NMR of Enzyme-bound Substrates of Rabbit Muscle Creatine Kinase

THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256, No. 4, Issue of February 25. pp. 1716-1721, 1981 Printed in U.S.A.

31P NMR of Enzyme-bound Substratesof Rabbit Muscle Creatine Kinase EQUILIBRIUM CONSTANTS, INTERCONVERSION RATES, AND NMR PARAMETERS OF ENZYME-BOUND COMPLEXES* (Received for publication, August 22, 1980)

B. D. Nageswara Raotand Mildred Cohn From the Departmentof Biochemistry and Biophysics,. University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 ”

The reaction catalyzed by rabbit muscle creatine ki- sively with the interconversion step E . SI.S2e E . PI.P2 of nase ATP + creatine + +4DP + P-creatine has been the reaction. The present paper reports a 31PNMR study of investigated by 31PNMR. At pH 8.0 and 4OC, the equi- enzyme-bound binary, ternary,quaternary and transition librium constant of the overall reaction [Pl][Pz]/[Si] state analog complexes of rabbit muscle creatine kinase and [&I is found to be 0.08, while that for the interconver- of the equilibrium mixture of enzyme-bound substrates and sion step between enzyme-bound substrates and prod- products. Creatine kinase catalyzes the reaction ATP + creucts [E-PI*P~]/[E*&-S~] is estimated to be -1; the atine e ADP + €”creatine with M e serving as theobligatory latter value is the same for all other kinases investidivalent metal ion (7). of enzyme-bound gated. The rate of interconversion Similarities between creatine kinase and the invertebrate substrates and products is -90 s” and is not the rate- enzyme arginine kinase have been noted previously (8) and limiting step of the overall reaction. Of the phosphate groups in enzyme complexes of reactants or products, have led to the suggestion that both enzymes catalyze the the ”P chemical shifts of b-P(ADP) and @-P(MgADP) phosphoryl transfer to the guanidino nitrogen by a similar change by -2 ppm downfield while all others change mechanism. The possible homology between these two enby t0.8 ppm. In the transition state analog complexes zymes was probed further by EPR and PRR’ measurements (9, 10) using Mn2+ as the activating cation in a variety of E*MgADP*NOs-*creatineandE-MgADP*HCOO-=creatine, the p-P(MgADP) signal shows a substantial up- complexes including transition state analog complexes. The field shift in the direction of the b-P(MgATP) resonance. PRR measurements show that as the binding sites of the ”he pattern of chemical shifts and line shapes of nu- different substrates at the active site are progressively occucleotide complexesof creatine kinase parallel those forpied, the active site becomes less accessible to the solvent, the corresponding complexes of arginine kinase, indi- while the Mn2+EPR measurements on the same complexes cating structural and/or conformational similarity of show that concomitant with the inaccessibiLity of the active the phosphate chainsof nucleotides bound to the two site to the solvent, increasing asymmetry and immobilization enzymes. However, a difference in activesites is indi- of the metal ligands result. These studies thus indicate a cated by the pH independence (pH 6.0 to 9.0) of the similarity in the topography of the active sites in the two chemical shift of the 8-P of MgADP bound to creatine enzymes. The enzyme-bound substrate complexes of arginine kinase, whereas with arginine kinase this resonance kinase have been studied in considerable detail by 31PNMR showed a pK, 7.5. (1, 2). The present paper presents a comparison of the ’”P NMR parameters of the different enzyme-bound substrate and transition state analog complexes for the two enzymes. Several phosphoryl transfer enzymes with ATP as substrate Since the 31Pchemical shifts signify electronic shielding of the nuclei contained in the moieties in the vicinity of the point have been recently studied by 31PNMR experiments with 3LP enzyme concentrations in excess of the substrates(1-6). These of enzymatic cleavage, a comparison of these parameters is experiments yield information on the changes in the NMR expected to shed light on the question of whether the topographical similarity of the active sites of these enzymes (9,lO) parameters, z.e. chemical shifts and spin-spin coupling constants of the different ”P-containing substrates in their var- is accompanied by similar electronic shielding effects associious enzyme-bound complexes which in turn reflect changes ated with the nuclei in the substrates. It may be noted that in conformation or the environment (or both)of the moieties the ”P chemical shift data for the enzyme-bound substrates containing 31P nuclei.In addition, it is possible to determine of different kinases, published earlier, do not reveal systematic in a straightforward manner from the 31Pspectra of equilib- changes that might indicate gross similarities in the environrium mixtures of enzyme-bound substrates and products, the ment or conformation (or both)of the phosphate chain of the equilibrium constant and exchange rates associated exclu- bound nucleotides (5, 11). A comparison of the NMR parameters of nucleotides bound to arginine kinase and creatine * This work was supported by National Institutes of Health Grant kinase should reveal whether these parameters for the two GM12446 and National Science Foundation Grant PCM 78-13633. guanidino kinases that areconsidered homologous to asignifThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby icant degree among the general class of kinases d i e r or not.

