THE JOURNAL OF BIOLOGICAL CHEMISTRYVol. 241, No. 13, Issue of July 10, pp. 3104-3115, 1966

Printed in U.S.A.

Magnetic Resonance Investigations of the Metal ComplexesFormed in the Manganese-activated Creatine Kinase Reaction*

(Received for publication, January 10, 196

W. J. O'SULLIVAN AND MILDRED COHN§

From the Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, Pennsovania 19104

SUMMARY

Stability constants for the manganese complexes of variouscomponents of the creatine kinase reaction, including possiblebuffers, have been determined by electron paramagneticresonance (EPR). EPR and proton relaxation rate (PRR)studies indicated that such interaction as occurred betweenmanganese and creatine kinase was nonspecific and rela-tively weak, unlike the interaction between manganese andpyruvate kinase (12). The maximum enhancement of thePRR induced by binding of manganese to the enzyme wasestimated to be 1.5, and this was considerably reduced athigh ionic strength.

Enhancements of the PRR of 19.4 at 25 mc per sec for theternary complex, creatine kinase-manganese-adenosinediphosphate, at pH 8.0 and 240 and of 9.8 at 25 mc per secfor creatine kinase-manganese-ATP at pH 7.5 and 24° wereobtained. Values of the dissociation constants of the metalnucleotides from the ternary complexes were obtained withthe use of PRR data alone or by a combination of PRR andEPR data. It was also possible to obtain estimates of thedissociation constants of the free nucleotides from theirbinary complexes with the enzyme.

The values for the dissociation constants were in satis-factory agreement with those derived from a limited studyof the activation of creatine kinase by manganese, consistentwith the enzymatic reaction obeying equilibrium kinetics.The values agreed with the results obtained by J. F. Morrisonand M. L. Uhr (personal communication) for the manganese-activated reaction and were similar to the dissociation con-stants obtained previously from kinetic and thermodynamicdata with magnesium as the activating ion. It was concludedthat the equilibria measured by magnetic resonance methodscorresponded to the equilibria among the kinetically activespecies.

Pyrophosphate and tripolyphosphate also formed ternarycomplexes with low values of et, 2.0 and 2.5, respectively.No evidence could be found for the formation of ternary

* This work was supported in part by United States PublicHealth Service Grant GM 12446 and National Science FoundationGrant G 23384.

$ This investigation was initiated during the tenure of anAustralian National University Travelling Scholarship and com-pleted during the tenure of a Research Fellowship of the AmericanHeart Association.

§ This work was carried out during the tenure of a CareerInvestigatorship of the American Heart Association.

complexes with AMP, inorganic phosphate, phosphocreatin%or creatine. However, evidence for the formation of theabortive quaternary complex, manganese-ADP-creatinokinase-creatine, was obtained. The enhancement, , the quaternary complex was less than et under the conditionsmeasured. The difference between et and was found btincrease as the temperature decreased, since the PRR dthe ternary complex had a negative temperature coefficieniand the PRR of the quaternary complex had a positive tealperature coefficient.

From the magnitude of the relaxation rates of the ternaqand quaternary complexes and the magnitude and signs dthe temperature coefficients, approximate calculations indcated that the large enhancement of PRR in these complexesresults predominantly from the fact that the rotational motionof water in the coordination sphere of the metal ion is greatblhindered in both complexes. On the other hand, the decrease in the magnitude of the PRR between the ternaland quaternary complexes appears to be caused by a decreasein the rate of water exchange between the metal coordinationsphere and the solvent for the quaternary complex.

The role of the essential divalent metal ion in the reactioncatalyzed by creatine kinase (adenosine triphosphate:creatiDphosphotransferase, EC 2.7.3.2) has been the subject of a number of studies in recent years. The emphasis has been on kinetstudies, with magnesium as the activating ion, although sol!thermodynamic studies of enzyme-substrate interactions halalso been carried out (1-3). An analytical survey (1) of thliterature led to the conclusion that the probable primary fulltion of the metal ion was to form an active metal-ADP or metlATP substrate and that direct interaction of the metal ion wAithe active site of the enzyme was not likely to contribute greatto the reaction.

More direct information was obtained from experiments witmanganous ion as the activating ion. The presence of the pamagnetic ion made the system amenable to magnetic resonasltechniques; the results of Cohn and Leigh (4) on the proton"taxation rate of the nuclear magnetic resonance of water in tcreatine kinase system provided direct evidence for the formatiOof a ternary enzyme-manganese-substrate complex. It was ahconcluded that the enzyme reacted with a manganese-nucleo

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,0 ,plex rather than with free manganous ion. The EPRt spec-Wm of the metal-ADP complex was unaltered on binding to theeomyne, suggesting that no additional ligands on manganese areintroduced in this process. It was pointed out in a previouspaper (5) that it should be possible to utilize PRR measurementsto determine the binding of MnADP- (and other substrates) toesatine kinase. It was found that the addition of increasingconcentrations of ADP to a solution containing manganese andcreatine kinase caused an increase in the PRR which passedtrough a maximum and then decreased. Analysis of such atitrationn" curve yields dissociation constants for the equilibriaset up between MnADP- and ADP3- and their respective com-plexes with the enzyme and also yields a value, et, the PRR en-hancement characteristic of the ternary manganese-nucleotide-enzyme complex.

This paper deals with the enhancement of the PRR of waterdue to various enzyme-manganese-substrate complexes. Dis-sociation constants for the equilibria between creatine kinase andthe complexes formed with its manganese-nucleotide substrateshave been determined from the values of the bound manganeseobtained from PRR measurements and from the measurementsof free manganese by EPR.

As previously noted (4), no ternary complexes of enzyme, man-ganese, and creatine or creatine phosphate could be detected byPRR. However, the formation of an abortive quaternary com-plex of creatine with enzyme-manganese-ADP could be detectedby the effect of creatine on lowering the enhancement of the PRRof the ternary complex. Thus, the dissociation constant forcreatine could be evaluated under these conditions.

A limited investigation of the kinetics of the activation of thecreatine kinase reaction by manganous ion was undertaken toascertain whether the complexes observed in the magnetic reso-nance experiments corresponded to the kinetically active ones, andto determine whether the kinetic behavior of the manganous ionwas essentially similar to that previously established for mag-nesium (3, 6). The kinetic study was minimal since an exten-sive analysis of the manganous ion activation was being carriedout by Morrison and Uhr.2

EXPERIMENTAL PROCEDURE

Materials and Methods

Manganese and magnesium chlorides were spectrographicallystandardized reagents from Johnson, Matthey and Company,Ltd., London. N-Ethylmorpholine, triethanolamine, and tetra-methylammonium chloride were purchased from Eastman. Thetetramethylammonium chloride was recrystallized from acetone-ether (7) and stored in a desiccator.

Nucleotides, phosphocreatine, and the Tris base were pur-chased from Sigma; bovine serum albumin, from Armour; andcreatine and sodium tripolyphosphate, from Fisher. OtherChemicals used were reagent grade.

Buffers (N-ethylmorpholine, triethanolamine, and Tris) were

'The abbreviations used are: EPR, electron paramagneticresonance; PRR, proton relaxation rate. In addition, throughoutthetext ES, EM, MS, and EMS are used to designate the enzyme-Substrate, enzyme-metal, metal-substrate, and enzyme-metal-substrate complexes, respectively. E, M, and S are used for thefree concentrations of enzyme, metal, and substrate, respectively,and the subscript T is used to denote total concentrations.

2J. F. Morrison and M. L. Uhr, personal communication.

made up at 0.5 M concentration after adjustment to the requiredpH with concentrated HC1. The pH on dilution (with car-bonate-free water) was checked on a Radiometer pH meter, typePAM 46.3

Creatine kinase was prepared by Method B of Kuby, Noda,and Lardy (8) and was twice recrystallized. We are grateful toDr. L. Noda for a gift of seed crystals. The crystals were col-lected, dissolved in 1 mM N-ethylmorpholine-HCI (pH 8.0), anddialyzed for 24 hours against a solution of the same buffer in thepresence of mM EDTA and then for 24 hours against bufferalone. Different preparations, as assayed by means of a pH-stat(9), had activities of 55 to 65 Kuby units (10).

