Biochem. J. (1970) 117,513-523 513Printed in Great Britain

Properties and Reaction with Iodoacetamide of Adenosine5'-Triphosphate-Creatine Phosphotransferase from Human

Skeletal MuscleFURTHER EVIDENCE ABOUT THE ROLE OF THE ESSENTIAL THIOL GROUP IN

RELATION TO THE MECHANISM OF ACTION

By I. KUMUDAVALLI,* B. H. MORELAND AND D. C. WATTSDepartment of Biochemi8try, G(uy's Hospital Medical School, London S.E.1, U.K.

(Received 1 December 1969)

1. The purification of creatine kinase from human and monkey skeletal muscleby horizontal electrophoresis on Sephadex blocks is described. 2. The purifiedenzymes are shown to have similar chemical and kinetic properties to the rabbitmuscle enzyme and a common mechanism is inferred. 3. Iodoacetamide has asimilar apparent second-order inhibition constant with the human and rabbitenzymes, but the inhibition does not go to completion with the former. This iseven more marked with the monkey enzyme, which has more reactive thiol groups,but inhibition is only about 50%. 4. Single substrates have little effect on theinhibition by iodoacetamide, but with the primate enzymes, in contrast with therabbit enzyme, high concentrations of ADP-Mg2+ plus creatine convert theessential thiol group from being pH-independent into one with a normal ionization.Low concentrations ofADP-Mg2+ plus creatine first enhance the rate ofinactivation,but cause protection as the reaction proceeds. These results are interpreted toindicate an activation of the thiol group on the subunit to which the substratesbind and a co-operatively induced decrease in the activity of the thiol group onthe other subunit which lacks substrates. 5. The effects of a substrate equilibriummixture on the rate of inhibition are essentially those of ADP-Mg2+ plus creatine.6. Since no substrate combination affords significant protection to the thiol groupassociated with the catalytic site to which the substrates are bound, it is concludedthat any mechanism involving the thiol group in a direct participation in thetransition-state complex of the catalytic reaction must be abandoned unless thetransition state is only a small part of the time taken for one catalytic cycle.

Most studies on mammalian creatine kinase(EC 2.7.3.2) have been on the enzyme fromrabbit skeletal muscle. The enzyme from mousemuscle appears to be essentially similar in itschemical and mechanistic properties (Hooton &Watts, 1966), whereas that from ox has asimilar amino acid composition with two reactivethiol groups located in similar amino acid sequences,although the overall 'fingerprint' pattern is ratherdifferent from that of the rabbit enzyme (Thomson,Eveleigh, Laws & Miles, 1968). A role for theessential thiol group per catalytic site in themechanism of action has been suggested by Watts& Rabin (1962). The oxenzyme hasnotbeenstudied,but that from chicken muscle appears to have

* Present address: Department of PhysiologicalChemistry, Medical School, University of Wisconsin,Madison, Wis. 53706, U.S.A.

17

properties incompatible with the suggested thiolfunction (Hooton, 1968). From an evolutionarypoint ofviewit appears improbable that the essentialthiol group has different mechanistic functions inthe kinases from different vertebrates or evenbetween vertebrates and invertebrates (Watts,1965, 1968; Thoai, 1968).The work now reported, which forms part of

our investigation into the nature of musculardystrophy, describes some chemical and kineticproperties of human creatine kinase that showsufficient differences from the rabbit enzyme to castdoubt on the validity of the Watts & Rabin (1962)mechanism.

Because of the difficulty of obtaining pure humancreatine kinase with a high specific activity theenzyme was also purified from the monkey Macacamulatta. It was found to be kinetically similar to

I. KUMUDAVALLI, B. H. MORELAND AND D. C. WATTS

the human enzyme and hence invaluable forchecking some experiments. Our available infor-mation on this enzyme is also reported.

MATERIALS AND METHODSMaterial8

Double-glass-distilled water was used throughout.Substrate8. Disodium ATP was obtained from Sigma

(London) Chemical Co. Ltd., London S.W.6, U.K., andfrom the Boehringer Corp. (London) Ltd., London W.5.,U.K. No difference was detected between the twopreparations. Other nucleotides were from BoehringerCorp. (London) Ltd. and creatine phosphate fromSigma (London) Chemical Co. Ltd. Creatine (BritishDrug Houses Ltd., Poole, Dorset, U.K.) was crystallizedfrom hot 1 mm-EDTA, pH7.5, and then from water, andfinally dried at 100°C.

Buffers. For electrophoresis, tris-acetate buffer, pH 8.6,and borate buffer, pH8.5, were prepared as described byKumudavalli & Watts (1968). Tris-citrate buffer, pH7.5,was prepared by dissolving 1.05g of citric acid and2.288 of tris base in a final volume of 1 litre. For thedetermination of pH optimum tris-HCl buffers, pH7.0,7.5, 8.2 and 8.8 and I0.1 at 30°C, and glycine-NaOHbuffers, pH8.8, 9.15, 9.6 and 10.3 and I0.1 at 30°C, wereprepared as described in Long (1961).

