ALLOSTERISM, REGULATION AND COOPERATIVITY: THE CASE OF RIBONUCLEOTIDE REDUCTASE OF LACTOBACILLUS LEICHMANNII DIWAN SINGH, YOSHIKUNI TAMAO and RAYMOND L. BLAKLEY Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242 INTRODUCTION

The classical examples of enzymes subject to regulation invariably consist of multisubunit enzymes which usually exhibit all of the phenomenon of regulation, allosterism and cooperativity. This has sometimes resulted in the assumption that these phenomena always accompany each other, and even in some confusion of the phenomena with one another. It may be that in fact the majority of regulated enzymes do exhibit both allosterism and cooperativity, and it is certainly true that most intracellular enzymes contain multiple subunits, but the three phenomena are distinct and do not always occur together. It is also possible, as we shall demonstrate, for one or more of the phenomena to occur without the presence of multiple subunits. Furthermore, many enzymes are known that have multiple subunits but do not exhibit cooperativity, showing instead Michaelis-Menten kinetics, that is to say, linear plots of reciprocal velocity versus reciprocal substrate concentration (double reciprocal plots). Well-known examples are aldolase and lactate dehydrogenase. It must be presumed that the modulation of enzyme activity by the binding of a regulatory ligand to the enzyme always involves a conformational response by the protein. In allosteric regulation the ligand binds at a site other than a catalytic site. It makes no difference from the point of view of this definition whether the allosteric site is on the same subunit as the catalytic site: it may just as well be on a different subunit which may have a specific role in regulation, as in the case of aspartate transcarbamylase. When the allosteric site is on the same subunit as the catalytic site, however, there is no a priori reason to assume that the conformational response to the regulatory ligand will cause subunit interactions. Allosteric regulation could therefore occur without subunit interactions and therefore without cooperativity, and o f course in the case of a monomeric enzyme subject to allosteric regulation this must be the case. From this perspective allosteric control involving subunit interaction is a special case of the general conformational response to the regulatory signal. It has been argued (1) that in the course of evolution the more general phenomenon of conformational response to ligand binding was developed earlier than cooperativity, the latter representing the secondary development of the fine tuning of regulatory control. 81

82

D. SINGH et al.

The examination of the regulatory behavior of an enzyme in the absence of cooperative effects has the advantage that the relative simplicity of the system facilitates the interpretation of the data. If, in addition, the enzyme is monomeric, the decreased size and complexity further simplifies the study of the regulatory process and the nature of the conformational response which it involves. One such enzyme is the ribonucleoside triphosphate reductase from L a c t o b a c i l l u s l e i c h m a n n i i (EC 1.17.4.2). This is a monomeric enzyme of molecular weight 76,000, having a single polypeptide chain (2). It does not aggregate over a wide range of protein concentration in the presence or absence of substrates or modifiers, except under denaturing conditions. It shows a specific pattern of activation of the reduction of ribonucleotide substrates by the deoxyribonucleotide products (3). Thus dGTP activates ATP reduction, dATP activates CTP reduction and, to a lesser extent, dCTP activates UTP reduction and dTTP activates ITP reduction. One of the problems which it becomes more feasible to study with such an enzyme is the question of whether regulatory effects are truly allosteric. In the great majority of cases the evidence that regulation is allosteric is very incomplete, often resting solely on some degree of chemical dissimilarity between modifier and substrate which are therefore assumed to bind to different sites. However, it is usually not clear how much commonality of structure would be required for the two ligands to bind at the same site. In the case of inhibitors, enzyme kinetics can readily distinguish between allosteric inhibitors and those binding at the catalytic site. A plot of slope of the double reciprocal plot versus the inhibitor concentration (secondary plot) will be hyperbolic in the former case and linear in the latter. Nevertheless, the erroneous idea that an altosteric inhibitor cannot exhibit competitive kinetics is surprisingly common. In practice the presence of cooperativity or other complicating factors may, of course, make it difficult to use secondary plots to determine whether inhibition is allosteric. While activators cannot compete with substrates for binding at the catalytic site it is possible that they could occupy some adjacent part of the catalytic site. Kinetics cannot distinguish this possibility from binding at a distant allosteric site. In order to determine with certainty the relationship between the regulatory site and the catalytic site more sophisticated techniques such as NMR or X-ray diffraction by crystalline complexes are required. In the following the interaction of ribonucleotide reductase with its modifiers and the consequent conformational response are discussed. The results to date indicate complex relationships between the binding of modifiers, substrates and coenzyme.

