THE JOURNAL OF BIOLOGICAL CHEMIBTRY Vol. 249, No. 5, hue of March 10, pp. 1445-1452, 1974

Printed in U.S.A.

The Interaction of 8-Anilino-l-naphthalenemlfonate

with Creatine Kinase

EVIDENCE FOR COOPERATIVITY OF NUCLEOTIDE BINDING*

(Received for publication, May 21, 1973)

ALAN C. MCLAUGHLIN$

From the Department of Biophysics and Physical Biochemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19174

SUMMARY

Creatine kinase has one tight binding site (& = 310 PM)

per subunit for &anilino-1-naphthalenesulfonate with an enhancement of fluorescence of ~100 and a blue shift of the emission maximum of 30 nm. Nucleotide substrates non- competitively interact with the dye-binding site causing a 50 % decrease in the fluorescence of the bound dye and a con- comitant red shift of the emission maximum of 7 nm, while guanidino substrates alone do not affect the fluorescence of the bound dye.

The fluorescence of the bound dye is used to determine the dissociation constants of nucleotide substrates from the enzyme in the presence or absence of other substrates or effecters, i.e. divalent metal ion or creatine. In the absence of a small planar anion, the presence of creatine or magne- sium (or both) have no significant effect on the Kn of ADP from the enzyme, while in the presence of the nitrate anion creatine induces a drastic tightening and a negative co- operativity in the binding of ADP. With MgADP, the tightening of binding and the cooperativity are further ac- centuated. Modified inactive forms of the enzyme do not show the tightening nor the cooperativity of ADP or MgADP binding. These results are consistent with the hypothesis that the small planar anion (e.g. nitrate) is acting as an analog of a planar phosphoryl group, thus mimicking the transition state of the enzyme-substrate complex, and further suggest that two of the distinguishing features of this transi- tion-state complex are a substantial interaction between the two protomeric subunits and the formation of a ligand from the protein to the divalent metal ion.

An impressive array of physical techniques has been utilized to determine the dissociation constants of nucleotide substrates

* This investigation was supported in part by Grant GM 12446 from the National Institutes of Health, United States Public Health Service, and by Grant GB 32168X from the National Science Foundation.

$ Present address Department of Biochemistry, Oxford Univer- sity, Oxford, United Kingdom.

from various creatine kinase complexes (l-8). Most of these methods, however, involve a number of limitations which would be useful to overcome. The proton relaxation rate method involves the obligatory use of either manganese-nucleotide complexes (1) or the inactive spin-labeled enzyme (2)) as does the electron paramagnetic resonance method (2, 3). The less re- strictive methods such as sulfhydryl reactivity (4, 5), suscep- tibility to trypsin digestion (6), kinetic inhibition (7), and pro- tein fluorescence (8) are, however, less convenient and less precise. The work presented here characterizes the binding of the fluorescent dye 8-anilino-1-naphthalenesulfonate to the en- zyme and illustrates how the fluorescence of the bound dye may be used to provide a convenient and precise method for meas- uring nucleotide and metal-nucleotide dissociation constants from any creatine kinase-substrate complex.

Although creatine kinase is dimeric (9), cooperativity of nucleotide binding has not been observed, either kinetically or in equilibrium titrations. Interactions between the nucleotide and the guanidino substrate-binding sites have, however, been observed under certain experimental conditions (57, lo), and it has recently been suggested (5) that monovalent anions play a key role in this interaction. The ANSl technique is used to further investigate the effect of the nitrate anion on the inter- action between the nucleotide and guanidino substrate-binding sites and on the cooperativity of nucleotide substrate binding.

MATERIALS AND METHODS

Materials-Creatine was obtained from Pfanstiehl Laboratories. HEPES was obtained from Calbiochem and Sigma Chemical Co. The sodium salts of ADP, ATP, DPNH, and TPN were obtained from Siema Chemical Co.. while the sodium salt of ADP was also obtained from P-L Biochemicals. P-enolpyruvate, beef muscle lactate dehydrogenase, yeast glucose-6-P dehydrogenase, and yeast hexokinase were obtained from Boehringer -Mannheim. Rabbit muscle nvruvate kinase was a gift from F. J. Kavne. Uni- versity of Pennsylvania. Iodoacetaide (Sigma Chemical Co.) was purified by recrystallization from hot water and thoroughly washed with ice-cold carbon tetrachloride directly before use. The purified ammonium salt of ANS was a gift of R. Hershberg, University of Pennsylvania. It was purchased from K and K Laboratories, recrystallized twice from hot water, and treated with acid-washed charcoal (Norit). Solutions were filtered hot

1 The abbreviations used are: ANS, %anilino-l-naphthalene- sulfonate; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesul- fonic acid.

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through paper to remove charcoal, and two additional recrystal- lizations from water were performed. The recrystallized ANS migrated as a single spot on silica gel thin layer plates with the use of several different solvent systems. All other reagents were reagent grade and were used without further purification. Quartz double-distilled water was used throughout. Creatine kinase was prepared from skeletal muscle of freshly killed rabbit as previously described (11).

