16 ml) were collected into counting vials. Under these conditions, all the enzyme activity appeared between the 2nd and 8th fractions (large molecular fraction), and ADP was eluted between the 9th and 35th fractions (small molecular fraction). The separation of a large molecular fraction from a small molecular fraction was achieved in times as short as a few minutes. The radioactivity in a large molecular fraction, which corresponds to the amount of ADP bound by the enzyme, was determined by the method described above.

RESULTS AND DISCUSSION

Enzyme Purification

All procedures were carried out at O-4”. Crude E&acts-Frozen cells (127 g) were suspended in 250

ml of 0.05 M potassium phosphate, pH 7.0, and were disrupted by sonic oscillation at 10 kc for 15 min. In all subsequent steps, potassium phosphate, pH 7.0, was’ used, which henceforth will be referred to simply as “buffer.”

Protamine Treatment-To 400 ml of crude extract were added 12 g of protamine sulfate, previously suspended in 40 ml of 0.5 M buffer, adjusted to pH 7 with KOH. After the suspension was kept overnight, the precipitate was removed by centrifugation at 8000 X g for 20 min.

Acetone Treatment-Two volumes of acetone, which had been kept at -2O”, were added to the above supernatant solution with stirring over a 30-min period at -15”, and the resulting precipitate was collected by centrifugation at 10,000 x g for 10 min and suspended in 300 ml of 0.05 M buffer. To this suspension were added 210 ml of a saturated solution of ammonium sulfate at pH 7.0. After stirring for 20 min, the precipitate was discarded, and 390 ml of the saturated solution of ammonium sulfate were added to the resultant supernatant solution. After 30 min, the precipitate obtained was dissolved in 50 ml of 0.05 M buffer. The solution was dialyzed overnight against a large volume of 0.05 M buffer.

Ammonium Sulfate Treatment-To 74 ml of the above dialyzed solution were added 60 ml of a saturated solution of ammonium sulfate at pH 7.0 with stirring. After 20 min, the precipitate


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1148 ADP Activation of Clostridial Threonine Deaminase Vol. 242, No. 6

was removed by centrifugation. To the supernatant solution were added 30 ml of the saturated solution of ammonium sulfate. After 20 min, the precipitate was collected by centrifugation at 10,000 X g for 15 min and dissolved in 15 ml of 0.05 M buffer.

First Chromatography on DEAE-Seph&x Column-A column of DEAE-Sephadex A-50 (5.7 cm2 X 17.5 cm) was prepared and equilibrated with 0.05 M buffer. Through the column were passed 100 ml of the ammonium sulfate fraction, which had been dialyzed overnight against 0.05 M buffer. The column was washed with 200 ml of 0.1 M and then with 200 ml of 0.15 M buffer. Protein was eluted with a linear concentration gradient of buffer, from 0.15 M to 0.50 M. The mixing chamber contained 500 ml of 0.15 M buffer, and the reservoir had 500 ml of 0.50 M

buffer. The flow rate was 50 ml per hour, and 15-ml fractions were collected. The enzyme, which was found in the 315- to 435-ml effluent volume, was collected and dialyzed overnight against 1 liter of 0.03 M buffer.

Second Chromatography on DEAE-Sephadex Column-The dialyzed enzyme solution was applied to a column of DEAE- Sephadex A-50 (2.6 cm2 x 11.6 cm) which had been equilibrated with 0.05 M buffer. Elution was carried out with a linear concentration gradient of NaCl solution in 0.05 M buffer, from 0.1 to 0.4 M (mixing chamber, 250 ml of 0.1 M NaCl in 0.05 M buffer; reservoir, 250 ml of 0.4 M NaCl in 0.05 M buffer). The flow rate was 80 ml per hour, and 12-ml fractions were collected. The enzyme in the 124- to 192~ml effluent volume wa+~ collected and dialyzed against 1 liter of 0.05 M buffer overnight.