Magnetic Resonance Measurements

Free manganese was measured by the intensity of its EPR spec-trum with a Varian model V-14500 A spectrometer at 9.5 gc persec. The bound manganese was determined by its effect on thePRR of water by a pulsed nuclear magnetic resonance methodat 25 me per sec, as described previously (11).

The observed enhancement of the effect of manganese on theproton relaxation rate, *, is defined (11) as

1 1

T, Tl(o)

where T, and T(o,) are the observed longitudinal relaxation timesof water in the buffer solutions with and without manganese,respectively. The symbols with asterisks represent the sameparameters in the presence of an agent participating in complexformation.

Unless otherwise indicated, all experiments were carried out in0.05 M N-ethylmorpholine-HCl buffer, pH 8.0, with a total con-centration of MnC12 of 0.1 mM and various concentrations ofnucleotide and creatine kinase.

Calculation of Enhancement and Binding Parameters

The procedures for determining the values of the enhancementof the PRR of binary manganese-substrate complexes, e,, anddissociation constants, K, have been presented in detail previ-ously (11, 12). A detailed analysis of the treatment of PRR andEPR data for an evaluation of the enhancement parameter ofternary EMS complexes, et, has been given for pyruvate kinase(13). The analysis of the data in the case of creatine kinase mustbe modified since in this system, unlike pyruvate kinase, thebinary metal-enzyme complex makes no significant contributionto the observed enhancement; experimental evidence in thepresent investigation and earlier investigations (1, 3-5) indicatesthat any interaction between metal ion and enzyme at the activesite is negligible. The ternary EMS complexes refer to man-ganese-ADP-enzyme and manganese-ATP-enzyme, since only thenucleotide substrates exhibit the enhancement phenomenon inthe presence of manganese and creatine kinase.

s Measurements of pH on a Leeds and Northrup 7664 pH meterwith a silver-silver chloride reference electrode gave values thatwere consistently too low with nitrogen base buffers. The devia-tions ranged from approximately 0.05 to 0.2 pH unit as a directfunction of the amount of base present in the free form.

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Interactions of Creatine Kinase Studied by Magnetic Resonance

Evaluation of Enhancement for TernaryComplexes, et

The general expression for the observed enhancement, *, is

[M] f+ MSI [EMI [EMS][MIT [MIT [MIb [eIT

(1)

where el, enhancement of free Mn2+, equals 1 by definition andea, eb, and t are enhancements of the MS, EM, and EMS com-plexes, respectively. In the case of creatine kinase, the term ineb is assumed to be negligible compared to the other terms inEquation 1. The error introduced by this assumption is dis-cussed later in this section; Equation 1 simplifies to

* [M [MS] I [EMS] 2MrIT [MIT a

+ [MIT ()

In order to evaluate et and the various dissociation constants, thefollowing additional relationships are used.

[M]I = [M] + [MS] + [EMS] (3)K = [M][S]/[MS] (4)K2 = [E][MS]/[EMSI (5)Ks = [EI[S]/[ES] (6)

From Equations 3, 4, and 5, Equation 2 may be expressed as

K1K2 K2

* [E][S] [El (7)

[E][S] [E+

It has previously been noted that e*, when measured as a func-tion of nucleotide concentration, exhibits biphasic behavior, firstincreasing with substrate concentration and then decreasing (5).As the substrate concentration is increased in excess of the metalion concentration, there is a competition between S and MS forthe enzyme. Consequently the concentration of EMS decreasesand the concentration of MS concomitantly increases, with theresult that the value of * approaches e,. In order to evaluateet from *, therefore, only the ascending limb of the titrationcurve with substrate is used, where [MS] is small. It is notfeasible to solve Equation 7 analytically to obtain et; the valueof et is obtained by approximate graphical methods as outlinedbelow.

First, the enhancement of the EMS complex at [S], o and finite[E], E*,, is found from extrapolation of a linear plot of 1/f* withrespect to 1 /[S]T of the data in the ascending limb of the titrationcurve. An approximate linear relationship may be derived fromEquation 7; if one extrapolates from the limited region describedabove, where [MS] is insignificant, then at high [S], IS] - [S]T andthe first term in the numerator becomes relatively small. There-fore the reciprocal of e* becomes

1 KK2 K2

1 [S] T [ +El +(8)-( (8)

e* K2[- + El[E]

In the limit, when [Si] -t x, the first terms in the numerator anddenominator of Equation 7 approach zero, so that

At high values of [E], [El - [E]T, the first term in the numerateof Equation 9 becomes small relative to et and the reciprocaform of Equation 9 expresses a linear relationship between l/fand 1/[E]T.

1 K2 1 1ec Et [E] (1l

If the values of 1 /e** obtained in the first graphical extrapolatiqare now plotted as a function of 1/[E]r, the second graphical estrapolation to [E]T = o yields

1 1

ec Et(10l

Determination of DissociationConstants K2 and Ks

Once et had been determined, the dissociation constant (Equation 5) could be evaluated by three different proceduroutlined below. Procedures I and II involve graphical solutionsProcedure III involves an analytical solution. Only PRR dataare used in Procedure I; in the other two procedures both PIhand EPR data are used. From Procedure III, a value of K(Equation 6) as well as K2 (Equation 5) is obtained.

Procedure I-The dissociation constant of EMS to yield and MS may be obtained from the half-maximal value of E*, =(et + ea)/2 in the plot of 1/e*, against 1/[EIT; half of the enzymemay be considered in the form of EMS, and half as free enzymeif the concentration of ES is assumed to be negligible; hence[E] = [EMS] = [E]v. Then, as there is no free [M] present

[MS] [ElK2 = [MS]

[EMS]

where [MS] = [M]T - [EMS] = [MIT - [E] T-

Procedure II-If the free metal ion concentration [M] immeasured by EPR in a series of experiments in which the nucleo-tide is varied at constant enzyme concentration, then the total 1lbound, [M]b, is known, and it is possible to plot 1/e* against1/[M]b. For each enzyme concentration, at the point on thecurve (*, + )/2, half of [M]b is in the form of [MS] and theother half as [EMS]. If we assume that the concentration ofES is negligible, then [MS] = [EMS] and

[MS][EK2 [EM = [E]

[EMS]

where [E] = [E]T - [EMS] = [EiT - [M]b.Procedure III-If the value of free metal ion concentration

is determined experimentally by EPR, the first term in Equatiol2 may be subtracted from E* to give the enhancement of the totalbound M, *(Mb).

(14[MS] [EMS]*(Mb) = [M + [I 2-

By using the known values of E, and et and the relationship[MS] + [EMS] = [M]T - [M] from Equation 3, it is possible tacalculate [MS] and [EMS]. [S] is obtained from Equation4

since K1 is known. Additional known relationships are

[ES] = [S]T - [S] - [MS] - [EMS] (l

[E] = [E] - [ES] - [EMS] (I;

K2[- e + t

+1

(9) and

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Thus the concentration of all the species is known, and it is possi-ble to calculate K2 and Ks, respectively.