NN-Di-(2-hydroxyethyl)glycine (bicine) was preparedby the reaction of sodium chloroacetate with diethanol-amine (Khromov & Remisov, 1953). The product was twicerecrystallized from 80% (v/v) redistilled methanol,1 mM-EDTA, pH 7.5, and water. Bicine buffers wereprepared by titrating 0.5M-NaOH with a concentratedsolution of bicine to the desired pH and diluting thesolution to the desired ionic strength (equal to the Na+concentration) and finally rechecking the pH.

Other chemicals. These were ofthe best grade obtainableand, except where mentioned otherwise, were used withoutfurther treatment.

MethodsEnzyme assay. Except where mentioned otherwise the

general assay procedure used was by determination of theacid-labile creatine phosphate formed in 10min at 30°C ina total reaction mixture of 0.4ml (Virden & Watts, 1964).If necessary, correction was made for the non-linearity ofproduct formed as a function of enzyme concentration.Enzyme inhibition experiments. These were carried out

at 30°C in a total volume of 1 ml in bicine-NaOH buffer,pH8.5 and I0.1, or other buffer as mentioned in the text.The reaction was started by the addition of 0.05ml ofiodoacetamide, and 0.1 ml portions were transferredimmediately and after the appropriate time-intervals to0.5 ml ofice-cold 5mM-cysteine, pH8.5, in the bicine bufferto stop the reaction. The enzyme activity remaining wasthen determined as described above. The iodoacetamidewas purified as described by Watts, Rabin & Crook (1961).

RESULTSPurification of creatine kinase. Human skeletal

muscle was obtained as soon as possible, theoptimum time being 1-2h after death, and stored at

-20°C until used, usually within 1 week. Monkeyskeletal muscle was dissected out immediatelyafter the animal had been killed with anaesthetic,cooled in ice for 30min and used at once.

Purification of the human enzyme was firstattempted by the ethanol fractionation method ofKuby, Noda & Lardy (1954a) for rabbit muscle, butthis method resulted in poor yields and a very lowspecific activity. Satisfactory purification wasachieved by electrophoresis on Sephadex G-100blocks.Human muscle (usually less than 100g) was

extracted twice with 3vol of cold tris-citrate buffer,pH8.6, containing lmM-EDTA in an M.S.E homo-genizer at 00C. The homogenate was centrifuged atapprox. 34000g for 30min at 4°C, and the super-natant was decanted through EDTA-washed glasswool to remove lipid and then concentrated toapprox. 1 ml by vacuum dialysis (Virden & Watts,1964) against the same tris-citrate buffer at 30C.The concentrated solution was centrifuged again at34000g at 4°C and the supernatant used for electro-phoresis.

Preparation of Sephadex block. Sephadex G-100[Pharmacia (G.B.) Ltd., London W.13, U.K.] (6g)was allowed to swell for 3 days in the tris-citratebuffer, pH8.6, at 30C and changed at intervals toremove fines. Finally the gel was allowed to settleand as much buffer as possible decanted off. Thethick slurry was then poured into a Perspex frame(20cm x 12cm intemal dimensions) resting on aclean glass plate (20cm x 20cm). The excess ofbuffer leaked out from the edge ofthe frame and wasblotted with filter paper, leaving a firm block ofSephadex about 0.5cm thick. A certain amount ofexperience is required to achieve a block of thecorrect consistency, as removal of too much bufferwill result in the proteins migrating very slowly andthe block may crack. The block was kept coldduring these operations.

Application of sample and electrophores8i. Thesample was applied as a band about 0.5cm wideacross the width of the block 3cm from the cathodeend, leaving 1.S-2.Ocm on either edge of the block.The Perspex frame was removed and thick filter-paper wicks soaked in borate buffer, pH8.5, wereused to connect the block to the buffer vessels, whichcontained the same borate buffer. The apparatuswas set up in the Phaerograph (Mini 1965) electro-phoresis machine with the glass plate carrying theSephadex block resting on a thick aluminium plate,to provide optimum heat transfer to the refrigeratedchamber, but electrically insulated from it by a thinsheet of polythene. Electrophoresis was carried outfor 5h at 200V (constant voltage) and with thePhaerograph set to -3°C the temperature in theSephadex block was maintained at 2-30C.

After electrophoresis the block was divided into

1970514

MECHANISM OF CREATINE KINASE

0.5cm fractions, each of which was separatelyeluted with tris-citrate buffer, pH 8.6, and theSephadex was removed by filtering through asintered-glass funnel. The fractions of optimumenzyme activity were combined and reconcentratedby vacuum dialysis but with tris-citrate buffer,pH 7.5, and then further purified by electrophoresisagain on the Sephadex block at pH 7.5 with tris-citrate buffer, pH 7.5, for the block and the boratebuffer, pH 8.5, for wicks and electrode vessels. Theenzyme fraction was eluted from the blocks asbefore, concentrated by vacuum dialysis againsttris-HCl buffer, pH 8.6 and 10.1, and stored at-200C.Human muscle creatine kinase purified in this