Hydroxylapatite Column-The dialyzed enzyme solution of the second DEAE-Sephadex fraction was applied again to a small column of DEAE-Sephadex A-50 (bed volume of 1.2 ml) and was eluted with 0.5 M buffer. After dialysis against 500 ml of 0.05 M buffer for 4 hours, 4.5 ml of the concentrated enzyme solution were passed through a column of hydroxylapatite (2.8 cm2 X 1.8 cm) which had been equilibrated with 0.05 M

buffer. Protein was eluted stepwise with 9 ml each of 0.05 M, 0.075 M, 0.10 M, and 0.125 M buffer. The two tubes of maximal specific activity were pooled. The combined fraction had a specific activity of 124 units per mg of protein, which was 730 times that of crude extracts. This preparation showed a pale yellow color and had an absorption maximum at 415 rnp, which might have been due to the bound pyridoxal-P, in addition to the usual absorption at 278 rnp at pH 7. The ratio of the absorption at 415 rnp to that at 278 rnp was 0.05.

Typical results of enzyme purification are shown in Table I. Because the most purified enzyme preparation could not be obtained in appreciable amounts, most of the kinetic experiments and all binding studies described in this paper were per-

TABLE I Purification of L-threonine deaminase

Fraction

mc wails Crude extracts.. . . . . 26,720 4,440 Protamine.............. 11,750 4,060 Acetone................ 3,220 3,030 Ammonium sulfate.. . . 1,230 2,110 First DEAE-Sephadex . 68 1,700 SecondDEAE-Sephadex. 12.3 1,030 Hydroxylapatite. . . 0.97 120

Specific activity Yield

mils/mg prolcin Y.

0.17 loo 0.35 92 0.94 68 1.7 48

25.0 38 83.8 23

124.0 2.7

L-THREONINE

FIG. 1. The rate of threonine deaminase reaction M a function of L-threonine concentration in the absence (O-O) and presence (O-0) of 10-S M ADP. The enzyme activity w&s determined with the standard assay system except that varying concentrations of L-threonine were used. The rate is expressed in micromoles of a-ketobutyrate formed per min per ml of reaction mixture with 10.7 pg of protein (specific activity, 13).

formed with the partially purified preparations, which had a specific activity of 12 to 27 units per mg of protein. With these preparations, no evidence for degradation of cr-ketobutyrate, ADP, or ATP was found under our assay conditions. A few kinetic experiments with the most purified preparation gave results identical with those obtained with the preparations of lower specific activities.

Kinetic Properties

E$ect of Threonine-When the rate of a-ketobutyrate formation was determined as a function of L-threonine concentration, a sigmoid curve was obtained instead of the usual hyperbolic one (Fig. 1). The rate of product formation was essentially zero until L-threonine concentration was raised to 10m3 M.

Thereafter the rate increased slowly, with a half-maximal velocity at 3.7 X lo* M L-threonine and a maximal velocity of 18.2 pmoles per min per mg of protein. When 10-s M ADP was present in the reaction mixture, however, a hyperbolic curve was obtained, with a K, of 3.5 x lo+ M and a maximal velocity of 21.6 pmoles per min per mg of protein.

In the absence of ADP, a sigmoid curve was also observed at pH 7.1, 8.1, and 9.1, whereas a hyperbolic one was obtained at pH 6.3 and 9.8. When L-serine, which could be utilized slightly by this enzyme, was used as the substrate instead of L-threonine, a sigmoid curve was also obtained, with an apparent K, for L-serine of 3 X 10-l M. Again, in the presence of ADP, a normal curve was observed, with a K, for L-serine of 2.2 X 103 M. EDTA at 10m3 M concentration had no effect on the relation- ships among rate, substrate, and activator. In addition, the rate, although normally measured by a single assay at 10 min, was shown to proceed linearly for 20 min, even at low concentrations of L-threonine. In view of these results, the sigmoid phenomenon does not appear to be’ due to the metal inhibitor or the instability of the enzyme at low concentrations of the substrate, but to the inherent nature of this enzyme.