Accuracy of Determinations of K2 and Ks

In addition to the mathematical approximations introduced inthe graphical solutions in Procedures I and II and the uncertaintyintroduced through the use of Equations 13 and 14 in ProcedureIII, the effect of three other possible sources of error should bediscussed. (a) For Procedures I and II, it was assumed that theconcentration of ES was negligible. As mentioned above, theactual extrapolations were limited to the data from the region ofthe nucleotide titration curves where e* is increasing; in that re-gion it is plausible to assume that the concentration of ES is smallrelative to EMS since Ks > K2. Justification for this assump-tion is obtained from the relative magnitudes of [EMS] and [ES]calculated by Procedure III (see Table III). Omission of theI[S] term introduced a maximum error of 20% in the value ofK2 as determined in Procedures I and II, and the effect was usu-ally much less. (b) The basic assumption for evaluation of etby all procedures was that the term in eb arising from the man-ganese-enzyme complex could be neglected. However, Mn2+complexes to some extent with the enzyme. The contributionof the manganese-enzyme complex would become less importantas the concentration of ADP increased. (c) Mn2+ has been re-ported to inactivate creatine kinase slowly (14). The time lagbetween the addition of Mn2+ to the enzyme and the measure-ment of free Mn 2+, sometimes as much as 5 hours, could havebeen sufficient for significant inactivation to have taken placeand thus could have led to spurious results. Some data bearingon this possibility were obtained by repeating the EPR measure-ments after 24 hours. - For those solutions in which there waslittle free Mn2+, i.e. in which the ADP concentration approxi-mated that of the total Mn2+ , there was little difference in themeasurements of free Mn2+ at the two times, but more randomresults were obtained with lower ADP concentrations.

Experiments were designed to minimize the effect of manganeseon the enzyme; PRR and EPR measurements were made ondifferent but identical solutions, in each case immediately afterthe addition of enzyme to the solution; such an experiment wasused to calculate the concentrations of all species present in thesolution (see Table III).

Kinetic Experiments

Measurements of the initial velocity of the enzymatic reactionwere made by following the release or uptake of protons with aRadiometer TTTlc pH-stat (9). The titrating solution wasfreshly prepared each day from a stock solution with carbonate-free water in the case of alkaline titrating agent. Tetramethyl-ammonium chloride was used to maintain ionic strength. Bovineserum albumin, which was used to prevent inactivation of theenzyme (9), was present at a concentration of 0.04% and had anegligible effect on the free manganese concentration.

RESULTS

Binary Complexes of Mn2+ with Components ofCreatine Kinase Reaction

As Mn2+ interacts to differing extents with all of the com-Ponents of the creatine kinase reaction, a preliminary investiga-tion of such interactions was carried out. The stability constantdeterminations were based on measurement of free Mn2+ by EPR,

the reduction of the Mn2+ signal on the addition of a complex-forming agent being taken as a measure of the formation of man-ganese complex (15). The enhancement values, ce, of the variousbinary complexes were determined from PRR measurements intitrations of manganese with the potential ligand.

Control Experiments on Mn2 + Binding to Buffers-Threebuffers, Tris-HC1, triethanolamine-HCI, and N-ethylmorpholine-HC1, were considered as supporting media for the enzyme experi-ments. The Mn2+ binding was tested at different pH values,corresponding to different concentrations of free base; the stabil-ity constant values in Table I refer to the free base. It is seenfrom this table that there was a small interaction between Mn2+and all three buffers. The interaction was least with N-ethyl-morpholine, as shown from the stability constants in Table I,and this buffer was used for subsequent experiments.

The possibility that the observed decrease in EPR signal in-tensity in the buffer solutions was due to Mn2+ interaction withchloride ion was tested by carrying out similar experiments withvarious concentrations of KC1. The measured values of freeMn2 + were found to decrease linearly with increasing KCI con-centrations, and corresponded to a value of 0.3 M- l for the ap-parent stability constant of the manganese-chloride complex.NaCI acted similarly to KC1, but no interaction with NaNOs at0.1 M was detected.

The formation of metal complexes with Tris was observed byHall et al. (16), who determined stability constants of the orderof 103 M for Cu2+ and Ni 2+. Indirect evidence for a weak inter-action between Mg2 + and Tris from measurements of the stabilityconstant of MgATP2- in this buffer has also been presented (7).It should be pointed out that any error introduced by neglectingthe interaction between Mn2+ and Tris would be small, particu-larly at pH values below 8 (cf. Mildvan and Cohn (11)).

Manganese Binding to Substrates-The stability constants forthe Mn 2+ complexes of phosphocreatine and creatine were deter-mined in the same way as described for the buffers and are in-cluded in Table I. The situation was more complex with ADP;

TABLE I

Stability constants of complexes formed between manganese andcomponents of creatine kinase system

The measurements with the buffer species, phosphocreatine,and creatine were carried out in 1 mM MnC12, and with ADP andATP in 0.1 mM MnC12, at 25°

i- 1. Other conditions were asindicated below. The stability constant is defined as K = [ML]/([M][L] ) for the reaction M + L - ML between manganese anda particular ligand. For the buffers, L is the free base, and thusthe K values are true stability constants. The values of K forthe other compounds are apparent stability constants for theconditions specified.

Compound pHa

K

-1

Tris ............................ 5Triethanolamine ............... 2N-Ethylmorpholine 1............. 1ADP ........................... 8.0 2.5 X 104ATP ........................... 7.5 ~105Phosphocreatine ................. 8.0 140Creatine ........................ 8.0 1.5

a In each case, where the pH is specified, the buffer was 0.05 MN-ethylmorpholine-HC1.

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Interactions of Creatine Kinase Studied by Magnetic Resonance

EPR measurements over a wide range of total ADP and totalMnCl2 concentration gave values of the stability constant forMnADP- varying from 2 X 104 to 7 X 104 M- ', depending onboth the relative and absolute concentrations of nucleotide andmetal ion. For the conditions used for the PRR measurements(MnCI2, 0.1 mM; total ADP, 0.02 to 0.2 mM), an average of theEPR data gave a stability constant of 2.5 0.7 X 104 M-1.This value of K1 is compatible with results from other techniques(7). Proton relaxation rate measurements (see below) gave avalue of 1.7 X 104 M- 1. Measurements with ATP gave stillmore variable results, and the value of 10 M-1 was used as rep-resenting an order of magnitude only for the stability constant ofMnATP2 . The values of the stability constants for all thebinary complexes investigated are listed in Table I.

The interpretation of the EPR measurements of complexformation between Mn 2+ and ADP and ATP is hampered by thefact that Mn+ can bind not only at the phosphate moiety butalso at the adenine ring, and it appears possible that more thanone type of complex is formed. In addition, the actual EPRspectra observed represent an average formed from the signalsdue to free Mn+ and to the manganese-nucleotide complex.The ratio of signal heights of free Mn2+ and MnADP- has beenestimated as approximately 16:1.4

An experiment was carried out to test the validity of equatingthe intensity of the EPR signal with the concentration of freeMn+ , i.e. to determine whether the MnADP- signal would makeany significant contribution to the EPR signal of solutions con-taining components in the same concentration range as used inthe PRR experiments. The spectra obtained with total Mn24

at 10, 20, and 200 yM in the absence of ADP were compared withthe spectra obtained with the same value of free Mn2+ (calcu-lated) in the presence of ADP; the ratio of MnADP- to Mn2+ inthe latter solutions was 4:1. The agreement of signal intensitieswas within 3 % in each case, indicating that any error introducedwas negligible. The actual values obtained were 0.98, 0.99, and0.97, respectively (average of two experiments) for the three con-centrations of Mn2+, compared to the expected values of 1.00.Similar results have been obtained by Mildvan' for manganeseand ATP in approximately the same concentration range.

Enhancement of Manganese-Nucleotide and Other Binary Com-plexes-A standard solution of MnC1 2 (0.1 mM) in 0.05 M N-ethyl-morpholine-HC1, pH 8.0, was titrated with a solution of ADP inthe range 0.01 to 0.2 mM, and measurements of the PRR weremade at each point. The enhancement, e*, reached a maximumof approximately 1.5. A double reciprocal plot of e* against[ADPIT was used to obtain a value of 1.6 for the enhancement,Ca, of MnADP-. This plot also yielded a value of 1.7 X 104 r-for the stability constant of MnADP-. MnATP2- at pH 7.5 in0.05 M N-ethylmorpholine-HC gave values of ea from 1.6 to 1.7and values of stability constants from the plots of 1 /* against1/[ATP]T of approximately 4 X 104 M- 1 .