way gave only a single protein band when analysedby starch-gel electrophoresis, and this coincidedwith the position of muscle creatine kinase activity.No other creatine kinase isoenzymes could bedetected. A single symmetrical protein peak wasalso obtained when the enzyme was passed down aSephadex G-100 column for molecular-weightdetermination. The monkey enzyme was purifiedin the same way with similar results.The specific activity of the human enzyme,

assayed by the procedure of Kuby et al. (1954a),was about 0.13 unit before purification, 16.5 unitsafter the first electrophoresis purification and25 units after the second electrophoresis purificationfollowed by a final dialysis against 1 mm-dithio-threitol (Calbiochem, Los Angeles, Calif., U.S.A.),pH 8.5. The purified monkey enzyme had a specificactivity of 89 units. These values compare with thatfor the rabbit enzyme of 75 units (Mahowald,Noltmann & Kuby, 1962).

Properties of human creatine kinase

Molecular weight. This was determined on acolumn of Sephadex G-100 by the method ofAndrews (1964), with horse myoglobin, ovalbumin,

human serum albumin and rabbit creatine kinaseas standards (assumed molecular weights 17800,45000, 68000 and 83000 respectively). Fourdeterminations gave values between 81280 and85000 with a mean of 82530. Throughout thispaper a molecular weight of 81000 is assumed forthe purpose of calculation. This is because thisvalue was first determined for the rabbit enzyme(Noda, Kuby & Lardy, 1954) and, although a highervalue has recently been reliably reported (Table 1),the value of 81000 is still used as a basis for calcu-lation so that comparisons with earlier workremain valid. In practice the difference is barelysignificant.

Molecular extinction coefficient. A sample of theenzyme (concentration approx. 15mg/ml) wasdialysed against several changes of water madepH 8.0 with dilute ammonia. Portions of thedialysed solution were used for measurements ofextinction at 280nm in a Unicam SP. 800 spectro-photometer for protein determination by the biuretmethod (Gornall, Bardawill & David, 1949),calibrated with Armour Pharmaceutical Co. (East-bourne, Sussex, U.K.) Standard Protein Solution,and for dry-weight determinations by freeze-drying to constant weight over phosphorus pen-toxide and using an EMB 1 electromicrobalance(Research and Industrial Instrument Co., LondonS.E. 22, U.K.). The dry-weight and biuret methodsgave identical results and for an assumed molecularweight of 81000 gave E'cn 7.1 x 104. This is identicalwith the value obtained by Noda et al. (1954) usingglycine buffer, pH 9.8 (Table 1). The molecularextinction coefficient of the monkey enzyme wasdetermined in a similar way (Table 1).

Nitrogen content and amino acid composition.These are compared with those of the monkey andrabbit in Tables 1 and 2.

(a) Nitrogen content (method communicated byDr G. R. Webster, Department of ChemicalPathology, Guy's Hospital Medical School). A

Table 1. Compari8on of some properties of creatine kinases from human, monkey and rabbit skeletal muscle

Details of methods and conditions used are given in the text. Tyrosine, tryptophan and thiol contents arecalculated for an enzyme molecular weight of 81000.

Human Monkey Rabbitenzyme enzyme enzyme

Nitrogen contentI cm

C280Molecular weightTyrosine (mol/mol of enzyme)Tryptophan (mol/mol of enzyme)Thiol groups/molecule of enzyme

FreeTotal in ureaTotal in guanidine hydrochloride

16.67.1x1048250019.55.5

1.676.666.66

7.95 x 104

19.55.5

17.07.1x 1048300019.07

(Noda etal. 1954)(Noda et al. 1954)(Yue, Palmieri, Olson & Kuby, 1967)(Noltmann, Mahowald & Kuby, 1962)(Noltmann et al. 1962)

2.0 1.6 (Bayley & Thomson, 1967)7.0 6.3 (Bayley & Thomson, 1967)7.0 7.7 (Bayley & Thomson, 1967)

Vol. 117 515

I. KUMUDAVALLI, B. H. MORELAND AND D. C. WATTS

known amount of enzyme was digested for 1 h with0.5ml of a mixture containing 7ml of concentratedsulphuric acid, 1 ml of syrupy phosphoric acid and100mg of potassium persulphate. The excess ofacid was neutralized with 5ml of 3.6M-sodiumhydroxide and the ammonia was determined by thephenol-hypochlorite method (Fawcett & Scott,1960). Ammonium chloride was used to prepare acalibration curve.

(b) Tyrosine and tryptophan. These weredetermined by the spectrophotometric method ofBeavan & Holiday (1952) with a Unicam SP. 800spectrophotometer. The absorption spectra of thenative and alkali-denatured enzyme are verysimilar to those for other phosphagen kinases(Virden, Watts & Baldwin, 1965) with the ratio ofextinction at 250nm and 280nm being 1:2.2.