M A T E R I A L S AND M E T H O D S Unlabeled dGTP was purchased from Sigma and radiolabeled [8 -14C] dGTP (50.6 mCi per mmole) from New England Nuclear. Contaminating dGDP and

REGULATION OF RIBONUCLEOTIDEREDUCTASE

83

dGMP were removed from dGTP (0.1 miUimole) by chromatography at 4 ° on a column (1.5 X 15 cm) of DEAE-ceUulose (bicarbonate form) with a linear gradient formed with water (1000 ml) in the mixing chamber and 0.5 M tdethylammonium bicarbonate buffer, pH 7.5, (1000 ml) in the reservoir. Eluates containing dGTP were pooled and freeze-dried. Triethylammonium bicarbonate was removed by dissolving in 1 mM phosphate buffer, pH 7.5, and freeze drying. This was repeated twice. The purity of the dGTP was routinely checked by chromatography on PEI-cellulose sheets (Baker) with 0.5 M potassium phosphate buffer, pH 3.5, as the solvent system. Nucleotides were located under ultraviolet light. Other materials were as described previously (4-6). Adeninylpentylcobalamin was a generous gift from Dr. H. P. C. Hogenkamp, Department of Biochemistry, University of Minnesota. Purification of ribonucleotide reductase from L. leichmannii. In the purification of reductase previously described (5), several low-activity forms are eluted from the 6-aminohexyldGTP Sepharose column by buffer before elution of the high-activity, tightly-bound form with 2 M urea or 0.5 mM dGTP. In some preparations these weakly-bound forms amounted to as much as 67% of the total activity eluted. Moreover, it could be shown that if the enzyme preparation was stored at 0 ° for several weeks prior to chromatography the amount of tightly binding form decreased to as little as 13% of the activity with a concomitant increase in the weakly-bound forms. Formation of weakly-bound forms was not prevented by the addition of protease inhibitors (5 mM NaHSO3, 1 mM phenylmethylsulfonylfluoride, 1 mM benzamidine, 1 mM O-phenanthroline, 50 mM EDTA) or by the presence of ligands (1 mM ATP, 1 mM dGTP, 1/riM adenosylcobalamin or 1 mM MgSO4). However, when the entire purification was carried out at lower pH, 50% of the activity was eluted from the affinity column in the tightly-bound, high-activity form. This suggested that conversion of high- to low-activity forms is due either to oxidation of sulfhydryl groups or to disulfide interchange ("scrambling"). This hypothesis was strengthened by the fact that if weakly-bound forms of the enzyme were treated with 5 mM dihydrolipoate at pH 7.3 and 0 ° for 30 min before affinity chromatography, the enzymic activity was subsequently eluted largely in the tightly-bound form. Furthermore, when tightly-bound form from the column was treated with 5 mM lipoate in the presence of 1 mM Cu 2÷ at room temperature for 30 min about 75% of the enzyme was converted to the weakly-binding forms. Treatment with 5 mM oxidized glutathione and 1 mM Cu 2 ÷ produced 30-40% of the low activity enzyme. Based on these observations the conventional purification of the enzyme up to the second ammonium sulfate step has been modified to include 1 mM 2-mercaptoethanol in solutions used throughout the procedure and to employ lower pH values throughout the procedure, as follows. According to the new procedure frozen cell paste (about 1 lb) was thawed and cells suspended in 0.1 M sodium citrate buffer, pH 6.3 (5°), containing

