270 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 12318

A D E N O S I N E T R I P H O S P H A T A S E A CTIV ITY OF

A D E N O S I N E T R I P H O S P H A T E - C R E A T I N E P H O S P H O T R A N S F E R A S E

T S U T O M U SASA* AND L A F A Y E T T E N O D A

Department of Biochemistry, Dartmouth Medical School, Hanover, N.H. ( U.S.A .)

(Received J u n e 4th, 1963)

S U M M A R Y

Creatine kinase (ATP-creatine phosphotransferase, EC 2.7.3.2) 1 has been found to have a low but constant amount of ATPase activity 2. The ratio of ATPase activity to creatine kinase activity remains the same during repeated crystallization and dnring repeated precipitations. Loss of kinase activity by a variety of treatments using phys- ical and chemical agents has been found to result in a proportionate loss of ATPase activity. Twice-crystallized preparations show no evidence of protein or activity heterogeneity on DEAE-cellulose column chromatography.

Kinetic studies like those carried out for the kinase activity 3 lead to the identical conclusion that MgATP 2- is also the true substrate for ATPase activity. It may be postulated that the trace ATPase activity is due to a water molecule acting as a nucle- ophilic agent instead of the creatine molecule.

INTRODUCTION

It was first suggested to us by Dr. M. COHN* that the crystallized creatine kinase (ATP- creatine phosphotransferase, EC 2.7.3.2) we had provided for her experiments contain- ed a trace of ATPase activity. Assay of other samples of enzyme preparations showed this to be generally true. This hydrolyzing activity is of such a low order of magnitude that by comparison with the creatine kinase activity, the ATPase activity may be considered absent as has been previously reported 5. It is only under conditions de- signed to detect very low catalytic activity that hydrolytic activity is apparent. The reversible kinase reaction is shown in Eqn. I and the ATPase activity in Eqn. 2.

M g A T P 2- + creat ine ~ - M g A D P 1~ + c rea t ine -P 2- + H + (i)

M g A T P 2- + H20 ---> MgAD Px- + Pl*- + H + (2)

This trace secondary hydrolyzing activity of a phosphate transferring enzyme recalls the very similar ATPase activity of hexokinase (EC 2.7.1.1 ) reported by

Abbrev ia t ions : PCMB, p -ch loromercur ibenzoa te ; NBS, N-b romosucc in imide ; NEM, N- e thy lmale imide .

* P re sen t address : The T o k u g a w a I n s t i t u t e for Biological Research, and the D e p a r t m e n t of Biochemis t ry , Un ive r s i t y of Tokyo, Tokyo (Japan) .

Biochim. Biophys. Acta, 81 (1964) 27o-279

ATPAsE ACTIVITY OF CREATINE KINASE 271

TRAYSER AND COLOWICK 6, a n d KAjI , TRAYSER AND COLOWICK 7. These a u t h o r s found

the ratio of ATPase to hexokinase activities to be about 5-8" io -8 and found no evidence on the basis of further experiments for the formation of an enzyme-phosphate inter- mediate. Similar to hexokinase, evidence on creatine kinase does not support the post- ulate of the formation of a phosphoryl-enzyme intermediate s. For creatine kinase, kinetic and other studies3, 8 support the postulate that there are two adjacent sites on the enzyme, one for binding the adenine nucleotide and the other for binding creatine or creatine phosphate. In terms of enzyme mechanism it appears that in ATPase activity, water merely substitutes for the creatine molecule.

Evidence is presented below that ATPase activity of creatine kinase is not a contaminant of the enzyme preparations but is a property of the enzyme molecule. Studies of some characteristics of the ATPase activity show close similarity to the properties of the kinase activity.

MATERIALS AND METHODS

ATP-creatine phosphotransferase

The enzyme was purified from rabbit skeletal muscle3,9,1° and crystallized at least twice. Specific activities under the standard conditions in the presence of MgSO 4 and the method of activity calculation using the second order rate constant were generally in the range 65-72 units/mg. The enzyme was stored for use as a concentrated solution (50-60 mg/ml) after thorough dialysis against o.ooi M glycine buffer (pH 9). Protein was determined by the biuret procedure of GORNALL, BARDAWlLL AND DAVID 11.

Reagents

Salts used were reagent grade.Glycine,gtycyigiycme,hlstidine and creatine hydrate were A grade (California Corporation for Biochemical Research). Nucleotides were the highest grade available from Sigma Chemical Company. Other chemicals and their sources included NEM (Schwarz Bio Research, Inc.), NBS (Matheson, Coleman and Bell), Tris (Sigma Chemical Co,).

Enzymic assay

Creatine kinase activity was me3:sured by determining the creatine phosphate formed under the standard conditions 9.