Kinetic Znhibition Studies-ANS was tested as an inhibitor of the reverse reaction at 24’, the creatine concentration being held fixed while the ATP concentration was varied from 0.19 to 2.50 mM. The conditions for the reaction were 0.1 N potassium glycine, pH 9.0,40 mM creatine, 5 mM magnesium sulfate, and 0.4 pg per ml of creatine kinase. The initial velocity was determined by measure- ment of the P-creatine produced at different time intervals after the addition of enzyme (12).

Enzyme Modification-Creatine kinase carbamylated at the es- sential sulfhydryl group was prepared by reacting enzyme (1.05 mM in sites) with iodoacetamide (1.15 mM) in 50 mM potassium acetate and 50 mM potassium glycine, pH 9.0, at 0”. After the ac- tivity had fallen to 4yo (k37,) of the initial value, the mixture was dialyzed at 5’ against 50 mM potassium acetate and 50 mM potas- sium glycine, pH 9.0.

Fluorescence Measurements-Fluorescence spectra were recorded on a Hitachi Perkin-Elmer MPF-2A spectrophotometer equipped with a thermostated cell holder. Titrations with ADP were car- ried out in a standard 4-ml cuvette. Titrations with ANS or en- zyme were carried out in a thermostated cell designed to use vol- umes of 100 to 500 ~1. A 5-mm outside diameter nuclear magnetic resonance tube was positioned in the center of a standard 4-ml fluorescence cuvette by means of spacers in the top and bottom. The cuvette was masked except for entrance and exit slits, both 1 mm wide and 5 mm high, and was filled with distilled water to allow efficient temperature control of the sample. While the smaller light path and aperture decreased the sensitivity they also de- creased the absorbance by the dye, thereby reducing the “inner- filter” effect.

Several precautions were taken to remove divalent metal ion impurities from the solutions used for the nucleotide titrations. Four milliliters of the solutions containing enzyme, ANS, and any additional substrates or anions present during the titration (i.e. creatine or nitrate) were incubated with 2 mg of Chelex-100 in the Na+ form. The solution was then filtered through a 0.65-/*rn diam- eter millipore filter and 3 ml used for the titration. Stock solu-

tions of ADP were passed over a Chelex-100 column in the K+ form. Titrations were done in the presence of 0.6 to l(r fi-mercaptoetha- nol. P-Mercaptoethanol did not change the binding constant of ADP to the enzyme but prevented the slight precipitation of en- zyme during the course of the titration, presumably by stabilizing the enzyme to air oxidation during stirring.

It was found that the KD of the nucleotide in the presence of excess Mgz+ (5 mM) did not depend on the solutions being pre- treated with Chelex. In the absence of magnesium, titrations using solutions that had not been pretreated with Chelex gave variable dissociation constants from 85 to 110 pM, while titrations using solutions that had been pretreated with Chelex gave repro- ducible results within 5%.

Titrations with ANS were performed by addition of small ali- quots of a concentrated ANS solution to 500 ~1 of a solution con- taining 50 mM potassium HEPES, pH 8.0, 50 mM potassium ace- tate, 25 PM creatine kinase, and 1% B-mercaptoethanol. Titra- tions with enzyme were performed by dilution of 100 ~1 of a solu- tion containing 50 mM potassium HEPES, pH 8.0,50 mM potassium acetate, 3.0 mM creatine kinase, 108 PM ANS, and 1% /3-mercapto- ethanol, with an identical solution minus enzyme. All titrations were performed at 24”.

Since the titrations with ANS extended to relatively large dye concentrations (a5 mM) it was necessary to minimize the absorp- tion of incident light by the dye. This was accomplished both by means of the smaller optical path length fluorescence cell described above, and by means of an excitation wavelength far to the red of that for maximum absorption (X,,, E 350 nm). Moving the ex- citation to longer wavelength made it necessary to monitor the emission at longer wavelengths to avoid artifacts from the Raman band of the excitation light. The combined effect is to decrease absorption by the dye and also decrease the fluorescence emission of the dye. The excitation wavelength used in these experiments was 440 nm. With the emission measured at 560 nm the calibra- tion curve of fluorescence against free ANS concentrations was linear up to 2 mM (Fig. 1). By fit#ting the data at higher dye con- centrations to a theoretical curve (see below) the absorption of the dye at any concentration can be determined. In this way, the fluorescence of the bound dye may be corrected for the absorption of the excess free dye at high concentrations of the free dye.

Calculation of Correction for Absorption of Incident Radiation by Fluorescent Dye-In a typical experiment fluorescence is measured at right angles to the direction of the incident beam. j(x) is de- fined as the fluorescence per unit path length (of the incident

X (ml ANS (mM1

FIG. 1 (left). The observed fluorescence of ANS as a function of dye concentration. 0, experimental points; -, theoretical fit to the data, as described in the text; - - -, relationship expected in the absence of absorption of incident light by ANS. Excitation wavelength, 440 nm, emission wavelength, 560 nm. Excitation and emission slits, 10 nm. The solution contained 50 mM potas- sium acetate and 50m~ potassium HEPES, pH = 8.0. T = 23.5”.