Curvature of Lineweaver-Burk Plot-Double reciprocal plots showed a straight line in the presence of ADP, whereas a down- ward convex curve was observed in its absence (Fig. 2). If the


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reciprocal of the reaction rate was plotted against the square of the reciprocal of L-threonine concentration, the data followed a straight line at lower concentrations of threonine but an upward convex curve was obtained at high concentrations of tlireonine (Fig. 2). When another plot was employed, namely [(VmL,/v) - l] (S) against l/(S) (where a represents reaction rate; VmaX, maximal velocity; and S, substrate), a straight line was obtained with the concentrations of substrate examined (Fig. 3). Thus, the curved Lineweaver-Burk plot of Fig. 2 can be represented by Equation 2, which may suggest that there are two binding sites for substrate on the enzyme protein.

1 - = 5.3 +

1.95 x 10-1

V w + 6.3 x lo-’

w (2)

Effect of pH on K,,, and V,,,az-In order to obtain some informa- tion about the ionizing groups of the enzyme or the enzyme-

FIQ. 2. Lineweaver-Burk plots for n-threonine in the absence (.- l ) and presence (O-O) of lCa M ADP. The data of Fig. 1 are plotted. Another plot, in which the reciprocal of the rate is related to the reciprocal of the square of n-threonine concentration, is also shown (X- - -X).

FIG. 3. The rate of threonine deaminase reaction as a function of n-threonine concentration (S) in the absence of CADP. [(Vm.Ju) - l] (8) was plotted against l/(S) from the results presented in Fig. 1. The Vm,, was obtained from the extrapolation of l/v at high substrate concentrations in Fig. 2.

I t I 6 7 8 9 IO II

PH FIG. 4. Effect of pH on the logarithm of the maximal velocity

and the negative logarithm of the apparent K, for L-threonine. The V,,, and apparent K, were calculated from Lineweaver-Burk plots in the absence (O-O) and presence (O-O) of ADP. The following concentrations of ADP were employed: 10-S r.s at pH 8.4 and 9.1; 5 X 10-Z M at pH 7.1; lo-* M at pH 6.3, 9.5, and 9.8; 10-l M at pH 10.4. The enzyme protein used was 10.7 fig (specific activity, 13).

I T

,-5

FIG. 5. Effect of temperature on the maximal velocity in the presence of 10-S M ADP (O-O) and in its absence (O-O). Maximal velocity was obtained from a Lineweaver-Burk plot. The rate of product formation proceeded linearly with time over the temperature range tested. The enzyme protein used was 10.7 pg (specific activity, 13).

substrate complex (lo), the apparent values of -log K, and log V,,, at various pH values were determined, and are summarized in Fig. 4. K, values vary with pH, as previously observed by Davis and Metzler with sheep liver threonine deaminase (11). These workers suggested that the uncharged amino group of the substrate anion combines with the enzyme, and that the free enzyme is reversibly converted to an inactive form by the loss of protons around pH 9.1 at 37”. Different pH profiles of maximal velocity were obtained with and without ADP, which may suggest that ADP influences the ionization of groups in the enzyme-substrate complex.

EfTect of Temperature-The effect of temperature on the maximal velocity at pH 8.4 in the presence and absence of ADP is shown in Fig. 5. In the absence of ADP, a linear Arrhenius plot


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was found over the temperature range of 4-45”, and the calculated activation energy was 13.9 kcal per mole. It is interesting that, in the presence of ADP, a new slope was established above 37”. The energy of activation was calculated to be 11.4 kcal per mole over the temperature interval of 4-37’, and 5.5 kcal per mole in the temperature range of 37-45”. Thus, lowering the activation energy is one of the unique features of the activation of this enzyme by ADP. The K, value at 37” was about one- tenth as much as that at 4” in the absence of ADP. It was, however, one-half in the presence of ADP.

Effect of ADP--When the reaction rate was determined as a function of ADP concentration at a fixed threonine level (lob3 M), a curve of the Michaelis-MenQn type was obtained, as shown in Fig. 6. The Michaelis constant of activation (K,) was estimated to be 2.3 x 10T6 M from this plot. In addition, plots of the reciprocal of the difference between reaction rates in the presence and absence of ADP against the reciprocal of ADP concentration (12) showed a bimodal character that yielded two K, values at higher concentrations of threonine (Fig. 7B). Be- low 10-s M threonine, the bimodality was not observed (Fig. 7A). The bimoclality was also observed at pH 7.1,8.1, and 9.1.