ATPase activi ty was measured by the inorganic phosphate liberated from ATP at 3 °0 , correcting for the blank mixture without enzyme incubated for the same t ime intervals. The io-ml final volume reaction mixture was composed of o.I M glycine buffer (pH 9.o), 6 mM magnesium acetate, i mM ATP added at zero time, and an en- zyme concentration of about 6 mg/ml. At o, 20, 30 and 4 ° min, 2-ml aliquots of the incubation mixture and the blank were each pipetted and rapidly mixed with i .o ml cold 24% trichloroacetic acid contained in separate tubes in an ice bath. Sample and blank were t reated in the same way. The denatured protein was rapidly removed by using Whatman No. 40 filter paper on pre-cooled glass funnels and the filtrate was

Biochim. Biopkys. Acta, 81 (1964) 27o-279

2 7 2 T. SASA, L. NODA

collected in tubes in an ice bath. Inorganic phosphate in 2 ml filtrate was determined by the KING procedure 1~ (added 6 ml 8% perchloric acid, I ml 5 % ammonium molyb- date, o.4 ml I-amino-2-naphthol-4-sulfonic acid solution and water to a total volume of IO ml in graduated test tubes). After standing at room temperature exactly IO min from the time of addition of the last reagent, the absorbancy at 66o m/, was read in a Beckman DU spectrophotometer and compared with the absorbancy of a standard containing o.5oo #mole phosphate.

Care was taken during trichloroacetic acid treatment to keep the solutions cold in order to minimize ATP hydrolysis. The initial rate of hydrolysis was determined by plotting the P1 values against time and it was found to be linear up to lO% hydrol- ysis of the total ATP.

R E S U L T S

The stoichiometry of the ATPase activity indicated in Eqn. 2 was shown by incubating for periods up to 3 h at 3 °o 0.07 mM enzyme with I mM ATP, 6 mM magnesium acetate, and o.I M glycine buffer (pH 9), followed by paper chromatography is. In- creasing amounts of Pl were shown by the inorganic phosphate determinations. In- cidentally, it was observed that ADPase activity of an enzyme preparation was less than 5 % of the ATPase activity, both being measured at 3 °° in glycine buffer (pH 9).

Activity ratio during purification

Creatine kinase and ATPase activities determined for the various fractions of the purification procedure ~ are shown in Table I. With increasing purity of the fractions and increasing creatine kinase activity the ATPase activity decreases. Early fractions compared to later purer fractions are very high in the hydrolyzing activity, possibly because of the presence of myosin. After crystallization the ratios of ATPase to kinase activities are constant with repeated crystallization. Table II shows the constancy of the activity ratio during repeated precipitations of the enzyme from a solution containing 3 mM MgSO, and 60 vol.% of 95% ethyl alcohol.

T A B L E I

CREATINE KINASE AND A T P A s E ACTIVITIES DURING ENZYME PURIFICATION*

I n c u b a t i o n c o n d i t i o n s f o r k i n a s e a c t i v i t y : i m~¢~ A T P , 6 m M M g S O , , o .o2 4 M c r e a t i n e , o . I M g l y c i n e , a t p H 9 a n d 3 o°. C o n d i t i o n s f o r A T P a s e a c t i v i t y : I m M A T P , 6 m M m a g n e s i u m a c e t a t e , o . I M g l y c i n e , a t p H 9 a n d 3 o°. T h e a m o u n t o f e n z y m e t a k e n w a s a d j u s t e d f o r t h e p a r t i c u l a r

a s s a y . Spec i f i c a c t i v i t i e s a r e e x p r e s s e d i n / * m o l e s P i p r o d u c e d ] r a i n / r a g .

Crea3ine kinase A TPase A TPase (units/rag) (unitslmg) Crealine kinase

F r a c t i o n I 5 .7 o . i 1 . 8 . lO -2 F r a c t i o n I I 29 0 .02 7 ° . 1o -5 F r a c t i o n I I I 45 o . o i 2 2 . lO -5 F r a c t i o n I V 54 i • lO -3 1 .9" lO -5 is* c r y s t a l l i z a t i o n 65 0 . 8 4 " IO -8 1 .3" IO-5 2 n d c r y s t a l l i z a t i o n 66 o . 8 6 . i o - s I . 3 • IO - s

* See re f . 9.

B i o c h i m . B i o p h y s . Ac ta , 8 i (1964) 2 7 o - 2 7 9

A T P A S E ACTIVITY OF CREATINE KINASE 273

TABLE II

CONSTANCY OF A T P A s E / C R E A T I N E KINASE RATIO DURING R E P E A T E D PRECIPITATION

AND DIALYSIS

The e n z y m e was prec ip i ta ted a t 3 m M MgSO 4 and 60 vol. % of 95 % e thano l a t -- i o ° and dia lyzed aga in s t cold o.ooi M glycine (pH 9.o), equa l in v o l u m e to a b o u t 3ooo × vo lume of e n z y m e

solut ion. Assay as in Table I.