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marked “adoertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $Part of the work presented in this paper was done during the tenure of a Research Fellowship of the Muscular Dystrophy Association ofAmerica,New York, NewYork 10019. Present address, Department of Physics, Indiana University-Purdue University at Indianapolis (IUPUI), Indianapolis, IN 46205.

EXPERIMENTAL PROCEDURF$

Materials-Creatine

kinase was isolated and purified from rabbit

’ The abbreviations used are: PRR, longitudinal relaxation rates of water protons; Hepes, 4-(2-hydroxyethyl)-l-piperazineethane sulfonic acid; ADPPS, adenosine 5”0-(2-thlodiphosphate).

1716

1717

31PNMR Study of Muscle Creatine Kinase Reaction muscle by method B of Kuby et al. (12). The specific activities ofthe preparations were 50-60 IU in the coupled assay with pyruvate kinase and lactate dehydrogenase (13). Protein concentrations were determined spectrophotometrically using an extinction coefficient of 8.96 a t 280 nm for a 1%solution of creatine kinase and a M,= 81,000 (14). The enzyme is normally stored at concentrations of -35 m g / d in 50 nm glycine buffer, pH 9.0. For the NMR experiments the enzyme is extensively dialyzed against the desired buffer (Hepes was used in most of the experiments) and concentrated using B-15 Amicon concentrators with 5 ml chamber volume to 200 mg/ml corresponding to 5 mM catalytic siteconcentration. The nucleotide concentrations were measured spectrophotometrically at 260 nm using a millimolar extinction coefficient of 15.4. ATP, ADP, creatine, P-enolpyruvate, and NADH were purchased from Sigma. Lactate dehydrogenase was obtained from Boehringer Mannheim GmbH, and pyruvate kinase was kindly supplied by D. Ash of the University of Pennsylvania. All other compounds were of analytical reagent grade. D2O was obtained from Thompson Packard Co. and redistilled in vacuum before use. ATP and ADP were extracted with 8-hydroxyquinoline to reduce metal ion contamination (1). NMR Measurements-The 31PNMR spectra were obtained on a JEOL PS-100 NMR spectrometer operating at 40.3 MHz. Sample tubes (10 mm outer diameter) were used with a Vortex suppressor. Sample volumes ranged from 1.0 to 1.5 ml containing a t least 10% D20 for a field frequency lock. Temperature was controlled using a variable temperature controller (JNM-VT-3B). The spectra were obtained in the Fourier transform mode using a pulse width -10 p (flip angle -45"), and signal averaging was performed using a Texas Instruments EC-100 computer. Broad band proton decoupling was used in obtaining some of the spectra in which the measurements of the areas enclosed by the signals were not important. All chemical shifts were expressed with reference to 85% H3P04 as the external standard; positive values of the chemical shifts represent signals upfield from the standard. pH values are direct readings on a Radiometer pH meter. Kinetic Measurements-The velocity of the overall creatine kinase reaction was determined spectrophotometrically under conditions similar to the NMR experiments, ie. T = 4OC, with a coupled assay system. The solution contained 150 mM potassium-Hepes, pH 7.8, 50 mM KCl, 6 mM magnesium acetate, 40 mM creatine, 4 mM ATP, 2 mM P-enolpyruvate, 0.2 mM NADH, 100 p g / d of pyruvate kinase, and 50 pg/ml of lactate dehydrogenase. The reaction was initiated by the addition of creatine kinase and A,,, was measured on a Cary 219 spectrophotometer. RESULTS