D. S I N G H et al.

84

1 mM 2-mercaptoethanol and 0.02% sodium azide. All subsequent steps were carried out at 5 ° unless otherwise stated. The cells were disrupted by two passages through a Manton-Gaulin mill at 7500 psi. After adjusting the pH of the extract to 5.6, the cell debris was removed by centrifugation (Sorvall GSA rotor, 12,000 rpm, 15 min). The supernatant was dialyzed against 0.1 M sodium citrate buffer at pH 5.6, containing 1 mM 2-mercaptoethanol and 0.02% sodium azide, for 1 6 - 2 4 hr. The dialyzed extract was then treated with protamine sulfate as previously described (2). The supernatant fraction from this step was then made 2 mM in EDTA before precipitation of enzyme with ammonium sulfate (360 g/l). The precipitate was recovered by centrifugation and suspended in and dialyzed overnight against 0.1 M sodium citrate buffer, pH 5.5, containing 1 mM 2-mercaptoethanol and 0.02% sodium azide. Any insoluble material was removed by centrifugation before applying the dialyzed solution to a Sephadex G-100 column (16 × 130 cm), which had been equilibrated with the same buffer used for dialysis. The active fractions from the column were combined and the enzyme reprecipitated by addition of solid ammonium sulfate (440 g/l) after preliminary addition of EDTA (to 2 mM). The precipitate was suspended in and dialyzed against 0.1 M sodium citrate buffer, pH5.0, containing 1 mM mercaptoethanol and 0.02% sodium azide for 3 6 4 8 hr if it was to be subjectad to affinity chromatography. When solutions were to be stored for several days mercaptoethanol was omitted from the dialysis buffer. A portion of the dialyzed solution containing about 35,000 units of activity was adjusted at 20 ° to pH 7.3 after addition of phosphate buffer to a final concentration of 0.05 M and then subjected to affinity chromatography on a 2.5 × 95 cm column as previously described (5). Fractions eluted with 2 M urea were pooled and dialyzed against 0.1 M sodium citrate buffer, pH 5.0, containing 0.02% sodium azide. The enzyme was then brought to the desired concentration (at least 10 mg per ml) by pressure dialysis and stored under N2 at 0 °. Weakly-binding activity eluted from the column by buffer was partially converted to the tightly-binding form by treatment with dihydrolipoate as described above. Table 1 illustrates the TABLE

1. P U R I F I C A T I O N

OF RIBONUCLEOTIDE L. L E I C H M A N N I I *

REDUCTASE

FROM

Purification step

Volume ml

Protein g

Activity units

Specific activity units/mg

Recovery %

Crude extract Protamine sulfate precipitation First ammonium sulfate Sephadex G-100 fractions Second ammonium sulfate Affinity column fractionst

1710 1700 170 1850 72 5.8

20.9 12.8 8.1 1.62 1.51 0.24

99,400 93,000 86,500 77,500 76,600 39,800

4.8 7.3 10.7 48.0 50.8 169

94 87 78 77 40

*Results for 459 g of cell paste. tCalculated from results with a portion of the material of the previous step. An additional 5.4% of the activity was obtained as high-activity enzyme by treating weakly-bound forms from the first affinity column with dihydrolipoate and rechromatographing.

REGULATION OF RIBONUCLEOTIDE REDUCTASE

85

results obtained with this procedure. When the enzyme purified in this way was stored under N2 in 0.1 M sodium citrate buffer, pH 5.0, the loss of activity was 23% in 15.5 months (Fig. 1). In 0.05 M potassium phosphate buffer, pH 7.0, 76% of the activity was lost in the same period.

i

E

i

i

i

IO0

o

tr 5C pH 7.010.05M Phosphote) *

Monlhs

FIG. 1. Stability of r~onucleotide reductase. The enzyme (13 mg/ml) was stored at 0 ° under nitrogen at pH 5.0 (0.1 M sodium citrate buffer) or pH 7.0 (0.05 M potassium phosphate buffer) in the presence of 0.02% sodium azide.

Measurement of enzyme activity and protein concentration. Activity was measured by the colorimetric method as modified by Orr et al. (7). Protein concentrations were determined by the method of Lowry et al., (8) as modified by Hartree (9). In this assay the reference protein solution was a stock solution of ribonucleotide reductase the concentration of which had been determined refractometrically in the analytical ultracentrifuge with a double-sector, synthetic-boundary cell (10). Protein concentrations were also measured spectrophotometrically using the absorption coefficient, E21% ao nm = 13.3-+ 0.1 (5). The two methods yielded the same results within experimental error. Measurement of ligand binding. Measurement of dGTP binding was usually carried out in equilibrium dialysis cells holding 0.1 ml of fluid on each side of a circular membrane (Sartorius SM11536) according to the procedure described earlier (6). The membrane was washed several times with 1 mM EDTA, and then with 0.1 M sodium phosphate buffer, pH 7.3, containing 0.02% sodium azide. A solution (0.1 ml) containing the buffered enzyme (12 to 45/~M) and labeled dGTP (5 to 10 X 106 cpm/p~ole) was added to one chamber from a Hamilton Syringe, and an identical solution, except for the absence of the enzyme, was added to the other. The cells were sealed with water-resistant, surgical tape, and rotated (8 rpm) on a drum submerged in a water bath maintained at the appropriate temperature. The concentration of dGTP was determined spectrophotometrically with the molar extinction coefficient 13.7 X 10 a M-lcm-1 (11).