Mutual Eflect of ADP and Threonine on K,,, and K,--With some other allosteric enzymes, the K, for the substrate decreases as the concentration of the added allosteric effector increases;

FIQ. 6. Effect of the concentration of ADP on the reaction velocity at 10-3 M n-threonine. The standard assay system was employed, with 10.7 pg of protein (specific activity, 13). The rate of reaction is expressed in micromoles of a-ketobutyrate formed per min per ml of reaction mixture.

(A) (a) - -

‘4 v 8

-. . 4

liIL.l 0 123

. lx 2

0 0.3 06 0.9

I/(ADp) X I@‘#’ FIG. 7. Plots of the reciprocal of the difference of rates of the

reaction in the presence and absence of ADP against the reciprocal of ADP concentration at 10-z M (A) and 5 X 10-z M (B) L-threonine. The standard assay system was employed. In A and B, 10.7 and 2.1 fig of protein (specific activity, 13) were used, respectively.

TABLE II K, for L-threonine at di$erent concentrations of ADP

Enzyme activity was measured by the standard assay system in the presence of varying concentrations of ADP and n-threonine, with 10.7 rg of protein (specific activity, 13). The apparent K, value was obtained from the intercept of the linear portion of a Lineweaver-Burk plot at high substrate concentrations with the abscissa.

Concentration of ADP

M x 108

0

0.001

0.01

0.1

1

10

100

1,m

P

L-

Lpparent Km for threonin~

H x 10:

37 36 24 5.5 3.7 3.3 3.5 3.6

18.2 18.2 18.2 18.2 19.7 20.7 22.7 21.7

0 These values are given in micromoles of a-ketobutyrate formed per min per mg of protein.

TABLE III K. for ADP at different concentrations of dhreonine

The enzyme activity was determined by the standard system in the presence of varying concentrations of n-threonine and ADP. K,, values were determined from double reciprocal plots as shown in Fig. 7. K. values in parentheses were obtained from the plot described in “General Discussion.” K., denotes the K, values which were obtained at high concentrations of ADP, and K,, those obtained at low concentrations of ADP.

x,-Threonine concentration

Y x 101 M x 106

1.0 2.3 2.5 1.0

5.0 1.1 (1.7) 7.5 1.2

10 1.3 (1.3) 25 1.1 50 2.0 (0.93) 75 1.9 (1.4)

6

Y x 106

2.3

0.20 (0.14)

0.08 (0.041)

conversely, an increase in the substrate concentration causes a gradual decrease in the K, for the allosteric effector (13, 14). In the present case, such a mutual effect could not be observed. The apparent K,,, value for threonine was essentially unaffected with ADP concentrations above 10ms M, as shown in Table II; however, the maximal velocity increased slightly from 15.2 to 22.7 pmoles per min per mg of protein. The Michaelis constant of activation (K,) was also determined at varying concentrations of threonine and is summarized in Table III. The constants at high ADP concentrations (Kal) were almost unchanged at various concentrations of threonine, although those at low ADP concentrations (K+.) were dependent on the concentration of threonine.

Inhibition Studies-The enzyme activity was inhibited by a number of compounds (Table IV). ADP did not protect the enzyme from inhibition by these compounds. The mode of inhibition was also tested with n-threonine as a substrate analogue;


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A. Nakazawa and 0. Hayaishi 1151 Issue of March 25, 1967

TABLE IV Inhibition experiments

Reagent added

n-Threonine CMB ............................ p-Chloromercuriphenylsulfate .... Mersarylic acid. ................. HgCl,. ......................... Iodoacetic acid ................. Hydroxylamine .................. Semicarbazide .................. Trinitrobenzenesulfonate., ....... N-Bromosuccinimide ............ Diisopropyl fluorophosphate ..... Pi ............................... PPt .............................

EDTA. .......................... NH&l ..........................

-

--

-

Concentration

M

5 x 10-8 10-7

10-T 10-7

10-1 9 x 10-X

10-a 10-a 10-4

10-b 10-a 10-s 10-a 10-a 10-s

-

I --

-

Zelative activity”

0.85 0.55 0.08 0.07 0.04 0.72 0.12 0.62 0.65 0.02 l.lOb 1.10 0.97 1.00 1.00

= Enzyme activity in the presence of the inhibitor relative to that in its absence. Standard assay conditions were used, with 2.1 pg of protein per tube.

b Preincubated for 1 hour at 23’ and pH 7.5.