The values of e for a number of other non-nucleotide com-pounds that form binary complexes with Mn+ were also deter-mined and are listed in Table IV. The enhancement with Piwas found to be less than 1. Pyrophosphate in the concentrationrange 0.1 to 1.0 m and tripolyphosphate at 0.2 to 2.0 m causedenhancements slightly greater than 1. However, outside theseranges an enhancement less than 1 was observed. The equilibriaset up between Mn+ and these compounds in solution appears to

4 M. Cohn, unpublished observations.A. S. Mildvan, unpublished experiments.

be complex, and the PRR results could be taken to indicate thexistence of more than one complex formed with each condemnsphosphate.

Binding of Mn2+ to Enzyme-Preliminary experiments indcated a slight binding between Mn 2+ and creatine kinase. view of the possibility that EM might be considered an intemediate along a reaction pathway (cf. Reference 3), this wgfurther investigated. Experiments were carried out, either witthe MnCl12 concentration held constant at 0.1 mm and with ezyme titrated over the range 0.02 to 0.2 mM, or with the enzymeconcentration held constant at 0.1 mM and the Mn2+ concentretion varied from 0.02 to 0.2 mM. Measurements of the protorelaxation rate and of free Mn2+ by EPR were carried out at eachpoint.

The proton relaxation rate data gave a value of approximately1.5 for b of manganese-enzyme. The data from both types oltitration were analyzed as described previously (11). Attemptswere made to plot all the available data according to the methodof Hughes and Klotz (17) to obtain n, the number of manganesebinding sites, and the corresponding constants. However, valuesof n ranging from 4 to 10 (cf. Reference 2) and values of the dissociation constant ranging from 2 X 10 - 4 to 10 - 3 M were deter.mined from this type of graphical analysis.

The binding of Mn2+ to the enzyme could be virtually saltedout (cf. Reference 18). Thus, in a particular experiment, KOat 0.5 M added to a solution 0.1 mM in MnC12 and 0.1 m increatine kinase reduced the measured enhancement from 1.4 to1.07, and the bound Mn2+, as measured by EPR, from 0.037 to0.01 m. It would appear that the binding of Mn2+ to theenzyme is nonspecific and relatively weak.

Ternary Complexes of Manganese, Nucleotides,and Creatine Kinase

Enhancement of Enzyme-Manganese-ADP Complex-The titra-tion of MnCl2 with ADP was carried out in the presence ofcreatine kinase. As previously reported (5), a considerable increase in the enhancement was observed with increasing ADconcentration. The value of * passed through a maximum andthen decreased. As predicted from Equation 7, it was found eMperimentally that the maximum observed was dependent on thenzyme concentration. Titrations with ADP were thereforecarried out at three enzyme concentrations.

The data were analyzed as described in "Experimental Procedure." Double reciprocal plots of the variation of (* - 1)where * is the measured enhancement,6 with total ADP colacentration, for three different concentrations of creatine kinasOare shown in Fig. 1 (cf. Equation 8). The extrapolation to i0finite concentration of ADP was made from the region of increaoing *, where ADP was bound to the enzyme in the form oMnADP and the binding of free ADP was negligible. The extrap-olated lines, fitted by the method of least squares, gave values ofe*,, the observed enhancement at infinite substrate and finitenzyme concentrations, of 7.6, 10.2, and 14.2 for enzyme concetrations of 21.3, 42.6, and 85.2 M, respectively. A plot of 1/eagainst 1/[E] (Fig. 2) yielded a value of 19.4 for the enhancement$

6 The data have been plotted both as 1/&* against 1/[ADP]r andas 1/(e* - 1) against 1/[ADP]T, with no significant differentbetween the results obtained. The use of 1/(e* -1) introduces 1

normalizing factor, as e* = 1.0 at [ADPT = 0, and usually givesslightly better fit to a straight line. Examples of both types Agraphical presentation of the data are given in this paper.

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e, of the ternary enzyme-manganese-ADP complex from the or-dinate at [E = (cf. Equation 11).

The results shown in Figs. I and 2 were obtained with a singleenzyme preparation that had an activity of 65 Kuby units.Subsequent experiments with other enzyme preparations of com-parable activity have given results which agreed to within 10 to20% of the value obtained from Fig. 2. It should be noted thatrelatively small variations in the slope of the line drawn in Fig. 2would give rise to relatively large errors in et, so that an error ofE10% in the estimation of et was regarded as satisfactory. Ex-

periments were also carried out at pH 7.5, close to the optimumpH of 7.0 for the reverse reaction (10), but no significant changein et was observed.

Dissociation Constant of MnADP- from Ternary Enzyme Com-plex-Determinations of the dissociation constant (K2) forMnADP- were obtained as described in "Experimental Pro-cedure." Procedure III allowed, in addition, the estimation of

0.8

0.6

I

E~/l0.4

0.2

0.00

[ADP]

FroIG. 1. Titration of 0.1 mM Mconcentrations of creatine kinas1/[ADPIT. The concentrations85.2 gM; Curve 2, 42.6 M; Curvecarried out in 0.05 M N-ethylmovolume of 0.1 ml, at 240.

0.15

0.10

,ElC

0.05

0.00

I

[E T

I'G. 2. Double reciprocal plotagainst creatine kinase concentgive a value of e equal to 19.4 a

TABLE II

Dissociation constants for equilibria between creatine kinase andmanganese-nucleotide complexes (K2) and free nucleotides

(Ks) for ADP and ATPThe average values determined from the PRR and EPR meas-

urements by the three procedures described in the text are com-pared with the values obtained from kinetic measurements (seeTable V).

III

III

AverageKinetic

0.4 0.20.7 + 0.40.5 f- 0.2

0.5 - 0.20.5 -i 0.2

6 0.4

1.6 -f- 0.46 2

Ks

0.9 0.6

1.3 4- 0.2

1.1 ±- 0.41.7 - 0.6

3 23 4 2

the dissociation constant of ADP 3- (Ks) from its enzyme com-plex. For the calculations of the dissociation constants, it wasassumed that there were two active sites per molecule of creatinekinase (1). A summary of the values of K2 and Ks and theirstandard deviations obtained by the various procedures is givenin Table II.

By Procedure I, K2 = [MS] at e*c = [Et + ea]/2 from Fig. 2;therefore K2 = 0.5 X 10- 4 M determined from e*C = 10.5. FromProcedure II, the plots of /e* against 1 /IM]b were used to givevalues of K2; an average value of 0.7 X 10- 4 M was obtainedfrom these determinations. Procedure III allows the determina-tion of K2 and Ks from each point on the titration curves in Fig. 1.The values reported in Table II are based on nine such determina-

2 4 6 tions for K2 and six for Ks. It was not always possible to evalu-ate Ks since in some cases the free ADP3- concentrations were

- X 1 0-4 M-' too low to be calculated accurately. The relatively large scatterFT in the calculated values of K2 and Ks listed in Table II is not sur-

nCl, with ADP at three different prising when the procedures, which involve a large number ofe. l/(e* - 1) is plotted against subtractions (Equations 13 and 14), are taken into consideration.of creatine kinase were: Curve 1, Determination of K2 and Ks from other experiments fell largely3, 21.3 Mu. The titrations were within the same range.zrpholine-1HC, pH 8.0, in a total From the results shown in Table II, it can be seen that the

PRR and EPR data have given values for the interaction ofMnADP- and ADP3- with creatine kinase that are in veryreasonable agreement with those from kinetic studies with Mn2+,as will be shown under "Kinetic Studies of Manganese-activatedCreatine Kinase Reaction," and also with those from kinetic andthermodynamic studies with Mg2+ (1, 3).