(c) Thiol content. This was determined with5,5'-dithiobis-(2-nitrobenzoic acid) (R. N. EmanuelLtd., Alperton, Middx., U.K.) as described byEllman (1959), in sodium phosphate buffer, pH 8.0and 10.003, assuming 4"' 13 600 for the thionitro-phenate ion. Guanidine hydrochloride was preparedas described by Hooton & Watts (1966) and urea

Table 2. Amino acid compo8itions ofhuman, monkeyand rabbit muscle creatine kina8es

Analyses of the human enzyme were carried out byDr A. R. Thomson, Wantage Research Laboratory,Berks., U.K.; those of the monkey enzyme were carriedout with the assistance of Dr M. D. Melamed, Departmentof Chemical Pathology, Guy's Hospital Medical School.Data for the rabbit are from Kuby & Noltmann (1962).-, Not determined.

Amino acidAspartic acidThreonineSerineGlutamic acidProlineGlycineAlanineValineMethionineIsoleucineLeucineTyrosinePhenylalanineHistidineTryptophanLysineAmide (NH3)ArginineCysteine

Nearest integral number of molof amino acid residues/81000g

of protein

Human

enzyme

73314175456648461925642026286

6653327

Monkeyenzyme

66284575426435471824621728

55

32

Rabbitenzyme

83344174376225531925701930337

6552348

was stored as a 1OM solution over Bio-Deminrolit(The Permutit Co. Ltd., London W.4, U.K.)mixed-bed ion-exchange resin.

(d) Amino acid composition. Samples of creatinekinase were exhaustively dialysed against waterand freeze-dried before hydrolysis at 110'C withconstant-boiling hydrochloric acid in vials sealedunder vacuum. For the human enzyme duplicatesamples were hydrolysed for 16, 46, 64 and 136h.For the monkey enzyme triplicate samples werehydrolysed for 24 and 72h. The human-enzymehydrolysates were analysed on a Technicon auto-analyser and the monkey enzyme hydrolysates ona Locarte autoanalyser. Table 2 shows the bestvalues for the nearest integral number of aminoacid residues.

(e) 'Fingerprinting'. Creatine kinase (2mg) wasdenatured by heating at 90°C for 2min and thendigested with 0.02mg of trypsin (3 x cryst.;Worthington, Biochemical Corp., Freehold, N.J.,U.S.A.) in 2% (w/v) ammonium hydrogen carbonatesolution plus a drop of toluene (final volume 2ml)for 24h at 37°C. The solvent was evaporated by astream of hot air, the residue treated with 0.25ml ofacetic acid and the mixture sublimed at 600C. Thedried peptide mixture was dissolved in 0.01ml of

o''w *I ( Origin

E (bJ0~~~~

OciO0 00.

-, OO

Origin-09 Electrophoresis Q

Fig. 1. Comparison of the 'fingerprints' of creatine kinasefrom (a) human and (b) rabbit skeletal muscle. Aftertryptic digestion the peptides were separated on thinlayers of silica gel G. Experimental details are given in thetext. Peptides that are clearly different between the twospecies are shown in black. The peptides inside the brokenlines could not be separated sufficiently accurately forprecise comparisons to be made.

516 1970

MECHANISM OF CREATINE KINASE

)tic acid-water (10:0.4:90, by vol.) 0.4mm thick. The dried plate was lightly sprayed4. Peptide separation was carried out with the pyridine buffer and placed on the coolingyer plate (20cm x 20cm) of silica gel G plate of the Phaerograph (Mini 65) electrophoresisE. Merck A.-G., Darmstadt, Germany) machine. The sample (approx. 0.5mg) was applied,

electrical connexions were made via thick pads offilter paper soaked in the same buffer and 400Vwas passed for 2h at 0-3°C. After electrophoresisthe plate was dried and chromatographed at right-angles in butan-l-ol-pyridine-acetic acid-water

Ck / & \(68:40:14:25, by vol.) as described by Wieland,Georgepoulos, Kempe & Wachsmuth (1964). Theplate was dried again and the peptides were stainedwith ninhydrin in the usual way. Fig. 1 compares'fingerprints' of human and of rabbit creatinekinase.

Kinetic properties (a). Optimum pH. This wasmeasured only in the direction ofcreatine phosphatesynthesis (Fig. 2). The rabbit enzyme has a similar

, , , , broad pH optimum (Kuby, Noda & Lardy, 1954b)7 8 9 10 1I but slightly displaced towards higher pH values

pH (Fig. 2).

urn pH of human muscle creatine kinase in(b) Michaelis Constants. These were determined

of creatofehumanpmuscleycreatineat30'C. in in the usual way from Lineweaver & Burk (1934)(final vol. 2ml) contained4smm-ATp-Mg23+T plots (Table 3). In the forward reaction measurede, enzyme and lml of tris-HCl buffer in bicine-NaOH buffer, pH8.5 and 10.1, at 300Cf glycine-NaOH buffer, IO.1 (0). The solid the Km for creatine (concentration varied betweenymbols show comparable data for the rabbit 4mM and 16mM) was measured in the presence ofculated from Kuby et al. 1954b). ATP-Mg2+, each 4mM; that for ATP-Mg2+ (ratio

Table 3. (ompari8on of 8Ome kinetic constants for human and rabbit creatine kinase

Details of the conditions used are given in the text.