FIG. 2 (center). The effect of creatine kinase and the ADP- creatine kinase complex on the fluorescence emission spectra of ANS. Excitation wavelength, 350 nm. Curve A, 20 PM ANS. Curve B, 20 MM ANS, 10 PM creatine kinase. Curve C, 20 PM ANS, 10 PM creatine kinase, 1 mM ADP. All solutions contained 50 mM

potassium acetate and 50 mM potassium HEPES, pH 8.0. T = 23.5”.

FIG. 3 (right). The fluorescence of ANS as a function of creatine kinase concentration. Excitation wavelength, 440 nm, emission wavelength, 560 nm. Curve A, 108 pM ANS. Curve B, 108 PM ANS, 5 mM ADP. Both solutions contained 50 mM potassium HEPES, pH 8.0, 50 mM potassium acetate, and 1% 8-mercapto- ethanol. T = 23.5”. Enzyme concentration is given as subunit concentration. The data shown are corrected for the fluorescence of the enzyme, as described in the text. p, theoretical satura- tion curves, with KD = 310 FM and 1.25 mM for Curves A and B, respectively.

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ANS (mM) P

FIG. 4 (left). Titration of fluorescence with ANS in the presence of creatine kinase. Excitation wavelength, 440 nm, emission wavelength, 560 nm. The solution contained 50 mM potassium HEPES, pH 8.0,50 mM potassium acetate, 1%) p-mercaptoethanol, and 24.3 pM creatine kinase (in sites). The data have been cor- rected for fluorescence due to the free ANS and the absorption of excitation light by the free ANS, as described in the text. -, sum of a theoretical saturation curve (Ko = 310 pM) and - - -.

FIG. 5 (right). Scatchard plot of the ANS-binding data for the tight binding sites. v is the observed fluorescence of the tight binding sites divided by the maximal fluorescence of the bound ANS. C is the free concentration of ANS. Conditions are as given for Fig. 3.

beam), per unit intensity of the incident beam, per unit concentra- tion, c, of the fluorescent dye. Z(Z) is the intensity of the incident light at anv nosition “1” along the incident beam (given by Beer’s Law). The total fluorescenceintensity,f(X), obtained byintegra- tion, is given by the formula

F(A) = B(Z - e-CT)

where

B ~ m)f(x) co’

y = l (X)L

and e(x) is the usual molar extinction coefficient, c is the molar concentration of dye, lo(X) is the incident intensity, and L is the optical path length of the incident beam in the cuvette.

The experimental data would be a titration of fluorescence at a particular wavelength, as a function of dye concentration. The data are analyzed by considering Z? and y to be empirical parame- ters which are varied to obtain the theoretical curve which most closely fits the data.

The expected relationship between fluorescence and dye concen- tration in the absence of absorption, F’(c), is then given by the limiting form of the above equation as c approaches zero

F’(c) = Brc

Using the values of B and y determined from the data given in Fig. 1, the limiting linear form of the equation, F’(c), is plotted on Fig. 1 as the dashed line. To correct the observed fluorescence at any dye concentration for absorption of incident light, it is only neces- sary to multiply the observed fluorescence intensity by the ratio of the value given by the dashed line to the value given by the solid line for the particular dye concentration.

RESULTS

Binding of S-Aniline-1 -naphthalenesulfonate to Creatine Ki- nase-Addition of creatine kinase to an ANS solution enhances

the dye fluorescence and shifts the emission maximum from

518 to 488 nm (cf. Fig. 2, Trace B) ; while the addition of ADP to a solution containing ANS and creatine kinase results in a de-enhancement and concomitant red shift of this emission max- imum to 495 nm (cf. Fig. 2, Trace C). Compared to the free

dye, the excitation maximum of the enzyme-bound dye is shifted 10 nm to the red.

To determine the dissociation constant of the dye from the enzyme, the fluorescence is titrated as a function of enzyme concentration in the presence of 108 pM ANS. The data shown in Fig. 3 is corrected for the fluorescence of an identical sample of enzyme without dye. Curve A is the fluorescence in t,he absence of ADP and Curve B in the presence of 5.0 mM ADP. The solid line in Curve A is a theoretical saturation curve with K, = 310 pM and a maximal fluorescence of 109.5 units. The solid line in Curve B is a theoretical saturation curve with a K, = 1.25 mM and a maximal fluorescence of 49 units.