In order to obtain the most reliable quantitative data, themeasurements from an experiment in which particular care wastaken to minimize artifacts resulting from changes in the enzymewith time, as described in "Experimental Procedure," were usedto calculate the concentrations of the different species presentand to evaluate K2 and Ks. The results of this experiment,which were analyzed by Procedure III, are presented in TableIII. It will be noted that the concentration of ES is small rela-tive to other components in the system, justifying its neglect in

x 10-4 M-l the calculations by Procedures I and II.Enhancement and Dissociation Constants of Ternary Complex,

of * values, obtained from Fig Creatine Kinase-Manganese-ATP--The experiments with ATPtration. The curve extrapolates were not as extensive as with ADP because of the difficulty intro-at infinite enzyme concentration. duced by the ATPase activity of the enzyme (19). Measure-

-

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TABLE III

Concentration of various species in solution and calculation of K2 and KsPRR and EPR measurements were made on identical solutions

immediately after addition of creatine kinase (37 uM = 74 M ac-tive sites) to solutions containing MnC12 (0.1 mM) and differentconcentrations of ADP. Experiments were carried out in 0.05 M

N-ethylmorpholine buffer, pH 8.0, in total volume of 0.1 ml at 22*All concentrations and the dissociation constants are expressed10- 4 M. K2 = ([E][MS])/[EMS], Ks = ([E][SI)/IES]. Calculations were carried out by Procedure III (see the text).

[ADP]T [MTa

[I *(Mb) [MS] IEMSI IS] [ES] [e K2 KS

0.20 0.90 3.26 0.72 2.54 0.05 0.13 <0.02 <0.01 0.61 0.3 (-2)60.40 0.92 4.30 0.60 3.70 0.14 0.18 0.08 0.02 0.56 0.4 2.20.80 0.94 5.76 0.43 5.33 0.25 0.26 0.23 0.06 0.42 0.4 1.61.00 0.96 6.16 0.37 5.79 0.31 0.28 0.33 0.08 0.38 0.4 1.6

Average 0.4 1.8

Total Mn 2+ , corrected for that bound to the enzyme.b Concentrations of S and ES were too low to give an accurate value of Ks.

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.00 1 2 3 4 5 6

x 10 '4 Ml

[ATP] X

FroIG. 3. Titration of 0.1 m MnCl 2 with ATP at three differentconcentrations of creatine kinase. /E* is plotted against 1/[ATP]T. The concentrations of creatine kinase were: Curve 1,71 MAM; Curve 2, 35 M; Curve 3, 18 uM. All titrations were carriedout in 0.05 M N-ethylmorpholine-HC1, pH 7.5, in a total volume of0.1 ml, at 24°.

ments of the enhancement produced with different concentrationsof creatine kinase and ATP in the presence of 0.1 mM MnC 2 werecarried out under conditions identical with those used for ADPexcept that a lower pH (0.05 M N-ethylmorpholine-HCl, pH 7.5)was used to minimize any effect due to the ATPase activity ofthe enzyme (19). Values of e*, of 2.7, 4.0, and 5.9 were obtainedat creatine kinase concentrations of 18, 35, and 71 juM, respec-tively (Fig. 3). A plot of 1/e*, against 1/[E] (Fig. 4) gave anextrapolated value of 9.8 (2) for the enhancement, et, of theternary complex, enzyme-manganese-ATP. The dissociationconstant for MnATP2 - from the ternary complex was determinedfrom this plot (Procedure I) as 0.4 X 10- 4 M.

EPR measurements were carried out on solutions identical withthose used for the PRR determinations at an enzyme concentra-tion of 35 iUM. The measurement was initiated within 2 min ofthe addition of the enzyme to the solution. The EPR spectrumof Mn2+ was swept through twice, the time of each sweep being3 min. On the basis that the amplitudes from the two sweepswere identical within experimental error, it was concluded that nosignificant change in free Mn2+ took place during the experimentand that the ATPase activity was insignificant under these con-ditions.

0.4

0.3

0.2

0.1

0.00 2 4 6

I 10E4IMT

FIG. 4. Double reciprocal plot of E,* values, obtained from q3, against creatine kinase concentration. The line extrapolalto give a value of t equal to 9.8 at infinite enzyme concentrate

Calculations of the dissociation constant of the ternary compifor MnATP2- from both the PRR and EPR data (Procedure 1gave a value of 1.3 X 10- 4 M. It was difficult to extract a vatfor the dissociation constant for free ATP4- from its enyIcomplex, as the free ATP 4- was always very small, sometimenegligible, under the experimental conditions, and, therefore, terminations of its concentration and that of E-ATP were subOjto large error. However, Ks for ATP was estimated to be inrange of 1 to 5 X 10- 4 M. The values of K2 and Ks for ATPlisted in Table II.

An attempt was made to repeat the ATP experiments at8.0. The results indicated that the enhancement was sligbincreased at this pH, and a value for e of approximately 14obtained. However, the proton relaxation time changed rapidin these experiments (presumably owing to the ATPase activeof the enzyme), and it was not possible to obtain accurate vafrom PRR measurements, nor was it possible to carry out reliesEPR measurements of free Mn2+.

Interaction of Other Compounds uith Creatine Kinase--Nohancement due to the formation of a ternary complex in the t

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nee of manganese and creatine kinase could be detected withphosphocreatine or creatine. No direct evidence for the forma-tion of a ternary complex with either AMP or Pi was obtained,although both have been reported as inhibitors of the creatinegaase reaction 7 (20). While both compounds reduced the en-hancement obtained with enzyme-manganese-ADP, the experi-mental results did not provide evidence for interaction of AMPor Pi at the active site, since the effect that they produced couldbe accounted for quantitatively by the formation of binary man-ganese complexes (MnAMP and MnPi), resulting in a lowerconcentration of enzyme-manganese-ADP.

A number of other compounds, either ones active in the reac-fion or ones that could act as inhibitors of creatine kinase, wereinvestigated with creatine kinase in the presence of manganese.The enhancements of the binary and ternary complexes obtainedare summarized in Table IV. Only for pyrophosphate and tri-polyphosphate could it be conclusively demonstrated that ternarycomplexes were formed. Estimates of the dissociation constantsfor the complexes formed with MnPP and MnPPP gave valuesof the order 10-2 and 2 X 10- a M, respectively. These are inreasonable agreement with the values of Kr determined kinet-ically by Nihei, Noda, and Morales (20).

Formation of Quaternary Complex, EMS-Creatine-While noternary EMS complex could be observed with creatine, additionof creatine to a solution containing creatine kinase, Mn2+, andADP was found to cause a significant decrease in the measuredenhancement. The effect was too large to be due to displace-ment of the equilibria in the solution through the binding of Mn2+

by reatine (see Table I) and was attributed to the formation ofan abortive quaternary complex, EMS-creatine, with a lower en-hancement, E%, than that of the ternary complex. It was possibleto titrate this effect, and the concentration of creatine at the half-maximal point was calculated to be approximately 10 mM, whichmay be compared with the value of 16 mM for the KM obtainedfrom kinetic experiments (1).

It was found that the respective temperature coefficients of therelaxation times for the ternary and quaternary complexes weredifferent in sign. As the temperature was lowered, lower valuesof the relaxation time (higher values of the PRR) were obtainedboth for the standard Mn2+ , as previously reported (21), and forthe solution containing the ternary complex. For example, inthe three-component system (e.g. MnC12, 0.1 mM; ADP 0.1 mM;creatine kinase, 54 /a; N-ethylmorpholine-HCI, pH 8.0, 0.05 ),a value of 0.195 sec for T at 23° was obtained. The value forTi decreased to 0.168 sec at 10. However, in the presence ofsaturating concentrations (50 mm) of creatine for the same solu-tions, the relaxation time increased (the PRR decreased) withdecreasing temperature, viz. from 0.212 see at 23° to 0.239 see at10. This difference in sign of the temperature coefficient of T1was reflected in a larger difference between e and e as the tem-Perature was lowered. The value for et decreased from 20 at230 to approximately 16 at 10, while approximate values of 15and 8 were obtained for e at the two temperatures, respec-tively.