Km (mM)

SubstrateATP-Mg2+ (1: 1)

Creatine

ADP-Mg2+ (1: 1)

Creatine phosphate

Substrate varied

ATP-Mg2+ (1:1)

Creatine

ADP-Mg2+ (1:1)

Creatine phosphate

Human

pH enzyme

9.0 3.138.5 3.138.09.0 1.478.5 1.68.07.0 1.747.0 -

7.0 3.57.0 -

Rabbitenzyme

0.5

0.4516.0

6.10.80.55.06.0

Phosphate K, (mM)

Human Rabbitenzyme enzyme

8.5 62.08.59.0

7.07.06.3

40.0

35.0130

ReferencesKuby et al. (1954b)

Morrison & James (1965)Kuby et al. (1954b)

Morrison & James (1965)Kuby et al. (1954b)Morrison et al. (1961)Kuby et al. (1954b)Morrison et al. (1961)

Reference

Nihei, Noda & Morales (1961)

35.040 Nihei et al. (1961)

Vol. 117

pyridine--acebuffer, pH6.on a thin-lay(nach. Stahl;

loor

80C6El6.0mCa4-i-0

0._P.-4

60

40 F20

6

Fig. 2. Optimthe direction oassay mixture32mM-creatin4IO.1 (e), or oflines without senzyme (recal

517

I. KUJMUDAVALLI, B. H. MORELAND AND D. C. WATTSkept 1:1 over the concentration range 0.25mM to2.5mm) was measured in the presence of 20mM-creatine. Measurements ofKm were also made in theforward direction at pH 9.0 and I0.1, withoutbuffer, by using the titrimetric assay of Hooton &Watts (1966) and the same concentration range ofsubstrates.Measurements ofKm values forthereversereaction

in tris-HCl buffer, pH7.0 and 10.1 (Long, 1961),were determined by assaying the creatine liberatedby using the method described by RosenbergEnnor & Morrison (1956) for arginine. A creatinecalibration curve was prepared and the extinctionof the coloured complex measured at 535nm(Morrison, O'Sullivan & Ogston, 1961).

(c) Inhibition by inorganic phosphate. This is ofinterest because in the rabbit enzyme inorganicphosphate appears to compete for the site occupiedby the transferred phosphoryl group in the catalyticreaction. Hence it is a competitive inhibitor for

0.08

0.06

0.04

-0.6 -0.4 -0.220.251

0.20 F

0.15F

0.10 f

0.05

0.2 0.4 0.6 0.8 1.01/[ADPJ (mM'1)

-0.4 -0.2 0 0.2 0.4 0.6 0.8 1.0

1/[Creatine phosphate] (mM-')

Fig. 3. Lineweaver-Burk plots to show the inhibition ofhuman muscle creatine kinase by orthophosphate at 3000.When creatine phosphate was the variable substrate theADP and MgSO4 concentrations were both 4mm. Whenthe creatine phosphate concentrations was held constantat 12mm the ADP and MgSO4 concentrations were bothvaried so that the ADP/Mg2+ ratio remained 1:1. *,

Control without phosphate; o, measured in the presenceof lOOmM-orthophosphate. Other experimental detailsare given in the text.

ATP and creatine phosphate but a non-competitiveinhibitor for ADP and creatine (Nihei et al. 1961).A similar pattern of inhibition was found with thehuman enzyme. Fig. 3 illustrates data for thereverse reaction. Because inorganic phosphateinterferes with the phosphate assay inhibition wasdetermined titrimetrically at pH 9.0 for the forwardreaction under conditions similar to those describedabove for the determination of the Michaelisconstants but with 25mM-inorganic phosphateadded. Inhibitor constants derived from Dixon(1953) plots are listed in Table 3.

Inhibition by iodoacetamide. The initial velocityof inhibition of human creatine kinase by iodoacet-amide is proportional to inhibitor concentrationsused in our experiments and followed apparentsecond-order kinetics with a rate constant of4701molPlmin-I at 300C in bicine-NaOH buffer,pH8.5 and 10.1. This value is lower than thosefound for the rabbit enzyme, 600 and 815lmol-Imin-' at 200C by Hooton & Watts (1966) and 7001mol1lmin-1 at 20°C by O'Sullivan, Diefenback &Cohn (1966), but similar to those found for the mouseenzyme, 440 and 5201mol'lmin-1 (Hooton &Watts, 1966). On the other hand the purifiedmonkey enzyme yielded an apparent second-ordervelocity constant of 13001mol-1min-1, whichindicates a much greater reactivity by the essentialthiol groups of the native enzyme than for anyother species so far reported.

(a) Effect of pH. Fig. 6 (control) shows that theinitial velocity of enzyme inhibition is independentof pH over the range in which the enzyme showsgreatest stability. Similar results were obtained in0.05M-tris-HCl buffers adjusted to 0.051 and 0.11with sodium chloride, showing that the pheno-menon was not a feature of the buffer type or aparticular ionic strength.