FIG. 8. Lineweaver-Burk plots for L-threonine in the presence of 5 X 10-* M n-threonine (A-A), in its absence (O-O), in the presence of 5 X W2 M n-threonine and lWa M ADP (A-A), and in the presence of 10-a M ADP (O-O). Veloc- ity is expressed in micromoles of a-ketobutyrate formed per min per ml of reaction mixture, which contained 10.7 pg of protein (specific activity, 13).

CMB,’ as an -SH inhibitor; and semicarbazide, as a carbonyl reagent which might attack the carbonyl group of pyridoxal-P of the enzyme.

n-Threonine was not utilized by the enzyme under the standard assay condition, but competitively inhibited its activity toward L-threonine, both with and without ADP, as shown in Fig. 8. The apparent inhibition constants (Ki) were 7.5 x lo+ M and 1.3 X 10” M in the presence and absence of ADP, respectively. Semicarbazide was found to act as a competitive inhibitor toward L-threonine with an apparent Ki of 3.0 x 10U3 M

1 The abbreviation used is: CMB, p-chloromercuribenzoate.

I I , I I ,

0 40 00 I

hs,

U

FIG. 9. Lineweaver-Burk plots for n-threonine in the presence of lo-7 M CMB (M), 5 X 10-z M CMB (A-A), and no CMB ( .o---• ), and in the presence of 5 X 10-8 M CMB and 10-* M ADP (A-A) and W3 M ADP (O-O). Velocity is expressed in micromoles of a-ketobutyrate formed per min per ml of the reaction mixture, which contained 2.1 pg of protein (specific activity, 13). As higher concentrations of n-threonine were used, straight lines were obtained by double reciprocal plots.

without ADP, and an apparent Ki of 2.3 X 10-a M with ADP, respectively. Hydroxylamine, a carbonyl reagent, also showed competitive inhibition.

CMB inhibited the deaminase strongly in a reversible manner. It was effective at a concentration as low as lo-’ M, its effect was not increased by preincubation for 1 hour, and its inhibition was completely and instantaneously reversed by adding 10-z M

/3-mercaptoethanol to the reaction mixture. As shown in Fig. 9, the effect of CMB was competitive with respect to n-threonine in the absence of ADP with an apparent Ki of 4 x 10-S M,

whereas it was noncompetitive in the presence of ADP. The activation constant for ADP was not altered by the presence of 5 x 10-r M CMB. The interpretation of these findings will be discussed below.

E$ect of ATP on Reaction Kinetics-The sigmoid nature of the rate-substrate curve became more pronounced in the presence of ATP. At low concentrations of n-threonine the activity was lower in the presence of ATP than in its absence, whereas at high levels of substrate the activity was about 2 times greater in the presence of ATP (Fig. 10). ATP was found to offset activation of the enzyme by ADP, with an inhibition constant of 7 x 10e5 M, when the enzyme activity was measured at low3 M threonine.

Binding of ADP

E$ect of ADP-The binding of ADP by the partially purified enzyme preparation has been reported (2). To rule out the possibility that ADP was bound by some protein other than the deaminase, the dissociation constant for ADP was measured by the method described in “Experimental Procedure.” From equilibrium dialysis, an average dissociation constant of the enzyme-ADP complex was calculated to be 3.0 X 10m6 M (Fig. ll), which agrees reasonably well with the K, value of 2 X 10m6 M obtained from reaction kinetics.2 The above results indicate that

* The K, value determined at 25’, the temperature which has been used in equilibrium dialysis experiments, was essentially identical with that determined at 37”.


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1152 ADP Activation of Clostr ‘idial Threonine Deaminase

* 0 04 0.8 Q8 1x10’ M

L-THREONINE

FIG. 10. The rate of the reaction as a function of n-threonine concentration in the presence of 1W3 M ADP (O-0) and W2 M ATP (+m), and in the absence of nucleotides (0-O). The standard assay system, containing 10.7 pg of protein (specific activity, 11) per tube, was used. The rate is expressed in micromoles of a-ketobutyrate formed per min per ml of reaction mixture.