Although sufficient data have not yet been obtained for a pre--me evaluation, preliminary calculations indicate that the binding

of lnADP- to the enzyme in the presence of saturating concen-ations of creatine is approximately twice that in its absence.

is is consistent with earlier results from kinetic studies, namely,tkatMgADP- binds more strongly to creatine kinase in the pres-ae of phosphocreatine than in its absence (3).

'T. E. James and J. F. Morrison, personal communication.

TABLE IV

Enhancements (PRR) of other compounds tested withmanganese and creatine kinase

All experiments were carried out in 0.05 M N-ethylmorpholine-HCI, pH 8.0, at an MnCI2 concentration of 0.1 m, at 21-23 °.

Other conditions are described in the text.

Binary complex ea Ternary complex

Mn-phosphocreatine 1.3 E-Mn-phosphocreatine 1.3Mn-creatine 1.0 E-Mn-creatine 1.2aMnAMP 1.2 E-MnAMP 1.3MnPi <1 E-MnPi 1.2aMn-pyrophosphate 1.2 E-Mn-pyrophosphate 2.0Mn-tripolyphosphate 1.3 E-Mn-tripolyphosphate 2.5

Under the conditions tested, this enhancement is the same aswith manganese and enzyme alone.

Kinetic Studies of Manganese-activated Creatine Kinase Reaction

Kinetics of Reverse Reaction-The results of a kinetic study inwhich the initial velocity was measured as a function of free man-ganese at various levels of free ADP3- at a fixed concentration ofphosphocreatine are shown in Fig. 5. The rationale of this ap-proach, from which kinetic constants are obtained for all stepsleading to the formation of an active enzyme-metal-substratecomplex, has previously been described in detail (3, 6).

The three pathways considered may be represented by the se-quence of reactions shown in Scheme 1. In this scheme, it is as-sumed that the constant concentration of phosphocreatine al-tered the velocity by the same factor under all conditions Theconcentration of EMS is independent of the pathway by whichit is formed, since

K,K 2 = KsK'A = K0K3

The velocity equation may be written

kEr

(15)

(16)KKs + K + K3 +

[M][Sl [M] []

where k is the velocity constant for the breakdown of the EMScomplex.

It is seen that the lines in Fig. 5 intersect approximately onthe ordinate. From these data, it follows that any interactionbetween the metal ion and the active site of the enzyme may beneglected (3). By inversion of the simplified form of Equation16, the results may be described by

1 1 [4K ±v ET[M [A1] J J (17)

so that the plots of 1/v against 1/[M] in Fig. 5 intersect theabscissa at -1/K', where

K' = K fKs + 1 (18)

s The presence of phosphocreatine on the enzyme has previouslybeen found to have a slight effect on the binding of MgADP-,and vice versa (3), and the evidence presented in this paper onthe abortive EMS-creatine complex would indicate a similar effectwith MnAD1- . However, neglect of this effect in the range ofconcentrations used in the experiments reported in this papershould not introduce any significant error.

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0.9

I 0.7V

0. I0.5

0. I

I I I . .

-18 -16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8

x 10 4 Ml1

[Mn2+]

FIG. 5. Double reciprocal plot of the observed velocity againstfree manganese concentration at various levels of free ADP -.

The experiments were carried out in a total volume of 2.0 ml withtetramethylammonium chloride, 0.05 M; bovine serum albumin,0.04%; and phosphocreatine, 0.01 M. The concentrations of freeADP3- were: Curve 1, 0.2 mM; Curve 2, 0.1 mM; Curve 3, 0.066 mM.Free manganese and free ADP3- concentrations were calculatedfrom the cubic equation derived from consideration of the equilib-ria set up between manganese and ADP3- and phosphocreatine,respectively, with the use of the stability constants reported inTable I. The reaction was initiated by the addition of 2 u1 ofenzyme solution (1.08 ,g); pH = 8.0, T = 250 1°.

M +S

KIMS

K5 KA IlK2 Ks KDE + S . ES EMS . ' EM . E + M

Products

SCHEME 1

A plot of K' against 1/[S] would intersect the ordinate andabscissa at K'A and -1/Ks, respectively. Thus, by usingEquation 15, it is also possible to calculate K2. The values ofK'A, Ks, and K2 so obtained are shown in Table V together withvalues for the magnesium activation of creatine kinase (3).

It is seen from this table that the value of K2, for the inter-action of the metal-nucleotide with the enzyme, appears to beindependent of the nature of the activating metal ion. Thisresult would be anticipated if the substrate were bound to theenzyme solely through its nucleotide moiety rather than throughthe metal ion.

A difference in the K'A values for manganese and magnesiumis not surprising in view of the fact that manganese binds muchmore strongly to ADP. The values for the dissociation constant(Ks) for the interaction between ADP3 - and the enzyme ob-tained from experiments with manganese and magnesium, re-spectively, are in reasonable agreement.

A maximum velocity of approximately 12,000 moles of creatineper min per mole of enzyme was obtained with the enzyme sat-urated with ADP3- and manganese ion (and thus with MnADP-)at a phosphocreatine concentration of 10 mM. Under similarconditions, magnesium as the activating metal ion gave a valueof 11,200, moles of creatine per min per mole of enzyme (3).

The Km of phosphocreatine was determined as 3 mM in the

TABLE V

Comparative kinetic constants for activation of creatinekinase by manganese and magnesium

Experimental conditions for the manganese activation aredescribed in the legend to Fig. 5, and the constants are definedin the text. All K values are expressed as 10- 4 M.

Constant

K2

KlA

ADP

Mn2+

0.5 i 0.2c d

0.600.6/

0.02 - 0.01 c

6 20100

mg2+

0.5 : 0.10.6 - 0.21.0 i 0.1

0.6 i 0.1

3 13= 1

ATP

Mn2f

1.7

Mg,+b

3

Taken from the corresponding experiments of Morrison andO'Sullivan (3).

b Taken from Kuby and Noltmann (1); MgATP2- was deter-mined at pH 9.0, and MnATP2- , at pH 7.5.

¢ Determined from the plots of 1/v against /[Mn2 + ] (Fig. 5).dMorrison and Uhr2 obtained values of 0.2 X 10- 4 and 0.7 )

10- 4 M for K2, depending on the experimental conditions.e Determined from the plots of inhibition by excess ADP'

(Fig. 6a).f Determined from the plots of inhibition by excess Mn

(Fig. 6b).

(a) (b)

63

2

-I 0 I 2 3

Xl0-4 M-[Mn ADP]

-2 -i 0 I 2 3

[ X10- 4 M I

[MnADP-]

FIG. 6. The inhibition of the reverse creatine kinase reactorby excess ADP or manganese. a, double reciprocal plot showirthe effect of excess ADPs- over MnADP- on the observed velocias a function of MnADP- concentration. The concentrations afree ADP - were: Curve 1, 0.1 mM; Curve 2, 0.5 mma; Curve 3, 1.0 mlOther experimental conditions were the same as for Fig. 5.double reciprocal plot showing the effect of excess Mn2+ o0MnADP- on the observed velocity as a function of MnAPJIconcentration. The concentrations of free Mn2+ were: Curve0.05 mra; Curve 2, 0.5 mM; Curve s, 1.5 mM; Curve 4, 2.5 mM. Othexperimental conditions were the same as for Fig. 5.

presence of 0.2 ma ADP and manganese. This is in reasonabagreement with previous determinations (1).

Inhibition of Reverse Reaction by Excess Manganese and ExMADP-As was found with magnesium as the activating ion, lo

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possible to obtain inhibition by sufficient excess of either man-ganese or ADP over the other. In Fig. 6, the results of experi-ments designed to determine the nature of the observed inhibi-tions are shown. The lines drawn show ADP3 - as a competitiveinhibitor and free manganese as a noncompetitive inhibitor withrespect to MnADP-. These results are similar to those ob-tained with magnesium as the activating ion (3) and indicatethat MnADP- and ADP 3- , but not free manganese, react at theactive site of creatine kinase.