To determine the number of binding sites on the enzyme for the dye, the observed fluorescence is titrated as a function of ANS concentration in the presence of 24.8 pM enayme.2 The data is first corrected for the fluorescence due to the free ANS and then corrected for the absorption by the free dye at high concentrations ( > 2 mM, see under “Materials and Methods”). The results are shown in Fig. 4. It is seen that, as well as the tight binding sites observed in the enzyme titration (K, = 310 PM), ancillary weak binding sites are also present. At high dye concentrations (> 2 mM), where the tight binding sites are ap- preciably saturated, the increase of fluorescence with increasing ANS concentration is essentially linear; it is then reasonable to assume that the weak binding sites are well below saturation. With this assumption the observed curve can then be analyzed as the sum of two components, a saturation curve with a binding constant of 310 pM and a linear component. The slope of the linear component is essentially determined by the slope of the observed curve at high dye concentrations, and the binding constant for the tight sites (K D = 310 PM) is determined by the titration with enzyme. Thus, there is only one adjustable pa- rameter in the theoretical fit, the fluorescence of the tight binding sites at saturation. The solid line through the data points shows the theoretical fit, with the resulting linear portion given in dashed lines. Subtraction of the linear portion from the complete curve gives the binding curve of the tight sites. With the use of the titration with the enzyme (cf. Fig. 2) to calibrate the fluorescence in terms of concentration of bound ANS, these data are plotted as a Scatchard plot (see Fig. 5) to obtain the number of binding sites. Within experimental error there is one tight binding site for ANS per subunit (the enzyme is dimeric). It is considered desirable to separate the contributions of the tight and weak sites before plotting the data as a Scatchard plot since it is known (13) that Scat,chard plots involving multiple sites are difficult to unequivocally interpret.

With the use of the dissociation constant determined above, the enhancement of fluorescence on addition of enzyme to ANS, calculated by taking the ratio of the normalized intensities at the wavelengths of maximum emission for enzyme-ANS solutions and ANS solutions (i.e. Curves A and B, respectively, in Fig. 2), is 105. This value of the enhancement is only approximate (&30%) as it ignores the small shift of the excitation band and the fiuorescence due to the weak binding sites. If the quantum yield of ANS in water is assumed to be 0.003 (14), then an en- hancement of 105 would make the quantum yield of the tightly bound ANS 0.31.

Interaction of Substrates with S-Anilino-i-naphthalenesuljonate- binding Site-The addition of a saturating concentration of ADP (5 RIM) both decreases the fluorescence of the bound ANS by a factor of -2 and increases the apparent dissociation con-

2 Enzyme concentration is given in terms of subunit concentra- tion.

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&ant of the dye from the enzyme by a factor of ~4 (Fig. 3, Curve B). This result is inconsistent with the hypothesis that ADP de-enhances the fluorescence by competitively displacing ANS from the enzyme. As will be shown, the dissociation con- stant for ADP under these conditions is determined to be 110 paa. If ADP directly competes with ANS, 5 mM ADP should increase the apparent dissociation constant of ANS by a factor of (1 + ADPIK,,,), which is equal to 46.

ATP produces a de-enhancement similar to ADP, but quan- titative titrations were not performed because of the ATPase activity of the enzyme (15). The effect of ANS on the binding of ATP was investigated by studying the inhibition of the reverse reaction by ANS. ANS (1 IIIM) increases the K, of ATP by 60% (from 470 to 770 pM) and decreases the V,,, by 30% (Fig. 6). This again is inconsistent with a direct competition between ATP and ANS for the same site on the enzyme, as under these conditions ANS should have increased the apparent K, of ATP by a factor of (1 + ANS/K&, 24.

Addition of creatine to the ANS-enzyme complex or the

-I -1.0 0 1.0 2.0 30 40 5.0

VATP

FIG. 6. Inhibition of the forward reaction by ANS with ATP as the variable substrate. Double reciprocal plots of velocity versus ATP concentration in the absence (+-CC) and presence (-O-@-O-) of 1 mM ANS. T = 25”. Experimental conditions are described in the text.

5ot A I ‘\

40 Cl rD t

I /

“q/y, ,,,,, ;o”oo yj ;\oi:lj (, ,,,,,,,, ,,,,,,,,,,,, Kl 100 IO 20 40

A'%., @'I I IO loo 1000

AF AD%ow [,uM]

FIG. 7 (left and center). A, titration of the decrement of S- means of the calculated dissociation constant and the total enzyme anilino-1-naphthalenesulfonate fluorescence, AF, with ADP in the concentration. The difference between the free ADP and the total presence of creatine kinase. The solution contained 8.2 PM crea- ADP was never more than 59&. Conditions were as described in A. tine kinase (in sites), 15 /*M ANS, 0.6% p-mercaptoethanol, 50 mM FIG. 8 (right). The decrement of ANS fluorescence, AF, as a potassium HEPES, pH 8.0, and 50 mM potassium acetate. T = function of ADP concentration in the presence of creatine kinase 23.5”. Excitation wavelength, 360 nm, emission wavelength, 460 and potassium nitrate. The solution contained 7.75 PM creatine nm. -, theoretical saturation curve as a function of the total kinase, 15 pM ANS, 0.6% 8-mercaptoethanol, 50 mM potassium ace- ADP concentration, with a KD of 110 PM. B, the decrement of tate, and 50 pM potassium HEPES, pH 8.0. T = 24’. Excitation &anilino-1-naphthalenesulfonate fluorescence (from A) plotted as wavelength, 360 nm, emission wavelength, 460 nm. -, theo- a Scatchard plot. C is the free ADP concentration and is obtained retical formation function for a cooperative binding process with from the totai ADP concentration by an iterative procedure by KI = 4 pM, KZ = 22 PM, as described in the text.