1 I

0 5 IO

b”dd ADP)X ~o-4~-’

FIQ. 11. Effect of ADP concentration on the binding of ADP by L-threonine deaminase. Concentrations of bound and unbound ADP were determined by equilibrium dialysis technique (see “Experimental Procedure”).

TABLE V Efect of various compounds on binding of ADP

Incubation and separation of bound ADP fraction were carried out according to the method described in “Experimental Pro- cedure.” The incubation mixture contained 5.6 wmoles of ADP-1% (82,100 cpm); 1.9 mg of protein (specific activity, 16.6) in the case of Experiment A or 5.0 mg of protein (specific activity, 12.9) in the case of Experiment B; and the various compounds, at the concentrations shown below, in 0.33 ml. From the incubation mixture, 0.20 ml was taken to determine the concentration of bound ADP. In Experiment A, incubation was carried out in 0.1 M Tris-HCl, pH 8.4, at 37”, and in Experiment B, in 0.1 M potassium phosphate, pH 7.0, at room temperature.

Reagent added Concentration ladioactivity il rrge molecular

fraction klative binding

M CM % Experiment A

None. . 3376 100 n-Threonine. . . 5 x 10-r 3139 93 Semicarbazide.. . . . . 10-z 3166 94 ATP . . . . 10-s 128 3.8

Experiment B None . . . 6850 cm3 . . . . . . 10-4 7070 Hydroxylamine . . . 10-Z 5820

100 103

85

Vol. 242, No. 6

ADP is actually bound by the deaminase, although they do not unequivocally rule out a possibility that ADP is bound by some other protein with very similar dissociation constant for ADP.

E$ect of Inhibitors-As the structure of ADP is distinct from that of threonine, it is hardly conceivable that the two compounds are bound at the same site. However, in order to obtain direct evidence, the effect of competitive inhibitors against n-threonine on the binding of ADP was investigated by the use of gel filtration.

Even when the competitive inhibitors such as n-threonine, CMB, semicarbazide, and hydroxylamine were included in the incubation mixture at concentrations well over the apparent K; values, the binding of ADP by the enzyme protein was not af- fected, as shown in Table V. On the other hand, 10d3 M ATP, which counteracted the activation by ADP, completely prevented the binding of ADP to the enzyme.

General Discussion

Kinetics in Absence of ADP-A concave curve of a Lineweaver- Burk plot is a characteristic feature of this threonine deaminase. A similar curvature was reported in the biosynthetic threonine deaminase from Escherichia coli (15), NAD-specific isocitrate dehydrogenase from yeast (16) and from Neurospora (17), thymidine kinase from E. coli (18), and aspartate transcar, bamylase from E. coli (19). However, the biodegradative threonine deaminase from E. coli (14, 20) and that from animal tissue (11) gave a classical linear plot at all substrate concentrations tested.

A well known example of the sigmoid curve is the oxygen saturation curve of hemoglobin, as already cited. Although hemoglobin is not an enzyme, several allosteric enzymes have been compared with it. For example, aspartate transcarbam- ylase of E. coli (19) was presumed to have several homologous active sites, but the attachment of 1 molecule of substrate to one site modifies the affinity of a different site for the second molecule of substrate. In those cases, the reaction obeys Hill’s empirical equation for hemoglobin,

vln*x(s)” ’ = K, + (S)”

(3)

and a plot of log [v/(V,,, - v)] against log (8) (Hill’s plot) gives a straight line with a slope of n, a parameter which is closely related to the average free energy of interaction of sites (21).

From Hill’s plot for the clostridial deaminase, a value of 1.1 for n was obtained as shown in Fig. 12. Since other allosteric enzymes, such as threonine deaminase and aspartate transcar- bamylase of E. coli, have n values between 1.4 and 2.8, the co- operative interactions between substrate sites are much weaker in this case than in other allosteric enzymes. A theoretical curve obtained from Equation 2 is also shown in Fig. 12, which shows that our data agree reasonably well with the theoretical curve.