86

D. SINGH et al.

The enzyme was checked periodically for any denaturation during experiments by assay of activity. After the attainment of equilibrium, the concentration of the free ligand was calculated from the radioactivity in the chamber lacking the enzyme, and the concentration of bound nucleotide from the difference in the radioactivity between the two compartments. Since the binding experiments were performed in buffer of high ionic strength the Donnan effect was negligible. The binding data were analyzed by the least-squares method according to the general form of the Scatchard equation (12) r

1 KDiss (r--n)

[L] -

and to the modified form of the Klotz equation (13) 1 r

1 n

1 KDiss n It]

where [L] is the molar concentration of free dGTP, n is the number of binding sites for the dGTP corresponding to the dissociation constant KDiss, and r is the average number of ligand molecules bound per enzyme molecule. Calculations of the binding ratio were based on a molecular weight of 76,000 daltons for the enzyme (2, 14). The two methods of analysis gave values of KDiss and n that were not significantly different. Equilibrium dialysis studies on the binding of adenosylcobalamin were carried out in essentially the same manner as for dGTP. Measurement of binding by ultrafiltration was carried out as previously described (6). O'rcular dichroism (CD) measurements. CD spectra were obtained with a Cary Model 60 recording spectr~photometer equipped with a model 6001 CD attachment and thermostatted cell holders. Measurements were made at 26 ° and 37 ° in 1 mM sodium phosphate buffer, pH 7.3, containing 0.1 M sodium sulfate. In these experiments sodium sulfate was used to provide high ionic strength since sodium phosphate at 0.1 M concentration absorbs significantly and hence interferes with the CD measurements. Solutions were deoxygenated and transferred to the cell under nitrogen. Determinations were made in a 1 mm cell in the far-ultraviolet region (200-250 nm) and in a 10 mm cell in the nearultraviolet region (250-330 rim). The mean residue ellipticity in units of deg-cm2 decimole-1 was calculated from: [01 X - (MRW) O° 10~c

REGULATIONOF RIBONUCLEOTIDEREDUCTASE

87

where 0~. is the observed ellipticity in degrees at wavelength k, ~ is the optical path length in centimeters, c is the concentration in grams per ml, and MRW is the mean residue weight which was calculated to be 110 g per mole of amino acid from the amino acid composition (2). Difference sedimentation studies. Sedimentation experiments were performed according to the method of Kirschner and Schachman (15) using a Beckman Model E analytical ultracentrifuge equipped with Raleigh interference optics. The optical system was aligned as described by Richards et al. (16) and the camera lens was focused at the two-thirds level in the cell. A precision Raleigh mask having an adjustable slit width (16) was constructed in the University of Iowa Medical Instruments Workshop. It was placed over the collimating lens and adjusted to its optimum position (16). A double-sector cell with a 12 mm optical pathlength, sapphire windows and a charcoal-filled epon centerpiece was used. A cell with suitable characteristics was found only after screening(15) twelve commercial double-sector centerpieces provided by the manufacturer and even the selected centerpiece gave good results only when the boundary was in the third of the cell nearest the meniscus. None of the other centerpieces in this batch were suitable. In a typical experiment 0.43 ml of solution was added to each sector by means of a Hamilton syringe fitted with a Cheney adapter. The solutions contained buffered enzyme (about 5 mg/ml) and were identical except that the solution in one compartment contained dGTP. The meniscus separation was always made 0.1 -+ 0.05 mm by withdrawing about 2/al of solution from one of the compartments. An AN-H rotor was used at 60,000 rpm. Photographic plates (Spectroscopic type IIG) were analyzed with the aid of a Nikon Model 6C microcomparator. Analysis of the data for calculation of As/g was made by a program written for the IBM 360 computer to fit the following equation (15):

f

rp Acrdr rm -

~m2Co

RESULTS

I(•-P) In

~m] AS -g

Ar m

(1)

rm

AND D I S C U S S I O N

Kinetics of Ribonucleotide Reductase in the Absence of Modifiers We have previously reported (3) that when the best substrate, GTP, is reduced in the presence of reductase, a low concentration of the coenzyme (adenosylcobalamin), and the thioredoxin reducing system, the kinetics are not of the classical Michaelis-Menten type (upper curve, Fig. 2). The double reciprocal plot is concave down, a result characteristic of substrate activation. This result can be

88

D. SINGH et al. !

KI F'+GTP.

i

K2

• E.GTP.