MgADP-ANS complex has no effect on the fluorescence within experimental error (*3%). The addition of creatine to the MgADP-ANS-enzyme complex in the presence of nitrate (10 mM) decreases the fluorescence by ~_7%. Approximately one- third of this effect can be attributed to the tightening of ADP binding in the simultaneous presence of creatine and nitrate. The addition of Mg* to the ADP-ANS-enzyme complex causes a slight increase in the fluorescence (~10 %), but the addition of Mg* to the AN&enzyme complex also causes a small increase in fluorescence (-5 %) .

The large de-enhancement of fluorescence of the ANS-en- zyme complex upon the addition of ADP is used to determine the dissociation constant of ADP from the ADP-enzyme com- pIex. The data for such a titration, plotted in Fig. 7A as a formation function and in Fig. 7B as a Scatchard-type plot, give KD = 110 PM. The presence of 5 mM magnesium acetate only slightly affects the binding of the nucleotide to the enzyme; the KD is found to be 90 PM. The presence of both saturating con- centrations of creatine (80 mM> and of magnesium acetate (5 mM) further decreases the KD of ADP from the enzyme by only 7% (KD = 84pM).

It should be noted that, although the measured experimental parameter is the decrease in the fluorescence of the ANS-enzyme complex on the binding of ADP, the dissociation constant meas- ured by this method is the dissociation constant of the nucleotide from the nucleotide-enzyme complex, not from the nucleotide- ANS-enzyme complex. This is because the ANS concentration is always well below the dissociation constant of ANS from either the ANS-enzyme complex or the nucleotide-ANS-enzyme com- plex. ’

It should also be pointed out that the observation of a simple saturation curve for substrate binding (see Fig. 7, A and B) does not necessarily imply that the enzyme exists in one unique con- formation. If the enzyme exists as an equilibrium between several different conformations, each with a different association constant for the substrate, then the observed saturation curve will still be a simple saturation curve, with the observed associa- tion constant being the weighted average of the association constants for each of the conformations.

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TABLE I DISCUSSION

Dissociation constant of ADP from various enzyme substrate complexesa

Complex KD

E’ _........................... 110 E + Mg2+................................. 90 E + Mg2+ + creatine. . . . 84 E + creatine + N03. . . . . . . . . . . . . 4.0, 22.2c E + Mg”+ + oreatine + NOa-. . . . 0.50, 11.W IAM-Ed.................................... 72 IAWE + Mg*+. . . 187 MM-E + creatine + NOa-. 126 MM-E + Mg*+ + creatine + NOa-. . . 238

ANS provides a useful probe for measuring conformational changes in creatine kinase which are related to binding of the nucleotide substrate. ANS has one tight binding site per mon- omeric subunit of the dimeric enzyme. Although the dissocia- tion constant is relatively weak (KD = 310 PM) it is similar to that found for ANS-glutamate dehydrogenase complexes (KD = 210 pM) (16) and for 2-toluidinylnaphthalene-6-sulphonate-

chymotrypsin complexes (K, = 200 pM) (17). The tight bind- ing site is characterized by a quantum yield of 0.31, suggesting that the binding site of the dye is hydrophobic (14). In addition to one tight binding site per subunit there are ancillary weak binding sites for ANS. These weak binding sites can only be characterized by a dissociation constant substantially greater than 3 mM and multiple binding sites per subunit.

o Conditions for the titrations are 50 mM potassium acetate, 50 mM potassium HEPES, pH 8.0, 7.75 PM creatine kinase, 15 PM

ANS, 5 mu magnesium acetate (when added), 12 mM potassium nitrate (when added), and 70 mM creatine (when added). !I’ = 24.5”.

) E, creatine kinase. 0 Under these conditions the binding is cooperative. The two

values given are the dissociation constants from one protomer when the second protomer is vacant and occupied, respectively.

d IAM-E, iodoacetamide-labeled creatine kinase.

E$ect of Nitrate on Binding of ADP to Native Enzyme--In the absence of creatine, potassium nitrate (100 mM) has no sig nificant effect on the dissociation constant of ADP or MgADP. The binding curves, plotted as Scatchard-type plots, are com- pletely linear (see Fig. 7B), indicating only one class of binding sites. However, the simultaneous presence of both creatine (70 mM) and nitrate (12 mM) produces significant curvature in the Scatchard-type plots of the data for both ADP and MgADP, suggesting the presence of more than one class of binding sites under these conditions. The data is analyzed with a cooperative model with the assumption that the binding of ADP to one of the nucleotide-binding sites of the dimeric enzyme changes the binding constant of ADP to the remaining site (see “Appendix”). Ki is defined as the dissociation constant of ADP from either of the two equivalent protomers in the dimeric enzyme, assuming that the nucleotide-binding site on the adjacent protomer is vacant. Kz is defined as the dissociation constant of ADP from one protomer, assuming that the nucleotide-binding site on the adjacent protomer is occupied. K1 and Kz were determined by fitting the theoretical curve to the data. Since the linear portion of the Scatchard-type plot at high ADP concentration gives the dissociation constant Ka and the limiting fluorescence change, AF,, fitting the data involves a single adjustable parameter, KI. Because of the nonlinearities, the data are plotted as a formation function, rather than a Scatchard plot (see Fig. 8). Table I gives the values for KI and Kz, determined in this manner for ADP and MgADP.