To describe the inhibition by excess ADP 3- (Fig. 6a), Equation17 may be rearranged in the form

1 1 K2 [S + t + t] (19)v kET [MS] Ks +

and plots of 1/v against 1/[MS] would have slopes of (K2/kET)([S/Ks + 1). Then a plot of the latter quantity against [S]would intersect the ordinate and abscissa at K2 and -Ks, re-spectively. The values obtained from such a plot are includedin Table V.

The reciprocal of the maximum velocities taken from Fig. 6b,plotted against concentration of free manganese, yielded a valuefor KI(Mn2+) of 4 m. Under similar conditions, K(Mg2+)for the noncompetitive inhibition of MgADP- by Mg2 + wasfound to be 16 mM (3). The smaller value for manganese couldbe due to stronger binding of manganese compared to magnesiumat some unspecified site on the enzyme.

Kinetics of Forward Reaction-Since the stability constant ofMnATP 2- is considerably higher than that for MnADP-, theexperimentally attainable range of concentrations of free Mn2+and ATP4- is insufficient to permit calculation of all the equi-

librium constants for the reactions leading to EMS in the for-ward reaction from determinations of reaction velocities.Therefore, kinetic experiments were limited to conditions suchthat both [Mn2+] and [ATP 4-] were negligible in comparisonwith [MnATP2-]. A double reciprocal plot of the velocity ofthe reaction with respect to MnATP 2- concentration in thepresence of 0.04 M creatine at pH 7.5 yielded a value of 1.7 X10-4 M for the K of MnATP 2- .

DISCUSSION

Earlier work on the enhancement of the proton relaxationrate of water due to manganese in its interaction with creatinekinase and the nucleotide substrates had established the existenceof ternary complexes of the three components with character-istic values of the enhancement factor (4, 5). In the presentinRestigation, the enhancement factors of enzyme-manganese-nucleotide have been determined and the dissociation constantsof MnADP and of MnATP from their enzyme complexes havebeen calculated with the use of the enhancement parameter,Et. The dissociation constants have been found to be quanti-tatively in agreement with the analogous constants determinedkinetically in the manganese-activated reaction, thus suggestingthat the complexes observed by the PRR technique are indeedthe kinetically active species.

further confirmation of the suggested identity of the ternarylplexes investigated by the PRR equilibrium technique and

in the kinetic experiments was derived from the observation ofCompetition between MnADP- and ADP3- for binding to theeve in both the equilibrium and kinetic studies. Analysisof the data from both types of investigation led to the same

ling constants for ADPS- to the enzyme. Two other in-

hibitors, inorganic pyrophosphate and tripolyphosphate, alsoform ternary complexes with manganese and creatine kinase;the dissociation constants of the manganese-inhibitor from theternary complex determined by PRR are in good agreement withpreviously reported inhibitor constants determined kinetically(20). It should be pointed out that the accuracy of the deter-mination of dissociation constants for the nucleotides and theirmetal complexes by the magnetic resonance techniques comparesfavorably with the accuracy of the more classical techniques ofequilibrium dialysis and sedimentation gradient (2).

The results presented in this paper substantiate earlier studieson the role of the divalent metal ion in the activation of creatinekinase. As reported previously (4, 5), there is only a slightchange in the PRR on addition of creatine kinase to manganese(eb = 1.5). The small interaction between enzyme and metalion could be largely salted out with 0.5 M KCL. In sharp con-trast, pyruvate kinase (12) binds manganese strongly at theactive site even at high ionic strength, giving rise to an enhance-ment of approximately 30 for the binary manganese-enzymecomplex. The absence of a specific, strong interaction of metalions with creatine kinase inferred from magnetic resonanceexperiments is in accord with kinetic and thermodynamic studieson creatine kinase (1-3).

The conclusion drawn from earlier work with respect to thestructure of the metal-nucleotide-enzyme complex (4), namely,that in the ternary complex the metal ion is bound to the nu-cleotide only and the nucleotide is bound to the protein directlywithout the metal acting as a bridge, is reinforced by the kineticexperiments with the manganese-activated enzyme. The ex-perimental findings of (a) similar kinetic behavior for activationby magnesium (1-3) and manganese, (b) similar dissociationconstants for the magnesium and manganese nucleoside di-and triphosphates from their respective ternary complexes,and (c) dissimilar dissociation constants for the two metal ionsfrom the ternary complex (cf. Table V) are consistent with thepostulated interaction of metal with nucleotide only and theabsence of a direct interaction between metal ion and enzymein the enzyme-metal-substrate complexes. Nevertheless, anincrease in the proton relaxation rate of water by a factor of 12when MnADP (eb = 1.6) is bound to the enzyme (et = 19.4)indicates that the environment of the water in the coordinationsphere of manganese is profoundly changed in the conversion ofthe binary MnADP to the ternary complex with enzyme. Aqualitatively similar relationship is found for the ATP binaryand ternary complexes.

An analysis of the molecular parameters which determine themagnitude of the enhancement of PRR in a particular complexof manganese, as discussed in detail elsewhere (4, 13), shouldlead to greater insight into the nature of the interactions in theternary complexes. For any molecular species, in this casewater, undergoing chemical exchange between two environ-ments, namely the solvent and the coordination sphere of aparamagnetic ion, it has been shown (22) that the paramagneticcontribution, 1/T 1p, to the observed longitudinal relaxation rateof the protons of water, 1/T,*, may be expressed as

1/Tp = T-I TI Tl(o) r n T a

(20)

where 1/T( 0) is the relaxation rate of pure water; P is the ratioof the number of protons in the first coordination sphere of themanganese which can equilibrate with the bulk water to the

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total number of protons in the solutions; r_ is the lifetime of awater molecule in the coordination sphere, i.e. the reciprocal ofthe chemical exchange rate of water coordinated to the metalion; and TIM is the longitudinal relaxation time of the waterprotons in the coordination sphere of the metal ion.

The term Ti1 is proportional to the reciprocal of r,, the cor-relation time for the dipolar interaction; in the case of the man-ganous aquocation, rc is the rotational correlation time of water.For aqueous solutions of manganous ions at 25 °, Bernheim et al.(21) have determined rm = 2.8 X 10-8 see and TiM = 2 X 10-6sec, so that rT is small compared to T and has a negligibleeffect in determining 1/Tp, which is therefore P/Ta (cf.Equation 20). The magnitude of the parameters in this caseis such that the rate of the chemical exchange is sufficientlyrapid for the observed relaxation rate of water to be the weightedaverage of the relaxation rates in the coordination sphere of themetal and in the solvent.

For ternary complexes, with the large increase in relaxationrates compared to the aquocation, probably both rM and TMcontribute significantly to the relaxation rate and hence the en-hancement factor cannot be simply equated to an increase inrc, the rotational correlation time, but rather yields only a lowerlimit on Tr. For example, in the ternary complex manganese-ADP-enzyme, the PRR is enhanced by a factor of 19.4 relativeto Mn(H20)6; i.e. et = 1/Tlp(t)/1/Tp(a2 o) = 19.4. Since twowater ligands of Mn(H 20)6 have probably been replaced in theternary complex, Pt/Pn2o may be taken as 4/6; it then followsfrom Equation 20 that

(,TM + TIM)H2 29.1 (21)(TM + T)t

If we make the reasonable assumption that the rate constant ofthe chemical exchange of water (1/rM) in a ternary complex isequal to or less than in the manganous aquocation, then thelower limit for rM(t) = rM(n20) = 2.8 X 10-8 see, and, fromEquation 21,

2 X 10-TIM(I) = - 2.8 X 10 -s = 4.1 X 10-8 see (22)

29.1

This value of TM(t) represents an upper limit, and thereforer, in the ternary complex is at least 49 times greater than it is

TABLE VI

Comparison of enhancements with creatine kinase and

pyruvate kinase

The results for pyruvate kinase are taken from the previouspapers of Mildvan and Cohn (12, 13).