• GTP.E.GTP

Products

Products

30

I~M AdoCbl t = 0"24ram

1

/k

.3145

20

V

10

1 GTP mM

FIG. 2. Analysis of kinetic data for GTP reduction in the presence of low or high concentration of coenzyme. Data from Vitols et al. (3). Curves calculated according to equation 2. anticipated if GTP binds not only at the catalytic site but also at the regulatory site, according to the following scheme:

K1

K2

E + GTP~--~E.GTP~GTP.E.GTP

lk

Products + E Products + GTIi.E where K 1 and K2 are the dissociation constants for GTP bound at the catalytic site and regulatory site respectively, and k, k' are the respective rate constants for product formation. The symbol E represents whatever form o f the enzyme GTP combines with; it may for example be the complex of the enzyme with the reducing substrate and/or the coenzyme. The appropriate rate equation for such a reaction scheme is

11 .

v

.

K 12+ .

----~-- -iV t l+-~K2~ .

.

,2,

89

REGULATION OF RIBONUCLEOTIDE REDUCTASE

k If the values Kt = 0.24 mM K2 = 4 mM,'~' = 0.3145, V = 0.3916 are inserted in this equation the upper curve is obtained, thus confirming that such a mechanism can account for the data. The results also indicate that under the experimental conditions the apparent dissociation constant for binding to the regulatory site is about 17 times higher than that for the catalytic site, and that the presence o f GTP on the regulatory site increases the reaction rate more than 3-fold. The points on the lower curve were obtained under the same conditions except that the coenzyme concentration was 12 ~tM. The solid curve is that calculated from the same rate equation with identical constants except that k = k'. The excellent fit to the experimental data suggests that the effect o f the higher concentration o f coenzyme is to increase k, the rate constant for the reaction without GTP at the regulatory site. This could occur if one major effect o f GTP binding at the regulatory site is to increase coenzyme binding. When a sufficiently high concentration o f coenzyme is present this effect o f substrate binding at the regulatory site is minimal because the coenzyme can saturate the enzyme even without the aid o f the regulator. In support o f the latter view it was found (3) that the apparent Michaelis constant for the coenzyme decreased as the GTP concentration increased. Similar substrate activation effects were seen with ATP and CTP. Determination o f the Michaelis constant for coenzyme in the presence o f the different ribonucleotide substrates (2 mM) gave values varying over a 10-fold range. Direct determination of the dissociation constant for the enzyme-coenzyme complex has shown that the constant is lowered considerably in the presence o f 5 mM GTP (Table 2).

TABLE 2. DISSOCIATION CONSTANT FOR THE ADENOSYLCOBALAMIN-REDUCTASE COMPLEX UNDER VARIOUS CONDITIONS Temperature

Other ligands present

KD (mM)

Method

5° 5° 5° 25 ° 25 °

None 2 mM dGTP 25 mM dihydrolipoate* 25 mM dihydrolipoate 2 mM dGTP and 25 mM dihydrolipoate 5 mM GTP and 25 mM dihydrolipoate

0.93 + 0.21 0.20 + 0.03

Equilibrium dialysis Equil~rium dialysis

0.62 ± 0.17

Equilibrium dialysis

37 °

1.27 ± 1.13 0.047 ± 0.008

Ultraf'dtration Ultraf'fltration

0.076

Ultrat~fltration

*The reducing substrate for r~onueleotide reduction. The physiological substrate is the thioredoxin system (17, 18).

90

D. SINGH et al.

Chen et al. (14) considered the possibiJity that the reaction sequence when substrate is self-activating in the absence of modifiers might be of the type:

K~ K2 E + GTP~GTP.E~GTP.E.GTP k' Product: + GTP.E and showed that in the case of CTP the measured apparent Km (10 -3 M) is consistent with the measured value of K1 (10-2 M) and an estimated value of K2 (10- 4 M). However, the rate equation for this type of sequence in which the catalytic binding site for ribonucleotide exists only when the regulatory site is occupied either by substrate or by modifier is of the form l_l ~+K2+~ v V A

(3)

This predicts a parabola, concave up, for the double reciprocal plot which was clearly not the finding in our experiments (3).