h

a-P IADPI

II

a-P IATPI

/I

~

-4

0-P

'

0

l

'

4

l

~

8

l

I2

'

l

16

~

20

l

'

l

'

l

CHEMICAL SHIFT (ppm) FIG. 1. 31P N M R spectra of equilibrium mixtures of creatine kinase, pH = 7.8, T = 4°C.The resonances of the different phosphate groups are labeled. A, catalytic enzyme concentration, initial concentrations: ATP, 15.1 mM;MgC12,18.7mM; creatine, 15.2mM; potassium-Hepes, 105 mM; enzyme, 150 pg. B, excess enzymeconcentration, initial concentrations: enzyme (sites), 4.2 mM; ATP, 3.9mM; Mg(CH&OO),, 5.1mM; creatine, 3.8 mM; potassium-Hepes 150mM. C, EDTA added to sample B to a frnal concentration of 14 mM. NMR parameters: A, 400 scans; bandwidth, 2 kHz; memory size, 16,000, line broadening, 0.3 Hz; pulse repetition time, 60 s. B, 200 scans; bandwidth, 2 kHz; memory size, 8,000, line broadening, 1.5Hz; pulse repetition time, 4.5 s. C, same as B.

Equilibrium Mixtures of the Overall Reaction and of Enzyme-bound Substrates and Products-A 31P NMR spectrum of an equilibrium mixture of the overall reaction catalyzed by plexes; the resulting spectrum is shown in Fig. 1C. Removal creatine kinase at usual catalytic enzyme concentrations at pH 8.0 and T = 4°C is shown in Fig. 1A. The resonances of divalent cation does not greatly alter the dissociation concorresponding to the six phosphate groups, a-P, p-P, and y-P stants of the nucleotides tothe enzyme(15) so that the of ATP, a-P and p-P in ADP, and P in P-creatine are labeled. percentage of enzyme-bound species is similar to that in the sample of Fig. 1B.The various resonances in the absence of The equilibrium constant of the reaction determined from the reaction (Fig. 1 0 are significantly sharper than in Fig. 13, areas under the different resonances (which are proportional to the respective concentrations) is K,, = [PI][P&[ SI][Sz] and some of the fine structure due to spin-spin coupling is = [MgADP][P-creatine]/[MgATP][creatine] = 0.08 f 0.02. discernible in Fig. 1C. Hence the characteristic line shapes in While this value is in general agreement with measurements Fig. 13 are due not to binding of the substratesto the enzyme reported in the literature (7), a detailed comparison is not but to theinterconversion of the substrates and products on attempted because of the variability in the conditions used in the surface of the enzyme, whichproduces chemical exchange different experiments. effects on the 31Presonance line shapes. A 31P NMR spectrum of the equilibrium mixture of the The equilibrium constant of the interconversion step E SI creatine kinase reaction set up with enzyme concentrations in SZe E. P I .P 2 and the associated rates of interconversion may excess of the substrates and products is shown in Fig. 1B. be derived by analyzing the spectrum in Fig. 1B in an analoExcept for the presence of a substantial signal from Pi at 2 gous manner to that of arginine kinase (1).However, two ppm, the spectrum is similar to that obtained for the equilib- complicatingfactors occur inthe creatine kinase experiments: rium mixture of enzyme-bound substrates and products of the (i) the irreversible accumulation ofPi (Fig. 1B) due to the arginine kinase reaction (1).The line shapes in the spectral ATPase activity inherent to the enzyme (16) and in part to a regions 4-7 ppm, 10-12 ppm, and 18-20 ppm can readily be contaminating activity, and (ii) the magnitude of the binding demonstrated to be due to chemical exchange effects on the constants of the substrates and products to creatine kinase are 31 P resonances arising from interconversion of enzyme-bound such (7) that appreciable concentrations of free substrates are substrates and products by stopping the reaction with the likely to be present in the sample of Fig. 13. Both factors addition of EDTA to sequester M e from the reaction com- adversely influence the accuracy of the determination of the