A TPase A TPase Precipitation Crea$ine kinase (units/rag x x o ~

and dialysis (units/rag) x z o * ) Creatine kinase

Original s ample 63 o,81 1. 3 Once 65 o.83 t .3 Twice 63 0.86 1. 4 Thr ice 63 0.80 i . 3

I I I. I I = l

.1o

~ 3.o 7 / / / , / 7 7 0.03 ~ ~ o

2O Tube number

Fig. I. C h r o m a t o g r a p h y of e n z y m e on 2 × I9 -cm colur r~ of DEAE-cel lu lose . F re sh ly p repa red a n d twice crysta l l ized enzyme . 17 ° m g was e lu ted b y O.Ol-O.O 5 M T r i s - a c e t a t e buffer (pH 8.05) ( total v o l u m e 30o ml) w i t h l inear gradient . F low ra te was a b o u t io m l /h and collector was se t for 5-ml f ract ions . E lua t e s were concen t r a t ed b y prec ip i ta t ion as in Table II . © - - ©, a b s o r b a n c y a t 280 m/~ ( lef t -hand ordinate) ; . . . . . , concen t r a t i on o f Tris--acetate buffer ( f igh t -hand ordinate) .

Tube Creatine A TPase A TPase kinase (units~rag x zo ~

No. (units/mg) x zo s) Creatine kinase

37-38 58 0.77 1.3 39 63 o.74 1.2 4 ° 6o o.75 1.3 41 64 o.86 1. 3 42 6o o.75 1.3

43-45 58 o.7o 1.2

B i o c h i m . B i o p h y s . A c t a , 81 (i964) 27o-279

274 T. SASA, L. NODA

DE AE-cellulose chromatography

In further a t tempts to separate ATPase activity from creatine kinase activity, DEAE-cellulose chromatography was run in a cold room using linear gradient elution, o.oi to 0.05 M Tris buffer (pH 8.05). A sample of twice crystallized enzyme containing 17o mg protein was placed on a 2 × 29-cm column which had been thoroughly washed and equilibrated. Absorbancy at 28o m# was measured on each 5-ml sample from the fraction collector. Since a comparatively large quanti ty of enzyme is required for the ATPase activity measurements, when it was deemed necessary, several fractions were combined and the protein was precipitated in 3 mM MgSO4 and 60 vol.% of 95% ethyl alcohol at --lO% After centrifugation the precipitate was dissolved in o.ooi M glycine buffer (pH 9.0) and dialyzed overnight against the same buffer. Conditions for creatine kinase and ATPase assay are given in Table I. In preliminary trials it was found that enzyme solutions which had been stored near o ° for long periods as a con- centrated solution in o.ooi M glycine (pH 9.0) showed small additional peaks having ATPase/kinase ratios greater than that of the main peak. Even in these cases, however, the ATPase/kinase ratios ~ of the main peak did not change on re-running the sample through a second column.

Fig. I shows the elution pat tern of a flesh enzyme preparation. A single protein peak is found. Enzymic activities of various cuts are indicated in the legend of Fig. I. The ratio of ATPase to kinase activity is essentially the same for all fractions, thus supporting the proposition that the ATPase activity is a secondary property of the creatine kinase molecule and not a contaminant of the enzyme preparation.

Effect of various treatments on enzymic activities

In the following experiments unless otherwise specified enzyme concentration was adjusted to a final concentration of 2o mg/ml in o.o5 M glycine buffer (pH 9.o) and temperature was maintained at o ° with an ice bath. I f other buffers than glycine or a chemical reagent had been used, the protein solution was dialyzed overnight against o.ooi M glycine buffer (pH 9.o). I f insoluble protein was present at the time of assay, it was removed by centrifugation at o °. Protein was determined by the biuret procedure 11 and enzymic activities measured under conditions given in Table I. Table I I I summarizes the data for inactivation by a varie~r of agents. The agents include those known to cause denaturation of proteins (heat, acid and urea) and group specific agents (NBS reacting with t ryptophan and other grbups14, ~5 and mercuric chloride, PCMB and NEM reacting with the sulfhydryl group). In the various t reatments it is seen in Table I I I that the ratio of the ATPase activity to creatine kinase activity is essentially constant with variation of the condition of t reatment and furthermore, that the ratio is very nearly the same for all the different kinds of inactivation proce- dures.

Some experiments were done using PCMB as the inactivating agent. At pH 7.4, o.oi M glycylglycine buffer, ATPase as well as kinase activity was decreased in the presence of PCMB, but on treating the enzyme at pH 9.o, glycine buffer, only the ATPase activity was inhibited. This is the only exception found to the constancy of the ratios of the two enzymic activities by t reatment of the enzyme.