Kinetics in the Presence o f Modifiers The substrate activation effects seen in the presence of low coenzyme concentrations disappear in the presence of the appropriate activator. Thus ATP and CTP give non-linear kinetics in the presence of low coenzyme concentrations ( < 4/aM), but linear plots with considerably increased velocities when 1 mM dGTP or dATP are present, respectively (3). The most obvious interpretation of these results is that the deoxynucleotide modifiers have a much higher affinity for the regulatory site, so that in their presence substrate is unable to bind there to any significant extent and substrate activation disappears. The deoxynucleotide modifiers also increased the binding of coenzyme to the catalytic site, as shown by decreased (18- to 23-fold) Michaelis constant for coenzyme in their presence, and a decreased dissociation constant for the coenzyme-enzyme complex in the presence of modifiers (Table 2). Effects o f Modifier Binding The enhancement of coenzyme binding when modifiers bind to the regulatory site can not be the only result produced in response to the regulatory signal since there would then be no specificity to modifier action - all modifiers would activate the reduction of all substrates provided the coenzyme concentrations were low. But each modifier activates the reduction of a specific substrate. Confirmation that the enhancement of coenzyme binding is not the only effect of modifiers was provided by the demonstration that in the presence

REGULATIONOF RIBONUCLEOTIDEREDUCTASE

91

of 40/aM adenosylcobalamin, dGTP still accelerated 0.5 mM ATP reduction 2.8-fold and dATP accelerated 0.4 mM CTP reduction 2.2-fold (3). That the modifier (or in its absence, activating substrate) may play a more crucial role in the enzymic mechanism than had been supposed from the kinetic data was apparently demonstrated when the partial reaction between coenzyme, enzyme and reducing substrate was studied by stopped-flow spectrophotometry(4) and by electron spin resonance (ESR) after freezequenching (19). The data seemed to demonstrate that enzyme-bound coenzyme in the presence of reducing substrate does not undergo any detectable reaction unless a nucleoside triphosphate such as dGTP is also present. In the latter case, however~ a rapid reaction occurs (rate constant about 38 sec- l ) with homolytic cleavage of the C-Co bond and generation of Cob(II)alamin (B12r) and a deoxyadenosyl radical. The radical pair disappears in the presence of ribonucleotide substrates at a rate indicating kinetic competence (4, 19) and a study of analogues of adenosylcobalamin(20) indicates that only those with coenzyme activity form the radical pair. This seems to be relatively good evidence that the radical pair is an intermediate in the overall enzymatic reduction of ribonucleotides and the fact that there is a requirement for a nucleoside triphosphate to be bound to the enzyme for appearance of the intermediate seems to put the role of nucleotide binding in a new light. It should also be noted that the presence of nucleotide is required for exchange of hydrogen between water and the methylene group of the coenzyme bound to cobalt, a phenomenon closely associated with intermediate formation (20) and for degradation of the coenzyme, a process apparently associated with formation of another type of radical pair (20). Binding Constants for Modifiers In considering these effects of nucleotide binding it is, of course, important to distinguish effects of binding at the catalytic site from effects of binding at the regulatory site. The available evidence is far from conclusive and is primarily based on dissociation constants. The fact that 1 mM dGTP causes only 14% inhibition of GTP reduction indicates that it cannot compete very successfully with GTP for the catalytic site (3), and since GTP has an apparent Michaelis constant for the catalytic site of 0.24 mM, this suggests a still higher value for the dissociation constant for dGTP bound at the catalytic site. Determination of the dissociation constant of the dGTP-reductase complex in absence of other ligands has recently been determined under a variety of conditions (Table 3). The values for the dissociation constant at 22 ° are in reasonable agreement with those at similar ionic strength reported earlier in the extensive studies of Chen et al. (14). At 37 °, the temperature for most reaction studies, the dissociation constant is of the order of 50/aM. Chen et al. (14) showed that the ribonucleoside triphosphates compete very poorly with dATP for this site with