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31PNMR Study of Muscle Creatine Kinase Reaction

1718

parameters of the interconversion step of creatine kinase compared to thatfor arginine kinase. Integration of the areas under resonance peaks in Fig. 1B indicates that the concentrations of enzyme-bound MgATP, MgADP, and P-creatine are approximately the same. In view of the factorsmentioned above, it may be concluded that the equilibrium constant for enzyme-bound substrates and products: K:, = [E.MgADP. P-creatine]/[ E MgATP. creatine] = 1. The accuracy of this value is approximately f 20-30%. As mentioned earlier, it is possible to deduce the rates of interconversion of enzyme-boundreactants and products from the line shapes in Fig. 1B. An approximate procedure for this, used earlier in the case of arginine kinase, is based on considering the excess line width of the ,f3-P(MgATP)resonance in Fig. 1B over that for one of the lines in the multiplet in Fig. 1C to be equal to the rate of phosphoryl transfer on the enzyme. Equating the rate with excess line width of the p-P resonance of MgATP was justified in view of the fact that this P is in slow exchange with that of p-P of MgADP by virtue of the large chemical shift between their resonances (1,17).Such a procedure implicitly ignores the effects of spin-spin interactions on the line shape. A computer program was recently developed onthe basis of a density-matrix theory of chemical exchange effects that allows computer simulation of spectra without any restriction on exchange rates or strength of spinspin couplings (18). Using this program, a value of 90 s-' was determined for the interconversion rates of creatine kinase (since K' z 1, the forward and reverse rates arenearly equal). The overall rate of the creatine kinase reaction at pH = 7.8 and T = 4°C is found to be 5.0 s-'. Thus, the interconversion of enzyme-bound substrates and products is not the ratelimiting step of the creatine kinase reaction. The computer-calculated spectrum is shown in Fig. 2 together with the experimental spectrum (taken from Fig. 1B). In obtaining the best fit for the calculated spectrum with respect to theobserved spectrum, the chemical shifts of some of the resonances that are not resolved in the experiment (e.g. the resonances of y-P(MgATP), p-P(MgADP), and P of Pcreatine in Fig. 1B) could be estimated. The chemical shifts used in the computer calculation are listed in Table I. Chemical Shifts of Enzyme-bound Nucleotide Complexes-The 31P NMR spectra of ATP and MgATP free and bound to creatine kinase are compared in Fig.3. The chemical shifts and spin-spin coupling constants measured from these spectra are listed in Table I. The chemical shifts of ATP and MgATP are affected little, c 0 . 5 ppm, and the 31P-3'Pspin-