Effect of Nitrate on Dissociation Constant of ADP to Modijied Enzyme-The enzyme is modified by stoichiometrically labeling the reactive sulfhydryl group of each monomeric unit with iodoacetamide. The dissociation constant of ADP from the modified enzyme (see Table I) is slightly lower than the cor- responding dissociation constant from the native enzyme. The dissociation constant of MgADP from the modified enzyme is, however, about twice as large as to the native enzyme. The addition of nitrate in the presence of creatine causes only a slight weakening of both ADP and MgADP binding.

The fluorescence of the tightly bound ANS is reduced at least a-fold by the addition of either nucleotide substrate (ATP or ADP), but no substantial de-enhancement of fluorescence is found for any other substrate or substrate combination lacking the nucleotide. It is not known if the binding of nucleotide alters the fluorescence of the weak binding sites. In the following discussion only the tight binding sites will be considered.

Although the relatively weak aflinity of the dye for the en- zyme makes it difficult to unequivocally determine whether the nucleotide substrate interacts competitively or noncompetitively with ANS, there is strong evidence for a noncompetitive inter- action. Such evidence stems from the effect of ADP on the dissociation constant of the ANS-enzyme complex and the effect of ANS on the K, of ATP for the forward reaction. If the inter- action were competitive, a large excess of ADP (5 mM) would increase the apparent dissociation constant of ANS from the enzyme by a factor of 46, but only a change of a factor of 4 is observed. Under the same conditions, the fluorescence of the bound ANS should continue to decrease as the ADP concen- tration is raised from 5 to 40 DIM if the interaction is competitive, but no further change is in fact observed. Also if the interaction were competitive, the presence of 1 mM ANS should increase the apparent K, of ATP by a factor of 4, but only a 60% change is found.

It is known from magnetic resonance studies (2) that addition of nucleotide substrates causes conformational changes in the spin-labeled enzyme. The same conclusion can be inferred for the native enzyme from the effect of the nucleotide on the disso- ciation constant of the guanidino substrate (6, 7, 10) and from the effect of the nucleotide on the reactivity of the essential sulfhydryl group (4), the susceptibility of the enzyme to trypsin digestion (18)) the intrinsic protein fluorescence (8)) and the rate of hydrogen exchange (18). It seems reasonable then to pos- tulate that the bound ANS is responding to a conformational change in the enzyme which is induced by the binding of the nucleotide substrate. Since, to within experimental error, no change in ANS fluorescence is observed following addition of creatine to the MgADP-enzyme complex, it is equivocal whether this implies that creatine does not cause a conformational change in the enzyme upon binding, or that the ANS fluorescence is insensitive to any conformational change in the enzyme upon binding, or that the ANS fluorescence is insensitive to any con- formational change which occurs. Small changes in fluoresence upon addition of magnesium to the ANS-enzyme complex occur both in the absence (AF N_ 5%) and in the presence (AF N_ 10%) of ADP. It is thus difficult to ascertain if the effect of mag- nesium is solely on the binding or quantum yield of ANS, or if the ANS is also sensing a conformational change in the enzyme

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induced by the binding of magnesium. Such a conformational The cooperativity is negative in the sense that the binding of change has been inferred from sulfhydryl reactivity studies (10) ADP to the first subunit of the dimeric enzyme weakens the and electron paramagnetic resonance and proton relaxation rate binding constant of ADP to the adjacent subunit. In the ab- studies of the spin-labeled enzyme (2). It is, however, inferred sence of NOa-, cooperativity of nucleotide or guanidino sub- from electron paramagnetic resonance (19) and sulfhydryl &rate binding to creatine kinase is not observed, either kinetically reactivity studies (5) that the simultaneous presence of nitrate, or by direct titrations, so it is reasonable to assume that the divalent metal ion, ADP, and creatine does cause a substantial conformational changes accompanying substrate binding in the conformational change in the enzyme. The fact that the addi- absence of NOa- do not involve substantial subunit interaction. tion of creatine to form this substrate combination only slightly It has been proposed (5), on the basis of the effect of NOa- in alters the fluorescence implies that the ANS fluorescence is decreasing the dissociation constants of MgADP and creatine relatively insensitive to certain types of conformational changes. from abortive quaternary MgADP-creatine-creatine kinase This, in turn, suggests that the conformational changes induced complex, that NOa- acts as an analog of a planar phosphoryl by the binding of different substrates (or substrate analogs, as group, converting the abortive quaternary complex into an in the case of nitrate) are structually distinct and are not merely analog of the transition state of the enzyme. The presence of different gradations in the transition between two conformations cooperativity of nucleotide binding and the presence of the (the active and the inactive conformations) of the enzyme. transition state of the enzyme thus appear to be closely related.