92

D. SINGHet al.

TABLE 3. EFFECT OF TEMPERATURE ON dGTP BINDING TO RIBONUCLEOTIDEREDUCTASE* Temperature

n

KD t~M

°C 4.5 15 22 27 30 34 37 39

1.070 1,025 0.992 1.157 0.931 1.254 0.910 0.985

± 0,018t ± 0,031 ± 0,057 ± 0,057 ± 0,093 ± 0,013 ± 0,148 ± 0,050

1.73 4.67 8.85 15.52 17.72 27.86 48.75 72.88

-+ 0.068 t ± 0,096 ± 0.33 ± 0.54 ± 0.26 ± 2.78 ± 5.08 ± 3.25

(2.73 (7.96 (15.67 (28.33 (32.69 (48.22 (84.44 (126.4

AG° Kcal/mole +- 0.16) ~ 0.52) -+ 0.61) ± 1.78) ± 1.09) ± 3.27) _+6.17) ± 9.5)

7.32 7.03 6.83 6.61 6.59 6.40 6.12 5.91

(7.07) (6.72) (6.49) (6.25) (6.22) (6.07) (5.78) (5.57)

AH° Kcal/mole

AS° e.u.

5.15 (16.3)

29.5 (33.4)

34.8 (35.4)

92.3 (95.3)

*From equilibrium dialysis in the presence of 0.1 M sodium phosphate buffer, pH 7,3. Values in parentheses obtained in 0.05 M sodium phosphate buffer, pH 7.0. ~'Mean from least squares fit in Klotz plot ± standard deviation.

apparent K D 2 to 27 mM whereas the deoxyribonucleoside triphosphates all have similar KD values (9--80/aM at 22°). We have confirmed very weak binding to this site in the case of ATP and GTP. It is noteworthy that KD values for ribonucleotides obtained by Chen e t al. are of the same magnitude as K2 calculated for GTP from the kinetic data in Figure 2. In both our work and that of Chen e t al. binding of dGTP to only one site was detectable. It is probable that in the presence of coenzyme and dihydrolipoate the dissociation constants are lower still. This would be consistent with the value of about 10/aM calculated for the dissociation constant for dGTP from the kinetics of its activating effect on ATP reduction (3). From these data it is clear that there is a very high affinity site for deoxyribonucleoside triphosphates to which ribonucleoside triphosphates bind weakly, and that this is probably the regulatory site. At the catalytic site, on the other hand, ribonucleoside triphosphates bind more strongly with apparent Km of the order of 0.2 mM whereas deoxyribonucleotides bind considerably more weakly. At which of' these sites is dGTP binding when it apparently initiates intermediate formation from coenzyme and associated processes? Although the effect of dGTP concentration on intermediate generation has not been studied in detail, it has been observed that lowering the dGTP concentration from 1 mM to 0.25 mM causes only a small change in rate constant at 37 ° (46 and 38 sec-1 , respectively) and causes only a 21% decrease in the equilibrium concentration of intermediate (4). In the case of the effect of dGTP on hydrogen-exchange between coenzyme and water an approximate value of 0.1 mM was obtained for the dGTP dissociation constant at 37 ° (21), but here the situation is even more complex because of the probable effect of dGTP on the off rate for coenzyme which may be rate-determining for the exchange process. These results are not clear-cut but on the whole the concentration effects suggest involvement of the regulatory site rather than the catalytic site. Assuming that it is the binding of modifiers or substrates to the regulatory site which initiates intermediate formation and the other associated reactions,

REGULATION OF RIBONUCLEOTIDEREDUCTASE

93

this seems to put the function of the regulatory site in a rather different light. Binding of ligands there apparently serves to initiate the enzymatic reaction as well as to modulate the reaction rate according to the substrate present at the catalytic site. However, when the binding data in Table 2 are used to calculate the concentration of bound coenzyme in experiments on intermediate formation (4, 19), it appears that in absence of nucleotide there would be a negligible amount of coenzyme bound, whereas in the presence of dGTP the calculated concentration of bound coenzyme closely approximates the concentration of intermediate which appears. Thus, the apparent initiation of reaction appears in reality to be due to the enhancement of coenzyme binding. This would certainly be consistent with the known effect of modifier binding to the regulatory site. One feature of the effects of modifiers which remains to be explained is the quantitative differences in the effects of various deoxynucleotides on intermediate formation (4, 19). Thus dGTP gives 5 - 1 0 times as much intermediate formation as dATP or dCTP, and dTTP gives 4 times less than dATP. It is possible, however, that this is explicable in terms of different responses of coenzyme binding to the different nucleotides.