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spin coupling constants remain virtually unaltered upon binding to theenzyme. The line widths of the resonances from the enzyme-bound complexes in Fig. 3 are broader than for the corresponding complexes of arginine kinase (see Fig. 1;Ref. 2) which has approximately half the molecular weightof creatine kinase. The 31PNMR spectra of ADP and MgADP free and bound to creatine kinase are shown in Fig. 4. The chemical shifts and spin-spin coupling constants (when measurable) from these spectra are included in Table 1. Just as in the case of arginine kinase (i) thep-P resonances of ADP and MgADP are shifted downfield by 2 ppm upon bindingto creatine kinase, (ii) the a-P resonances of ADP and MgADP showsmall upfield shifts, and (iii) resonances of bound ADP and MgADP are significantly broader than those of bound ATP and MgATP (2). p H Dependence of the 31PChemical Shifts of Some Enzyme-bound Nucleotide Resonances-The chemical shift of p-P of MgADP bound to arginine kinase showed a variation with pH with an apparent pK, of 7.5 & 0.3, whereas the pK, of p-P(MgADP) free in solution is 5.9 (2). Furthermore, the signal from the enzyme-bound complexshifted upfield as the pH was increased, a direction in reverse to that for the p-P signal of free MgADP. In viewof the suggested homology between arginine and creatine kinases and the fact that P-

-

C H E N l C A L SHlFl

IPPN)

FIG. 2. Comparison of experimental and computer-calculated "P NMR spectra for the equilibrium mixture of enzymebound substrates and products of creatine kinase. A, experimental spectrum, same as Fig. 1B. B, computer-calculatedspectrum using the spectral parameters listed in Table I, and an interconversion rate of 90 s-' in either direction.

TABLEI 31Pchemical shifts (8) in parts permillion from 85% HPO4 as external reference, and spin-spin coupling constants (J)in Hertz for free and enzyme-bound complexes of creatine kinase substrates Temperatures were either 4-6°C or 12-15°C. The parameters were independent of temperature inthis range. Typical concentration ranges were: freesubstrates 10 to 15 mM, bound substrates 2 to 4 mM, and enzyme4 to 5 mM. The solutions were bufferedin potassium-Hepes, 50 mM to 150 mM, pH 7.8. s J Complex

ATP a-P

ADP

P-P

v-p

10.6 19.2 21.0 19.0

6.0 5.5 5.7 5.4

a-P

P-creatine

ADP

ATP"

P-p HZ

PPm

21.5 6.3 Free substrates Free substrates + Mg E ATP E * MgATP E * ADP E-MgADP Equilibrium mixture + EDTA 21.2 Eauilibrium mixture (linewidth

10.9 10.6 10.9 10.9

3.8 10.9 11.2

'Values obtained from computer calculation.

19.0

5.8 5.8' (10) (55)

10.2

6.0

10.2 11.0 20.0 10.0 10.7 (20)

4.8 19.0 4.6 3.8'

3.1 3.1

3.1 3.5O (55)

19.5 15.5 19.0 16.0

22.0 18.2

15.0

18.0

31PNMR Study of Muscle Creatine Kinase Reaction

4 m 1 - T : - : 6

CHEMICAL SHIFT

-F"2212 Ipprn!

FIG.3. "P NMR spectra of ATP and MgATP complexes, free and bound to creatine kinase, pH 7.8 and T = 4°C. Signals of a-P, p-P, and y-P of ATP are labeled. A, free ATP, 19.5 mM; B, ATP,

1719

erable broadening of the P-P(MgADP)resonance. The spectrum of the same sampleat 31°C is shown in Fig. 5B in which the two signals coalesce into a single resonance. It appears, therefore, that there are two MgADP sites of nearly equal populations and unequal chemicalshifts such that thesignals are in slow exchange at 4°C and the exchange rate is sufficiently enhancedat 31°C to produce a coalescence of the two resonances at anintermediate position. Addition of Nos- to form the E -MgADPsNO~-.creatine complex produces a significantupfield shift of the broad doubled p-P resonance (at 4°C) as shown in Fig. 5C. This upfield shift is in accord with the results on arginine kinase and demonstrates the formation of a transition state analog complex in which the electronic shielding of P-P(MgADP) assumes a value leading toward the chemical shift of enzymebound P-P(MgATP).Once again, as in the case of the arginine kinase system(2), by recording spectra of complexesin which one or more components is absent, it is shown that for the upfield shift to occur and therefore for the formation of the transition state analog complex, the presence of all the components of the quinary complex is mandatory. In particular the spectrum Fig. 5D shows that when EDTA is added to remove Mg2+ fromthe transition state analog (Fig. 5C), the P-P(MgADP) resonance is shifted back approximately to its position in the E .ADP complex. Note further that in the