Changes in fluorescence of the enzyme-bound ANS on binding This suggests that in the transition state of the enzyme-sub- of nucleotide may be used to determine the dissociation constant strate complex there is substantial interaction between the two of the nucleotide from the enzyme under various experimental subunits, while in the normal state of the enzyme the subunits conditions. This method is particularly useful in that it does are independent. This would also explain why cooperativity is not require the presence of other components (i.e. a divalent not observed kinetically, since the transition state of the en- metal ion). Thus, multiple equilibria which are typically in- zyme-substrate complex would not be the dominant species volved in such measurements (1) are obviated, and titrations may under these conditions. be analyzed as a simple saturation curve. This also means that the dissociation constant for the nucleotide in the absence of the divalent metal ion or in the presence of any saturating com- bination of substrates may be directly determined. Dissocia- tion constants determined by this method agree very well with those measured under comparable conditions by other methods. Reed et al. (1) found a value of 112 FM for the KD of the ADP- enzyme comp!ex by analysis of proton relaxation rate data, and Price (8) obtained a value of 96 PM by means of quenching of enzyme fluorescence. These values are consistent with the value of 110 pM obtained in this investigation.

Recent work has indicated that certain small anions (NOa-, N01, HCOi-) have a profound effect on the structure of the abortive metal ADP-creatine-creatine kinase complex. These

The dissociation constants of ADP and MgADP from com- plexes involving the inactive carbamylated enzyme are similar to those obtained for the native enzyme. However, addition of nitrate to the abortive quaternary complex involving the earbamylated enzyme neither reduces the dissociation con- stant of ADP or MgADP from the complex nor introduces cooperativity into the ADP or MgADP binding. This evidence is consistent with the hypothesis that the effect of NOa- is to produce, to some extent, the transition state of the enzyme, as the inactive enzyme would not, be expected to be able to proceed to a transition state. The lack of cooperativity with the in- active modified enzyme also strengthens the link between the transition state and the presence of subunit-subunit interaction between the two protomers. It is consistent with the observa-

effects include a substantial change in protection of the essential tion that the conformational change characterized by the gross sulfhydryl group by the abortive substrate complex (5) and a distortion of symmetry around the divalent ion on the addition gross change in the symmetry of the electronic environment of of NO3 to the abortive quaternary complex does not occur if the divalent metal ion, as shown by electron paramagnetic the essential sulfhydryl is blocked with a spin-label or with iodo- resonance spectroscopy (19). Titrations of the protection of acetamide (19). inhibition by iodoacetamide have also indicated that the disso- Nitrate is also found to produce, as well as an apparent inter- ciation constants of MgADP and of creatine from the abortive action between the two nucleotide-binding sites in the dimeric quaternary complex were considerably decreased in the pres- enzyme, an interaction between the nucleotide and the guanidino ence of NO3 (5). The results presented here demonstrate the substrate-binding site (presumably on the same subunit). The tightening of nucleotide binding and also show that NOa has effect of nitrate on the binding of ADP to the enzyme in the little or no effect on the dissociation constant of ADP from any presence of creatine, reported in these studies, is paralleled by enzyme-substrate complex unless creatine is simultaneously the concomitant effect of NOa- on the binding of creatine to the present. enzyme in the presence of MnADP (11). However, if acetate

Not only does nitrate tighten the binding of ADP to the en- is used in place of nitrate, the presence of creatine has no sig- zyme-creatine complex, but it also produces an apparent CO- nificant effect on the binding of ADP or MgADP to the enzyme. operativity in the binding of ADP to the enzyme-creatine com- This is consistent with the observations that acetate does not plex.3 With MgADP the tightening of binding to the enzyme- produce the tightening of binding of MgADP in the presence of creatine complex and the cooperativity are further accentuated. creatine (from kinetic evidence) and that in the presence of

3 It should be emphasized that because the nucleotide-binding acetate the abortive quaternary complex provides no protection

data in the presence of creatine and nitrate cannot be adequately for the essential sulfhydryl group (5). Previous experiments fit by a simple saturation curve but can be very nicely fit with the which have demonstrated a substantial interaction between the cooperative model (see Fig. 8), this does not unequivocally imply that the binding is cooperative under these conditions. Strictly,

nucleotide and the guanidino substrate-binding site have been

it can only be said that the binding data is consistent with the done in the presence of chloride (6)) which has been demonstrated

cooperative model. However, in the following discussion the to act qualitatively in the same manner as nitrate (5). An

term “cooperative” will be used in the operational sense to imply important thermodynamic corollary to the observation that “being consistent with the cooperative model.” creatine does not affect the binding of MgADP to the enzyme

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in the presence of acetate is the reciprocal relationship that MgADP does not affect the binding of creatine to the enzyme in the presence of acetate. Thus, the dissociation constant of creatine from the enzyme-MgADP-creatine complex, measured in the presence of acetate, will be approximately equal to the dissociation constant of the enzyme-creatine complex. There are several reports which suggest that the dissociation constant of the enzyme-creatine complex is relatively large (K, = 50 to 110 mM) (6, 10) and that only in the presence of MgADP does the dissociation constant approach the value for the Michaelis con- stant. It is known, however, from inhibition of the forward reaction at high substrate levels (~100 mM), that guanidino substrates of creatine kinase have ancillary weak binding sites to the enzyme (11). It is quite plausible that the methods used to measure creatine binding (protection of sulfhydryl reactivity and susceptibility to trypsin digestion) are insensitive to the binding of creatine at the active site of the bare enzyme, but are monitoring instead the binding of creatine to the ancillary weak sites.