The concave curve of a Lineweaver-Burk plot might also result from a substrate acting as an activator (22, 23). In the present case, the assumption was made that a single substrate could be bound to two distinct sites of the enzyme: one, the catalytically active site; the other, the activating site. Unless substrate was bound both at the activating and at the catalytic sites of the enzyme, the substrate at the catalytic site would not be degraded at all or perhaps would be broken down at a very slow rate.


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The following equation can be derived.

E+S=ES,, Kt

ES1 + S s ES&, olK2

ES2 + S ti E&f& Cl&

k E&S2 _-) ES1 + product

where ES2 represents the enzyme, with the catalytic site occupied by the substrate; ES,, the enzyme, with the activating site occupied by the substrate; and ES&; the activated form of the enzyme, which has sites occupied by substrate molecules. K1 and Kz are the dissociation constants for activating and catalytic sites, respectively, and CY characterizes any change in the dissociation constant by binding of one substrate molecule on the other site. The term k is the rate constant for the breakdown of E&S2 to ES1 and product, which is assumed to be the rate- limiting step in the over-all reaction. By assuming equilibrium conditions, the reciprocal rate expression is

V msx - =1+ ci(K~ + Kz) + CZKIK,

(S) 692 (4)

V

which may be rearranged as

(5)

I f [(V,,,/v) - l] (S) is plotted against l/(S), a straight line with the slope of CYK~K~ will be obtained, and the intercept with the ordinate will be a(K1 + KJ, which is the apparent Km obtained from a Lineweaver-Burk plot. As was shown in Fig. 3, a straight line was obtained in such a plot with an intercept of 3.7 x lo* M, which agrees well with the apparent K,,, obtained by a Line- weaver-Burk plot (Fig. 2).

Assuming (I! = 1, KI and Kz are calculated from the experimental data to be 3.3 X lo* M and 3.5 X 10-s M, respectively.

Kinetics in Presence of ADP-In the presence of 10-a M ADP, normal kinetics was obtained with a K, value for threonine of about one-tenth of that in the absence of ADP (Fig. 2). The binding experiments revealed that ADP was bound at a site distinct from the sites for threonine. In order to simplify the calculations, reactions which were conducted under the following conditions only were analyzed. Since the dissociation constant (K1) for the substrate is 3.3 X 10” M, and the K, for ADP is about 2 x 10ms M, reactions conducted with threonine concentrations up to 10e2 M and 10e3 M ADP should be activated pre- dominantly by ADP. Under these conditions, presumably only a small portion of the enzyme has both catalytic and activating sites occupied by threonine. Thus the mutual interaction between ADP and threonine sites need not be considered under these conditions, and the following sequence can be formulated.

E+A=EA, K,

E+S=ESz, Kz

EA + S G= EASz, Kz

ES2 + A = EASz, K,

k’ EA& - EA + product

log(S)

FIG. 12. Activity of threonine deaminase as n-threonine concentration. V,,, was obtained

a function of from a Line- through these weaver-Burk plot. I f a straight line is drawn

points, the slope of the line which defines n in Equation 3 is 1.1 (- - -). The curve (----) in the figure could be drawn accordine to Equation 2, which might also cover the points obtained.

where K, is the dissociation constant for ADP, and k’ is the rate constant for the breakdown of EA&, which is considered to be the rate-limiting step of the over-all reaction. ADP is assumed to act like threonine, which is bound at the activating site. Since the rate of product formation from E& is unquestionably slower than that of the formation from EAX2, the binding of ADP to the enzyme might be considered of major importance in allowing product formation, The reciprocal velocity expression is

+.E = [1+f$][lffg 03)

Thus the double reciprocal plot of l/v against l/(S) should be a straight line, and Km might equal Kz. With increasing concentrations of threonine up to lo- M, the KS value was estimated to be 3.5 x 10e3 M, which agrees well with the K, value calculated from the results in the absence of ADP.