4.1 mhi; enzyme (sites), 4.5 mM; potassium-Hepes, 150 mM; C, ATP, 15.1 mM; MgCh, 17.1 mM; potassium-Hepes 50 mM. D, magnesium acetate added to sample B to a final concentration of 5.5 m ~NMR . parameters: memory size 8,000.A, 100 scans; bandwidth, 1 kHz; line broadening, 0.3 Hz; pulse repetition time, 50 s. B, 2000 scans; bandwidth, 2 kHz; line broadening, 1.5 Hz; pulse repetition time, 2.2 s. C, same as A except pulse repetition time, 10 s; proton noise decoupling with a bandwidth of 10 kHz was used to eliminate spin-spin coupling between a-P and CS protons. 0, same as B.

P- P

a-P

m P(MgADP) bound to creatine kinase showsa downfield shift very similarto that with arginine kinase,the pH-dependence of this signal was measured in the pHrange 6.0 to 9.0. However, no dependence of the chemical shift on pH was measurable in this pH range withinthe experimental error of about f0.2 ppm for the /3-P resonance of MgADP bound to creatine kinase. With MgATP bound to creatine kinase, the /3-P resonance also did not shift with pH. It has been demonstrated recently that thiophosphate analogs of the nucleotides exhibit a much larger range of chemical shift change withpHin the range appropriate for titration (19). The chemical shift of/3-Pof creatine kinase-bound MgADPPS was, therefore, measured in the pH range 6.0 to 9.0 and was also found to be unchanged. Transition State Analog Complexes of Creatine Kinase with Nitrate and Formate Ions-In the 31PNMR studies with arginine kinase, it was demonstrated that the chemical shift of /3-Pof enzyme-bound MgADP monitors the formation of the transition state analog complex E MgADP NO3- Arg by exhibiting an upfieldchemical shift of /3-P in this complex with respect to E -MgADP and E .MgADP Arg (2). The formation of such a transition state analog complex for creatine kinase was proposed by Milner-White and Watts (20). For creatine kinase considerable evidencewas provided for the formation of such complexes with Nos-,HCOO-, and SCN- both by PRR and EPR studies (9, 10). Results of the 31PNMR experiments on creatine kinase-ADP complexesto monitor the effects ofNOS- and HCOO- similar to those performed on arginine kinaseare shown in Fig.5. The 31P NMR spectrum of the E .MgADP creatine complex shown in Fig. 5A was obtained at 4°C. Note that the addition of creatine caused an apparent doubling and consid-

-

.

e

-

-

C LL-.

r i l ' l ' 2 4 6

z

l

8

'

l

IO

'

I

I!?

lb

CHEMICAL SHIFT ( ppm) FIG.4. "P NMR spectra of ADP and MgADP complexes,free and bound to creatine kinase, pH 7.8, and T = 4°C. Signals of a-P and p-P of ADP are labeled. A, free ADP, 15 m ~ potassium; Hepes, 150 mhi. B, ADP, 3.8 mM; enzyme (sites), 4.5 mM; potassiumHepes, 150 mM. C, MgClt added to sample A, fiial concentration 20 mM. D, magnesium acetate added to sample B, final concentration 5.5 m ~ NMR . parameters: memory size, 8,000.A, 100 scans; bandwidth, 1 kHz; line broadening, 0.3 Hz; pulse repetition time, 8.0 s. B, 200 scans; bandwidth, 2 kHz; line broadening, 1.5 Hz; pulse repetition time, 2.2 s; proton noise decoupling with a bandwidth of 10 kHz was used to eliminate spin-spin coupling between a-P and CS, protons. C, same as A. D, same as B.