(b) Effects of substrates. Apart from ATP-Mg2+(Fig. 4a) single substrates (substrate-magnesiumsulphate complex, always in the molar ratio 1:1,is considered as a single substrate) and analogueshad no effect on the rate of inhibition or the humanenzyme by iodoacetamide either in bicine-NaOHbuffer, pH8.5, or in tris-HCl buffer, pH 7.0. Theconcentrations used were: ATP-Mg2+, ADP-Mg2+and AMP-Mg2+, 0.25mM and 4mM; creatine, 8mM;creatine phosphate-Mg2+, 1mM. Single substrateshad no effect on the inhibition of the rabbit enzymeby iodoacetamide (Watts & Rabin, 1962).With ATP-Mg2+ plus creatine phosphate an

unusual effect was observed in that the enzyme wasimmediately activated by about 40% (Fig. 4b).However, the subsequent inhibition by iodo-acetamide was faster, with the enzyme being 50%inhibited in 3min rather than 5min as found with-out substrates. This substrate combination has notbeen investigated with the rabbit enzyme.

518 1970

7

MECHANISM OF CREATINE KINASE60

-I.. 40-0

,s 205

(a)

n U

i4 |(b)01S 0.5

cd 0.4

~40

03

4

> 0.2 -

.Sn 0. I

zI - _ '

0 5 l0 i5 20Time (min)

Fig. 4. Effects of ATP-Mg2+ and of ATP-Mg2+ pluscreatine phosphate on the rate of inhibition of humanmuscle creatine kinase by iodoacetamide at pH8.5 and300C. For graph (a) the reaction mixture containedenzyme and iodoacetamide (0.0625mM): e, alone; o, plusATP and MgSO4 (both 0.25mg); A, plus ATP and MgSO4(both 4mM). For graph (b) the reaction mixture containedenzyme and iodoacetamide (0.25mM): *, alone; 0, pluscreatine phosphate (Imm), ATP and MgSO4 (both 4mm).Experimental details are given in the Materials andMethods section.

.1404X0

~2020

(b)

80 _

60 -

_.-04.o40 -

20 -

0 5 lo 15 20

Time (min)

Fig. B. Effects of ADP-Mg2+ plus creatine on the inhibi.tion of human muscle creatine kinase by iodoacetamide atpH8.B and 30°C. For graph (a) the reaction mixturecontained purifiedenzymeandiodoacetamide (0.0625mM):*, alone; o, plus ADP and M9SO4 (both 0.25mm) andcreatine (8mm); A, plus ADP and M9SO4 (both 4mm) andcreatine (8mm). For graph (b) the reaction mixturecontained the dialysed muscle Ejupernatant as preparedfor electrophoresiis and iodoacetamide (0.0625mm): *,alone; o, plus ADP and M9S04 (both 0.2B5mm) andcreatine (8 mm).

The other dead-end complex, ADP-Mg2+ pluscreatine, has been reported to afford strong pro-tection to the rabbit enzyme against inhibition byiodoacetate and iodoacetamide (O'Sullivan et al.1966) and against digestion by trypsin (Lui &Cunningham, 1966; Jacobs & Cunningham, 1968).The human enzyme behaves differently and theeffectvarieswiththenucleotide-Mg2+ concentration.With 0.25mM-ADP-Mg2+ plus 8mM-creatine atpH 8.5 iodoacetamide enhanced the rate of inhibi-tion during the early phase of the reaction butafforded progressively more protection as thereaction proceeded (Fig. 5a). This result wasconsistently obtained in six experiments with twoenzyme preparations and also with a crude muscleextract (Fig. 5b). With the crude muscle extract asenzyme source the rate of inhibition was nearly

twice that of the purified enzyme and approachedmuch more closely to zero activity. However, inthe presence of the dead-end complex the inhibitioncurve levelled off with about 40% of the activityremaining for both enzyme preparations (Fig. 5).When the ADP-Mg2+ concentration was raised to4mM but with the same creatine concentration alarge enhancement of the rate of inhibition byiodoacetamide occurred and there was now noprotective phase (Fig. 5a). The effect ofpH on thisprocess was then investigated and revealed thatin the presence of 4mM-ADP-Mg2+ plus 8mM-creatine the essential thiol groups had become freelyionizing with pK about 8.6 (Fig. 6).The effect of an equilibrium mixture ofsubstrates

Vol. 117 519

I. KUMUDAVALLI, B. H. MORELAND AND D. C. WATTS

_h a* a*

50

40

8~-30-0

t20

.,I00

0 5 10Time (min)

15 20

0L7 8 9 l Fig. 7. Inhibition of human muscle creatine kinase byiodoacetamide in the absence and presence of an equili-

pH brium mixture of substrates at pH8.5 and 300C. Eachreaction mixture contained bicine buffer, pH 8.5 and IO.1,