Evidence of the Conformational Response to Modifiers A conformational response may be inferred from the enhanced binding of coenzyme and the specific activation of substrate reduction. More direct evidence has been obtained in two ways. The degradation products which are formed from the coenzyme intermediate are 5'-deoxyadenosine and cob(II)alamin (9). They bind tightly to the catalytic site, and since cob(II)alamin is paramagnetic, the ESR spectrum of the latter provides a sensitive probe for conformational changes at that part of the catalytic site where it is bound (22). Small but significant and reproducible differences in the hyperfine structure of the low-field region of the spectrum are obtained when different modifiers are present. Further evidence of the conformational response was obtained from studies of circular dichroism. The CD spectrum in the far-ultraviolet region (200 to 250 nm) for ribonucleotide reduetase showed the usual minima at 208 and 220 nm (Fig. 3). The method of Chen and Yang (23) gave a value of 26% of ~-helix, and almost identical results were obtained (23%c t-helix) by the procedure of Greenfield and Fasman (24). This region of the spectrum was the same at 26 ° and at 37 ° and was unaltered by the presence of dGTP, so that the binding of modifier causes no major alteration of the secondary structure of the protein. In the near ultraviolet region (250 to 330 nm), which is strongly dependent on the asymmetry of aromatic sidechains, the spectrum is identical at 26 ° and

94

D. SINGH e t

al.

8.0 t 4.0 ¢P

~

-4.0

x

-8.0

-12.0 200

220 240 Wavelength ( nm )

260

FIG. 3. Circular dichroism of r~onucleotide reductase in the far ultraviolet. Superimposable curves were obtained at 26° or 37° and in the presence or absence of 22.6 t~M dGTP. The concentration of reductase was 9.5 uM.

37 ° and shows sharp negative bands at 260, 286 and 292 nm, sharp positive bands at 258 and 265 nm and a poorly-defined broad positive band at about 273 nm (Fig. 4). Although addition of 34/zM dGTP to the enzyme did not change the general shape of the latter spectrum, it caused the disappearance of the positive 258 nm and negative 260 nm CD bands (Fig. 4). The ellipticity for the enzyme in the presence of the ligand is also more positive in the 260 to 320 nm region and this effect is more pronounced at 26 ° than at 37 ° presumably due to increased dGTP binding at 26 °. In addition, the bands centered at 265 and 273 nm broadened and the latter showed a 2 - 4 nm red shift. A linear relationship was found to exist between the ellipticity changes and the fraction of the enzyme in the binary complex. These CD effects of dGTP binding are presumably due to perturbation of tryptophyl, phenylalanyl and possibly tyrosyl residues. The effects might be produced directly by dGTP perturbing aromatic residues at the regulatory site, or indirectly if there is a large conformation response that causes relative movement of aromatic residues at a distance from the regulatory site, or both.

REGULATION OF RIBONUCLEOTIDE REDUCTASE

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FIG. 4. Circular dichroism of ribonucleotide reductase in the near ultraviolet. The concentration of reductase was 20 uM and the concentration of dGTP 34 #M. Reductase in absence of ligand at 26° and 37° ( ), in presence of dGTP at 26° ( - • -) and 37° (---).

Effect of dGTP on the Sedimentation Behavior of the Enzyme The conformational response to modifier binding might be a small, localized conformation change produced at a regulatory site that is close to the catalytic site or overlaps it. On the other hand it might be a relatively massive change, involving much of the protein molecule, which is necessary to transmit a regulatory signal from a binding site distant from the catalytic site. One method which might distinguish between these possibilities is the determination of changes in the sedimentation coefficient by the method of Kirschner and Schachman (15). The data from a typical experiment, shown in Figure 5, give a value of - 1 . 1 4 % for As/g. The control experiment with no dGTP in either sector yielded a value of - 0 . 0 3 6 % for As/g which reflects the limit of the precision of the technique with the available centerpiece. Extrapolation to a saturating concentration of dGTP gave a maximum corrected change in sedimentation coefficient at 22 ° o f As/g = - 1 . 1 6 --!-0.02%. At 37 ° the corresponding change (in the presence of 500#M dGTP) was As/g = - 1 . 1 4 % , which is essentially the same. These observed values must be corrected for the fractional change in s due solely to the effect on buoyant weight of ligand binding. This is given (15) by the expression ML(1--VJLp) -

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96

D. SINGH et al. ,

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