1720

31P NMR Studyof Muscle Creatine Kinase Reaction a- P

A

and Knowles (23) suggested that the equilibrium constant of the interconversion step approaches unity for an optimally evolved enzyme. As a corollary to this, it follows that the interconversion step would not be the rate-limiting step of the reaction for a perfect enzyme. On the basis of this criterion, the kinases studied could be considered to have attained a high degree of maturity in their evolution. In previous determinations of interconversion rates from exchange rates of enzyme-bound substrates and products from 31PNMR, the increasein the linewidth of ,&P(MgATP) due to the interconversion process was equated to the reciprocal lifetime of reaction complexes containing ATP, i.e. the rateof phosphoryl transfer on the enzyme (1, 17). This procedure overestimates the exchange rate because the spin-spin coupling effects are ignored (18). In the presentwork a computer program based on a density-matrix theory of exchange was used tosimulatetheexperimentalspectrum fordifferent C exchange rates and the calculated spectrum that agrees best with the experiment was chosen to determine the interconversion rate of 90 s-I for the creatinekinase reaction (18). Binding of ATP or MgATP to creatinekinase causes negligible chemical shift changes for the three "P signalsof ATP. This was the case for all the kinases studied except for /IP(MgATP) bound to adenylatekinase (11, 17). Of all the six phosphate groups among the substrates and productsof cre/Iatine kinase,only the chemical shiftsP-P(ADP)and P(MgADP) undergo largechange uponbinding to theenzyme. The magnitudes of these changes (-2 ppm downfield)for creatine kinase are similar to the corresponding values obtained in 31PNMRstudies withargininekinase reported previously (2). Among the 31PNMR measurements made on nucleotide and metal-nucleotides bound to different kinases r ' l ' l ' l ' l thus far, only the chemical shifts and line width patterns of 0 4 8 12 16 these complexes of arginine kinase and creatinekinase resemble each other closely. It may be noted that the p-P(ADP) CHEMICAL SHIFT ( p p m ) resonance was not shifted when ADP is bound to pyruvate FIG. 5. "P NMR spectra of dead-end and transition state analogs in complexes of creatine kinase containingMgADP, kinase (5) andwas shifted upfield when boundto 3-P-glycerate pH 7.8. A, E.MgADP.creatine at 4°C: enzyme (sites),4.8 mM; ADP, kinase (4). Thechemical shifts may possibly be related to the of different kinases imposed 4.2 mM; magnesium acetate, 6.9 mM; creatine, (saturated solution), distinctions among the active sites > 60 m);potassium-Hepes, 150mM. B, sample A at 31'C. C, E . by the specificity for the second substrate, the phosphoryl MgADP.creatine.NO3- at 4OC: enzyme (sites),4.2 mM; ADP, 3.6 mM; acceptor. Arguing along these lines, the close similarity in the magnesium acetate, 5.1 mM; creatine, 4.8 mM; NaN03, 6.8 mM. 0, chemical shifts of nucleotides and metal-nucleotides bound to sample C with added EDTA, final concentration 6.8 mM. E, sample arginine kinase and creatinekinase may be related to the fact B with added sodium formate, final concentration, 60 mM; T = 25'C. phosphoryl transfer to a NMR parameters: bandwidth,2 kHz; memory size, 8,000, line broad- that these twoenzymescatalyze ening, 1.5 Hz.A, 300 scans; pulse repetition time, 4 s. B,same as A . C, guanidino nitrogen. 4000 scans; pulse repetition time, 2.2 s. 0,1860 scans; pulse repetition Further evidence for the possible homology between argirate, 2.2 s. E, 4450 scans; pulse repetition time, 4.0 s. nine kinase and creatine kinase is revealed by the 31PNMR results on transition state analog complexes. The pabsence of Mg2+, the broadening and doubling of the p- P(MgADP) resonance from E. MgADP Nos-. creatine comP(MgADP) resonance exhibited in Figs. 5, A and C, are no plex shows a significant upfield shift in the direction of the longer observed. Fig. 5 E shows the spectrumof the transition p-P(MgATP) resonance, and for this shift to occur, the presstate analog complex formed by adding formate rather than ence of all the components of this complex is mandatory. nitrate, E .MgADP HCOO- creatine, which shows asimilar Exactly the same result was obtained for the EeMgADP. upfield shift of p-P resonance as in Fig. 5C; the resonance has N03-.arginine in the studies with arginine kinase (2). The a single peak since the spectrumwas obtained a t 25°C. shift of the P-P(MgADP) resonancein the direction of the pP(MgATP) is cogent evidencefor the formationof a transition DISCUSSION state analog complex since the electronic shielding of the pIn spite of some complications in the experiments arising P(MgADP) resonances alters in the direction of its value in from the ATPase activity of creatine kinase and from the the final product, uiz. P-P(MgATP). The 31PNMR spectraof E . MgADP creatine and the tranunavoidable presence of some free substrates and products under the experimental conditions used, the results that the sition state analog complex show another feature thesignifiequilibrium constant of enzyme-bound reactants and products cance of which is somewhat unclear. The p-P(MgADP)resonance in both these complexes shows two broad peaks of is approximately equal to unity, and that the interconversion nearly equalintensity(see Fig. 5) at low temperatures step of the central complexes is not the rate-limiting step conform to what seems tobe a general featurecommon to all (-4°C). At temperatures above 25°C these two peaks merge reaction (22) into a single peak at an average position possibly due to kinases (5,6, 11,21), and the one adenyl transfer studied thus far. On the basis of kinetic arguments, Albery chemical exchange. This observation was independently ree