Plot of the apparent second-order rate enzyme, creatine (8mm) and either ATP plus MgSO4k2, for the inhibition of human muscle creatine (both 0.25mm) (0) or ATP plus MgSO4 (both 4mM) (A).a function of pH in the presence and absence of The enzyme reaction was allowed to proceed to equilibriumt+ plus creatine at 300C. The reaction was (15min) before the addition ofiodoacetamide (0.0625mM).at in bicine-NaOH buffer, 10.1, at the pH 0, Control without added substrates.

values shown. *, Enzyme plus iodoacetamide (0.0625mM)alone; o, enzyme plus ADP and MgSO4 (both 4mM) andcreatine (8mM); the iodoacetamide concentration variedfrom 0.0625mm to 0.00625mm at the higher pH values.The solid line is a calculated curve for an ionizing grouppK 8.6.

was investigated by incubating either 0.25mM- or

4.0mM-ATP-Mg2+ with 8mM-creatine and enzyme

for 15min before adding the iodoacetamide. Theinhibition progress curves were similar to thoseobtained with ADP-Mg2+ plus creatine but lesspronounced (Fig. 7). With the low nucleotide-Mg2+concentration the initial velocity was the same as

the control, but the reaction progressively deviatedin the direction of protection; with the highnucleotide-Mg2+ concentration the rate of inhibi-tion was slightly enhanced. These findings are

again different from those obtained with the rabbitenzyme, where a substrate equilibrium mixture gaveabout 35% protection against inhibition (Watts &Rabin, 1962).

T'he effects of ADP-Mg2+ plus creatine and thesubstrate equilibrium mixture were also tested on

the inhibition of the monkey enzyme by iodoacet-amide. Essentially the same results were obtainedas with human creatine kinase (Fig. 8), and in thepresence of the dead-end complex a freely ionizingthiol group was formed (Fig. 9).

DISCUSSION

The significant discovery about human creatinekinase is the markedly different way in which itssubstrates affect the rate of alkylation by iodo-acetamide as compared with the rabbit enzyme.

One would expect the enzymes from two mammalsto be fairly similar in their properties, and inparticular those properties concerned with thefundamental features ofthe mechanism of catalysis.The differences in chemical properties (Table 1)amino acid analysis (Table 2) and 'fingerprints'(Fig. 1) are no greater than would be expected tooccur between two different mammalian groups.

Kumudavalli & Watts (1968) have shown thehuman enzyme to be composed of two dissociablesubunits just like that from rabbit. The 'finger-print' evidence supports this finding.The similarities also extend to the general kinetic

behaviour (Fig. 2). Although differences exist forthe Michaelis constants, that for creatine beingtenfold smaller for the human enzyme and that forATP+Mg2+ being sixfold larger,muchless differenceexists between those for the substrates for the backreaction (Table 3). This is understandable, sinceit is in the breakdown of creatine phosphate toreplenish the ATP supply for muscle contractionthat creatine kinase fulfills its main function andthus where the stabilizing influence of selection

28r

24

20 -

16

0

12

8

4

Fig. 6.constant,Xkinase as E

ADP-Mg2carried ot

520 1970

MECHANISM OF CREATINE KINASE

7

6

5

4

3

2

0

7 8 9 10

pH

Fig. 9. Effect of pH on the apparent second-order rateconstant of monkey muscle creatine kinase in the absenceand presence of ADP-Mg2+ creatine at 30°C. *, Controlwithout substrates; 0, plus dead-end complex as describedin Fig. 6. The solid line is a theoretical ionization curve

for a group with pK8.5. Experimental details are thesame as for Fig. 6.

Time (min)

Fig. 8. Effects ofan equilibrium mixture of substrates andthe dead-end complex, ADP-Mg2+ plus creatine, on theinhibition ofmonkey muscle creatine kinase at pH 8.5 and300C. Graph (a) shows the effect of substrate equilibriummixtures prepared with low (o) and high (A) concentra-tions ofATP plus MgSO4 as described in Fig. 7. Graph (b)shows the effect of the dead-end complex prepared withlow (0) and high (A) concentrations of ADP plus MgSO4as described in Fig. 5. *, Control curves obtained in theabsence of substrates.

would be greatest. A lower Michaelis constant forcreatine would make the enzyme more efficient inthe direction of creatine phosphate synthesis when,under physiological conditions, the availability ofATP would not be a problem. Hence changes in theMichaelis constants are indicative only of evolution-ary adaptation rather than any fundamental changein the catalytic mechanism. The inhibition charac-teristics of inorganic phosphate, which appears

to act by binding to the site of phosphoryl transferin the enzyme from both species, provides additionalsupport for the view that the mechanism ofcatalysisis the same in both. How, then, are the iodoacet-

amide inhibition differences explained, particularlywhen it is borne in mind that in the absence ofmodifiers the essential thiol groups of both enzymesshow the same unusual character of having a

reactivity that is independent of pH (Fig. 6).The greatest disparity lies in the behaviour with