In the case of allosteric activation, the decrease in the Km of the substrate has been attributed to an increase of affinity, which is due to an effector-induced conformational change in the enzyme protein. However, in our case, it is more attractive to assume that ADP and threonine at the activating site modify

$ P

I

I

IO-

l

2

S-

1/ 1 > +3 -I-

O T

0.3 09 x~l0%- J/ OS * (ADPI

FIG. 13. Effect of ADP on the reaction rate at 5 X 1O-3 M n-threonine. The data in Fig. 7B were plotted again. The velocity in the absence and presence of ADP is represented by v0 and va, respectively. V,,, and V’,,, are maximal velocities obtained from a Lineweaver-Burk plot in the absence and presence of 10-s M ADP, respectively.


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the conformation of the enzyme protein and thereby allow the enzyme-substrate complex to break down more rapidly. The flexibility in enzyme proteins has been predicted by Kosh- land (24).

At concentrations of threonine up to lo+ M in the absence of ADP, the catalytic site, which has a dissociation constant of the order of 10m3 M, will first be filled. However, substrate breakdown is negligible because the activating site, which has a dissociation constant of about lo-* M, will not yet be occupied by threonine. Above 10-2 M threonine, the enzyme activity appears normally, as the activating site is beginning to be saturated. Because ADP is bound with the dissociation constant of the order of 10m6 M, the enzyme is fully activated in the presence of 10v3 M

ADP. Therefore, even at low concentrations of threonine, the rate of the reaction follows typical Michaelis-Menten kinetics, with a K, of the same value as Kz. As the Kz value is one-tenth of K1, the apparent K, value should decrease in the presence of ADP to one-tenth of its prior value. By this mechanism, therefore, the binding of the effector does not change the actual affinity of the enzyme for the substrate, although the K, may vary.

Allosteric Site-The following findings indicate that the allosteric site is distinct from the substrate sites: (a) mutual interaction was not observed between allosteric and substrate sites; (b) although n-threonine is considered from kinetic analysis to be bound at both substrate sites, it did not interfere with the binding of ADP; (c) CMB inhibited the enzyme competitively toward n-threonine in the absence of ADP, but noncompetitively in the presence of ADP (Fig. 9). I f CMB were bound only at the activating site, or if it cut off the process of activation by threonine at the activating site, CMB would not affect the K,

for the substrate in the presence of ADP, because the activating site would not be necessary for enzyme activity in the presence of ADP. If the sites for substrate and ADP were identical, the results shown in Fig. 9 could not be interpreted simply.

The mechanisms of activation by ADP and by threonine, although probably similar in kind, do not seem to be identical for the following reasons: (a) each of these compounds is bound at a distinct location in the enzyme; (5) maximal velocities in the presence of ADP and in its absence are different; (c) the energy of activation in the presence of ADP is 17% lower than in its absence.

Estimation of the dissociation constant (K,) for ADP from kinetic data is often difficult. As described above we used a plot of the reciprocal of the difference in the reaction rate in the presence and absence of ADP against the reciprocal of ADP concentration. With low levels of n-threonine, K, can be obtained from such a plot. However, at a high concentration of n-threonine, another plot will be preferable.

When Equation 6 is subtracted from Equation 4, assuming (Y = 1, the following equation can be obtained.

v msx --+q1+$][$-$1 (7)

VO

I f the difference of V,,, /v in the absence and presence of ADP is

plotted against l/(ADP), a straight line with a slope of -K, [I + K2/(S)] will be obtained. The intercept of the abscissa is KI/K,(S). As K1, Kz, and (8) are known values, K, can be calculated from the experimental data. With this plot, almost the same values were obtained as those calculated from the above plot (Fig. 13 and Table II). Bimodality was also observed in this plot. The fact that the activation constant estimated from this kinetic treatment agrees reasonably well with that from the binding study suggests that above kinetic treatment is plausible.

AcknowZedgments-We are indebted to Dr. J. A. Olson for his valuable aid in the preparation of this manuscript. Thanks are also due to Drs. Y. Tonomura, S. Kuno, H. Chiba, and E. Sugi- moto for their generous advice and helpful discussions of this work.

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Atsushi Nakazawa and Osamu Hayaishi by Adenosine Diphosphatetetanomorphum

ClostridiumOn the Mechanism of Activation of l-Threonine Deaminase from

1967, 242:1146-1154.J. Biol. Chem.

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