-

-

31PNMR Study

of Muscle Creatine Kinase Reaction

ported earlier by Milner-White and Rycroft (24). Creatine kinase is a dimer, and possible dissimilarities between two MgADP binding sites on this enzyme were noted previously (25, 26). The results maybe interpreted as a distinction between the binding sites on the two subunits and possibly some intersubunit interaction (24). However, that may not be the case since a similar line shape of the 8-P(MgADP) resonance was noted in the experiments with arginine kinase which is' a monomer (2). At any rate, these spectra reveal some conformational heterogeneity and immobilization inthe vicinity of the active site even in the absence of the planar anion suitable for the formation of the transition state analog complex. It is interesting to note that the characteristic line shape described above occurs only in the presence of Mg2+.It might be useful to explore this point further using the effect of paramagnetic Mn2+in place of M$+ on the spin relaxation rates of 31P or other nuclei in the vicinity of the active site and comparing 31Presonances of diastereomers of ADPPS, @17 or 0 ADP/3170, Although the nucleotide chemical shifts of the various binary, ternary, quaternary, and .transition state analog complexes of arginine kinase and creatine kinase show significant resemblance, the similarity does not obtain in the pH-dependence of the @-P resonance of the E .MgADP complex. For MgADP bound to arginine kinase this resonance shows a pK, of 1.5 units higher than free MgADP, whereas the chemical shift was independent of pH in the range 6.0 to 9.0 for the complex with creatine kinase. The chemical shifts of the 31P resonances from MgADPPS' boundto creatine kinase are also unchanged in the pH range 6.0 to 9.0. Thus, while there appears to be a broad similarity in the structure or conformation (or both) of the phosphate chain of the nucleotides in the complexes bound to arginine kinase and creatine kinase (to theextent that these features arereflected by the chemical shifts), theenvironments of these moieties seem to be signifcantly different as indicated by their protonation behavior. The pH dependence of the chemical shift of MgADPPS bound to arginine kinase has been determined (C. L. Lerman and M. Cohn, unpublished) and the pK,was found to be below pH 6.5, and furthermore, the change with pH is in the opposite sense from that of the MgADP complex. Consequently, the original suggestion that thepK. of the chemical shift was due to an ionization of an amino acid residue at theactive site is an oversimplification.The problem is being further investigated.

1721 REFERENCES

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