ADP-Mg2+ plus creatine, which gives substantialprotection to the rabbit enzyme but produces a freethiol group in the human and monkey enzymes(Figs. 6 and 9). The only reasonable inference is thatthe behaviour of the thiol group in the presence ofthis dead-end complex is not an important featureof the transphosphorylation mechanism and reflectsinteractions involving the species-variable parts oftheenzyme. Withtheprimateenzymesthe type ofin-teraction varies with the ADP_Mg2+ concentrationin the reaction mixture. The low ADP_Mg2+ concen-

tration provides an effective concentration ofADP-Mg2+ complex in the reaction mixture of 0.095mM,as calculated from the stability constant of4OOM-1(Morrison & Uhr, 1966), and this would on averagesaturate only one of the two catalytic sites on eachenzyme molecule. In the reaction (Figs. 5 and 8) aninitial enhancement of inactivation by iodoacet-amide is followed by protection as the reactionproceeds. With the high ADP-Mg2+ concentration(effective concentration of ADP-Mg2+ complex3.11 mM) a high proportion of both sites on each

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Vol. 117 552 1

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522 I. KUMIUDAVALLI, B. H. MORELAND AND D. C. WATTS 1970

enzyme molecule would be saturated and enhance-ment of inhibition occurs throughout the reaction(Figs. 5 and 8). Clearly at low ADP-Mg2+ concen-trations the dead-end complex must activate onlythe essential thiol group in the subunit to which itbinds and at the same time, or on alkylation, thethiol group in the substrate-free subunit becomesstrongly protected, presumably as the consequenceof a conformational change. A small residualactivity (about 10%) after alkylation under similarconditions is found with the rabbit enzyme but waspreviously attributed to partial oxidation of thethiol groups (Watts et al. 1961). Partial oxidationcould not account for the high values of residualactivity observed with the very active monkeykinase (Table 1 and Fig. 8) and a conformationalchangeseemsthe onlyreasonable inference. Further,this conclusion is compatible with the report thatafter maximal inhibition of the rabbit enzyme thesmall amount of activity left can only be destroyedby the total denaturation of the enzyme (Noda,Nihei & Moore, 1961). That such conformationalchanges may involve some loss of activity issuggested by Fig. 4(b) in which the other dead-endcomplex, ATP-Mg2+-creatine phosphate, facilitatesactivation.An equilibrium mixture of substrates affects the

rate of inhibition of the primate enzymes by iodo-acetamide in a similar way to ADP-Mg2+ pluscreatine, but to a smaller extent (Figs. 7 and 8).There is possibly a small contribution by theenzyme-ATP-Mg2+ complex (Fig. 4a), but themajor influence is clearly the formation of theenzyme-ADP-Mg2+-creatine complex (Figs. 5 and8b). With the rabbit enzyme Watts & Rabin (1962)observed protection against iodoacetamide inhibi-tion by a substrate equilibrium mixture and usedthis finding as the basis for an enzyme mechanismin which the essential thiol group was protected inthe transition-state complex of the reaction. Theydid not investigate the effect of ADP-Mg2+ pluscreatine, and the strong protection these gave wasonly shown subsequently (O'Sullivan et al. 1966).The work reported here shows that there is now noevidence for protection of the essential thiol groupin the transition-state complex of the catalyticreaction and consequently no evidence for themechanism proposed by Watts & Rabin (1962).However, such a mechanism is not completelyeliminated, because if the transition-state complexrepresented only a small fraction of the time takenin the total reaction pathway then the measurableprotection would be very small. From this point ofview the formation of dead-end complexes may beadding further confusion to an already complicatedsituation. It must be concluded that at the presenttime the nature of the catalytic mechanism is stilla completely open question.

Some general evidence has already been forth-coming for conformational changes on the bindingof substrates to the rabbit enzyme (Samuels, 1961;Watts, 1963; Jacobs & Cunningham, 1968). It isnot surprising that such conformational changescan also be co-operative with the modification ofthe essential thiol group of one subunit causing theessential thiol group ofthe second subunit to becomeprotected. Co-operativity appears to be muchmore marked in the primate kinases than in therabbit enzyme, and this should provide a goodmaterial for further study of this phenomenon.Although alkylation of the thiol groups was not

determined quantitatively it seems inconceivablethat both thiol groups per molecule could have beenfully alkylated under the various conditions usedfor Fig. 8 and yet result in such different degrees ofresidual activity. However, this point has still tobe checked formally.The problem remains as to the role ofthe essential

thiol groups. The most attractive alternative todirect participation in the catalytic mechanism isa supporting role involving the mediation of aconformational change. The studies by O'Sullivanet al. (1966) on the properties of the alkylatedenzyme and the observation that alkylation withiodoacetamide renders the enzyme considerably lessstable than when iodoacetate is used (Watts, 1966)strongly suggest that this is at least part of theirfunction.

This research was supported by grants to I. K. andB. H. M. from the Muscular Dystrophy Group of GreatBritain. Equipment grants from the Medical ResearchCouncil and Science Research Council are also gratefullyacknowledged. We are indebted to Professor S. Cohen forproviding the monkeys and to Dr B. McArdle, Dr K.Mant and Dr H. F. Lunn for making the human musclesamples available.

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