Enzyme Conditions Complex et

Creatine 0.05 M N- E-Mn 1.5kinase ethylmor- E-MnADP 19

pholine-HCI, E-MnATP 10pH 8.0; 24° E-Mn-creatine 1.2

E-Mn-phosphocreatine 1.3

Pyruvate 0.05 M Tris- E-Mn 33kinase HCI, pH 7.5; E-MnADP 20

0.1 M KCI; E-MnATP 1325° E-Mn-pyruvate 11

E-Mn-P-enolpyruvate 2.3

in the manganous aquocation, indicating that a large hindranceof the rotational motion of the water in the coordination sphereof manganese is induced by binding of MnADP to the enzyme.

Evidence that the chemical exchange rate has, in fact, notchanged very much in the ternary complex compared to theaquocation and, furthermore, that the actual value of T ofthe ternary complex is of the same order as its upper limit cal-culated above may be adduced from the preliminary experimentson the temperature dependence of the PRR presented in thispaper. Since rM decreases with increasing temperature andTIM increases with increasing temperature, the paramagneticcontribution to the relaxation rate, 1/T1i (f. Equation 20), willhave a positive temperature coefficient if TM is the predominantterm but a negative one if TIM is the predominant term. In themanganous aquocation, the temperature coefficient is negative(21), consistent with the predominance of TM in Equation 20;in the ternary MnADP-enzyme complex it decreases almost tozero although it remains slightly negative; and in the quaternarycomplex, MnADP-creatine-enzyme, the temperature coefficientbecomes positive. The tentative calculations of TM and i

in the ternary complex yielded values of the same order for bothparameters, neither term dominating in Equation 20, so thatthe temperature coefficient of 1/T,, although still negative,should be less so than in the aquocation. In the quaternarycomplex, 1/Tip(q) is less than 1/TlP(t); therefore (M + TM) >(rM + TM)t.9 If TM(,) was greater than TIM(t), the temper-ature coefficient of the relaxation rate would be more negativefor the quaternary complex. However the observed tempera-ture coefficient for the quaternary complex is actually positivefrom which it may be concluded that TM(q) > M(t). Therefore,the addition of creatine to the MnADP-enzyme complex de-creases the rate of exchange of water remaining in the metalcoordination sphere of the complex; i.e. the manganese is leaaccessible to the solvent water in the quaternary than in thwternary complex. We do not have sufficient information todecide whether there has also been a change in TIM, i.e. in ,

but since the change in the relaxation rate is small and the increase in TM is in the right direction, any change in r, would bsmall, unlike the large change in T, which occurs in the formstion of the ternary complex from the binary complex. Mondetailed temperature studies of both T and T2 will be undertaken in an attempt to obtain values of rM and TIM for eadcomplex (cf. Reference 22).

The complexes formed with creatine kinase differ qualitativelyfrom those formed with pyruvate kinase. Unlike creatine kinasin which a strong interaction of metal can be detected only the ternary nucleotide complexes, pyruvate kinase formsbinary metal-enzyme complex with 2 metal ions tightly boul

9 It has been assumed that the number of manganese ligandand therefore P, is the same for both the ternary and the quateunary complexes. There is no experimental evidence that a cretine-manganese bond is formed in a ternary complex with thenzyme. The possibility that the formation of the quaternacomplex produces a conformational change so that a water ligaleof the manganese ion is now displaced by a creatine or protelligand cannot be excluded. However, a change in P would h3ano effect on the temperature coefficient of 1/T 1P. The dominalof the contribution of TM, the chemical exchange lifetime, in tdetermination of 1/TIP for the quaternary complex, in contrato the ternary complex, follows from the observation that temperature coefficient of 1/TI, is positive for the quaternsacomplex and negative for the ternary complex and is independedof the magnitude of P.

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W. J. O'Sullivan and M. Cohn

per molecule, characterized by an enhancement of about 30

(12). Each substrate, not only the nucleotides, effects a con-ierable decrease in e upon addition to manganese-pyruvate

kinase (13). A comparison of the values of eb and t for theenzyme-manganese and enzyme-manganese-substrate com-plexes, respectively, shown in Table VI, illustrates the differencesin the patterns of the two enzymes. The similarity in values oftfor the two enzymes is probably fortuitous since other enzymesinvestigated, either of the pyruvate kinase or of the creatinekinase type, form ternary complexes of enzyme, metal, andnucleotide which yield quite different values of t.1°

Acknowledgments---We are indebted to Mr. J. S. Leigh for hisconstant advice on the magnetic resonance measurements. Weshould also like to thank Dr. J. F. Morrison for communicationof results from his laboratory prior to publication.

REFERENCES

1. KuisB, S. A., AND NOLTMANN, E. A., Enzymes, 6, 515 (1962).i2. KUBY, S. A., MAHOWALD, T. A., AND NOLTMANN, E. A., Bio-

chemistry, 1, 748 (1962).

10 For example, the ternary complex formed between man-ganese, ATP, and myokinase (ATP:AMP phosphotransferase,EC 2.7.4.3) has been found to have a value for et(MnATP-myo-kinase) > e t(MnADP-myokinase) which is the reverse of the orderfound for the ternary complexes formed with MnATP - andMnADP-, respectively, with both creatine kinase and pyruvatekinase (W. J. O'Sullivan, unpublished experiments).

3. MORRISON, J. F., AND O'SULLIVAN, W. J., Biochem. J., 94, 221(1965).

4. COHN, M., AND LEIGH, J. S., Nature, 193, 1037 (1962).5. COHN, M., Biochemistry, 2, 623 (1963).6. MORRISON, J. F., O'SULLIVAN, W. J., AND OGSTON, A. G.,

Biochim. Biophys. Acta, 52, 82 (1961).7. O'SULLIVAN, W. J., AND PERRIN, D. D., Biochemistry, 3, 18

(1964).8. KUBY, S. A., NODA, L., AND LARDY, H. A., J. Biol. Chem., 209,

191 (1954).9. MAHOWALD, T. A., NOLTMANN, E. A., AND KUBY, S. A., J.

Biol. Chem., 237, 1535 (1962).10. KUBY, S. A., NODA, L., AND LARDY, H. A., J. Biol. Chem., 210,

65 (1954).11. MILDVAN, A. S., AND COHN, M., Biochemistry, 2, 910 (1963).12. MILDVAN, A. S., AND COHN, M., J. Biol. Chem., 240, 238 (1965).13. MILDVAN, A. S., AND COHN, M., J. Biol. Chem., 241, 1178

(1966).14. WATTS, D. C., Biochem. J., 89, 220 (1963).15. COHN, M., AND TOWNSEND, J., Nature, 173, 1090 (1954).16. HALL, J. L., SWISHER, J. A., BRANNON, D. G., AND LIDEN,

T. M., Inorg. Chem., 1, 409 (1962).17. HuGHES, T. R., AND KLOTZ, I. M., Methods Biochem. Anal., 3,

265 (1956).18. MALMSTRM, B. G., VXNNGARD, T., AND LARSSON, M., Bio-

chim. Biophys. Acta, 30, 1 (1958).19. SASA, T., AND NODA, L., Biochim. Biophys. Acta, 81, 270 (1964).20. NIHEI, T., NODA, L., AND MORALES, M. F., J. Biol. Chem.,

236, 3203 (1961).21. BERNHEIM, R. A., BROWN, T. H., GTOWSKY, H. S., AND

WOESSNER, D. E., J. Chem. Phys.. 30, 950 (1959).22. Luz, Z., AND MEIBOOM, S., J. Chem. Phys., 40, 2686 (1964).

f$,gue of July 10, 1966 3115

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W. J. O'Sullivan and Mildred CohnCreatine Kinase Reaction

Magnetic Resonance Investigations of the Metal Complexes Formed in the Manganese-activated

1966, 241:3104-3115.J. Biol. Chem. 

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