It is interesting that the divalent metal is not necessary for the cooperativity of ADP binding. However, the fact that the divalent metal ion enhances the cooperativity and tightens the binding of ADP in the presence of NO3 strongly favors the hypothesis that under these conditions the metal ion must have at least one ligand to the protein, and not just be complexed to the nucleotide (20). In the absence of N03, however, the presence of magnesium does not substantially alter the binding constant of ADP to the enzyme, neither in the presence nor in the absence of creatine. This suggests that in the MgADP- enzyme or MgADP-creatine-enzyme complexes the divalent metal is not liganded to the protein, but that it is only in the transition state that the protein forms a ligand to the metal. This suggestion is consistent with the electron paramagnetic resonance data for the manganese complexes (19) which show that the most dramatic change in the electronic symmetry around the divalent metal ion occurs on the addition of nitrate to the abortive quaternary complex to form an analog of the transition State.

Acknowledgments-I am pleased to acknowledge the invaluable assistance of Dr. A. H. Caswell in the preliminary experiment.s and the generous gift of ANS from Dr. R. Hershberg. I also wish to thank Professor Mildred Cohn, Dr. George Reed, and Dr. J. S. Leigh for many helpful discussions, and Dr. George McDonald for help with the computer programs.

APPENDIX

Computer Fitting of Data for Nucleotide Titrations of Fluorescence by Means of Cooperative Model

The dimeric enzyme is assumed to have one nucleotide-binding site per protomer. Ki is defined as the dissociation constant of the nucleotide from one protomer, assuming that the nucleotide- binding site on the adjacent protomer is vacant. Kz is defined as the dissociation constant of the nucleotide from one protomer, assuming that the nucleotide-binding site on the adjacent pro- tomer is occupied. If I$t is the total concentration of dimeric enzyme, then the total concentration of nucleotide-binding sites that are occupied, N(X), for any given free nucleotide con- centration, S, is given by the expression (21, 22)

N(S) = 2EtS(S + Kz)

KIK, + 2K,S + S2 (1)

Equation 1 can be shown to be formally equivalent to the equa-

tion derived from assuming a two-state model for the enzyme (23) if the effective dissociation constants K1 and Kz are written in terms of assumed intrinsic dissociation constants from the two forms of the enzyme and the assumed equilibrium constant between the two forms of the enzyme.

It is assumed that the change m fluorescence of the dye bound to any protomer is affected only by the nucleotide binding to that particular protomer. In obher words, we assume that the interaction between the two protomeric subunits affects only the nucleotide-binding sites and not the fluorescence of the dye- binding sites. This is reasonable, as it is experimentally found that the addition of creatine to the MgADP-ANS-enzyme com- plex in the presence of nitrate (which produces the interaction between the nucleotide-binding sites on different protomers) does not substantially alter the fluorescence of the bound dye ( 15%). Thus, the decrement in fluorescence, AF, in the pres- ence of nucleotide will be given by the expression

AF = AFoS(S + KJ

K,K, + 2SK, + S2 (2)

where AFo is the decrement of fluorescence on saturation with nucleotide. Since S is the concentration of free nucleotide, it is necessary to correct the total nucleotide concentration, St, for the amount bound. From Equation 1 we have

s = St - BETS(S + KS)

KlKz + 2KzS + S2 (3)

For any given values ofKl and Kz Equation 3 can be analytically solved to obtain a value for S. This value for X can then be inserted into Equation 2, and the parameters AFo, K1, and KZ can be varied until the best fit is obtained. However, since it is complicated to give a general analytical solution for Equation 3 which could be easily used in the computer program, it was found more convenient to solve Equation 3 for given values of K1 and Kz by iteration.

The computer program which iterates Equation 3 and then transfers the solution into Equation 2 is used to generate the- oretical curves of AF as a function of total nucleotide concen- tration for given values of the three parameters AFo, KI, and Kt. It is easy to show that in the limit A/K1, A/K2 >> 1, i.e. near saturation, that Equation 2 reduces to the equation

AFoS AF = -

S + Kz (4)

In these experiments the value of Et was such that in this limit the free nucleotide was very nearly equal to the total nucleotide. The data at high nucleotide concentration may then be analyzed using Equation 4 to obtain unequivocal values for AFo and Kz. Thus, the fitting of the computer-generated curves to the experi- mental data points over the complete range of nucleotide con- centration involves only one adjustable parameter, KI.

1.

2.

3.

4.

5.

6.

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Alan C. McLaughlinEVIDENCE FOR COOPERATIVITY OF NUCLEOTIDE BINDING

The Interaction of 8-Anilino-1-naphthalenesulfonate with Creatine Kinase:

1974, 249:1445-1452.J. Biol. Chem. 

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