These experiments clearly show that t reatment leading to the decrease of one of

Biochim. Biophys. Acta, 8i (I964) 270-279

A T P A s E ACTIVITY OF CREATINE KINASE 275

TABLE III

T H E E F F E C T OF VARIOUS TREATMENTS ON ENZYMIC ACTIVITIES

Enzyme samples were treated for 3 ° min except as indicated, while at a concentration of zo mg/ml in o.o 5 M glycine buffer (pH 9) unless otherwise indicated and at o ° (except 5 °o heat treatment), followed by exhaustive dialysis against o.ooi M glycine buffer (pH 9) and centrifugation at o c

if precipitation occurred. Assays were run under conditions indicated in Table I.

Condition Creatine kinase A TPase A T P a s e Treatment varied (units/rag) (units/rag x x o s

× zo s) Creatine kinase

Heat (5 o°) Acid (pH 2.I)** U r e a

bIBS***

NEM

Mercuric chloride

PCMB

o.o5 M glycyl-glycine

(pH 7.4)

PCMB

5 m i D * 2 0 O.23 1 .2

60 in.in* 35 0.45 1.3 7.o M* 21 0.27 L3 NBS

io* 19 o.26 1. 4 enzyme

NEM 8* lO o.13 1.3

enzyme HgC1,

3" 20 0.30 1.5 enzyme PCMB

o 56 0.67 1.2 enzyme PCMB

I.O 33 0.43 1.3 enzyme PCMB

1. 5 25 0.28 I.I enzyme PCMB

2.0 14 o.17 1.2 enzyme PCMB

o 62 o.8o 1.3 enzyme PCMB

I.O 60 0.60 I.O enzyme PCMB

1.5 65 0.44 0.67 enzyme PCMB

2.0 61 0.26 o.4z enzyme

* Four or five lower values including control, not reported here, gave similar values of decreased creatine kinase and decreased ATPase activities compared to the control but with essentially the same ATPase/creatine kinase values,

** o.0 5 M glycine-HC1. *** o.o 5 M acetate (pH 5.2).

t h e c a t a l y t i c p r o p e r t i e s of t h e e n z y m e s i m u l t a n e o u s l y r e su l t s in a c o r r e s p o n d i n g loss

o f t h e o t h e r c a t a l y t i c p r o p e r t y , t h u s s u g g e s t i n g t h e ove r l ap , i f n o t t h e i d e n t i t y o f t h e

e n z y m i c s i t e fo r t h e t w o ac t iv i t i e s .

Kinetic properties of ATPase compared with creatine kinase

A p p l y i n g t h e a n a l y s i s p r e v i o u s l y u s e d for t h e c r e a t i n e k i n a s e f o r w a r d r e a c t i o n 3

t o t h e A T P a s e r e a c t i o n , we f o u n d c o m p l e t e l y a n a l o g o u s resu l t s . I n Fig. 2 t h e v a r i a t i o n

of t h e r a t e o f r e a c t i o n w i t h v a r i a t i o n o f A T P c o n c e n t r a t i o n a t 1.0 a n d 0.3 m M m a g n e -

s i u m a c e t a t e are s h o w n as e x p e r i m e n t a l p o i n t s a n d t h e s m o o t h cu rv es are d r a w n f r o m

a t h e o r e t i c a l c o n s i d e r a t i o n a s s u m i n g t h a t M g A T P 2- is t h e t r u e s u b s t r a t e for t h e re-

a c t i o n a n d a s s u m i n g v a r i o u s d i s soc i a t i on c o n s t a n t s u s e d p r e v i o u s l y 8. T h e c a l c u l a t e d

B;ochim. Biophys. Acta, 81 (t964~ 270-279

276 T, SASA, L. NODA

~D .>

I00

80

60

40

20

O

' ' I I 0 .5 I 2:

ATP(mM)

Fig. 2. E x p e r i m e n t a l and ca lcula ted init ial ra tes of A T P a s e reac t ion as effected by added A T P a t o . i M glycine buffer (pH 9.o) a t 3 o°. S moo t h curves , I m M and 0.37 m M m a g n e s i u m ace ta te , and b roken line, 0.3 ° m M m a g n e s i u m acetate , r ep resen t ca lcula ted theore t ica l values. Exper i -

m e n t a l po in ts : O, i m M m a g n e s i u m ace ta te ; O, o.3o mM m a g n e s i u m ace ta te (see text) .

curve assuming a total of o.3 mM magnesium acetate as added, gives a poor fit, but calculations using a total of 0.37 mM magnesium acetate give a much better fit. Since measurements of ATPase are run at very high enzyme concentration (about o.o 7 raM) and the ash content of enzyme is about o.11% (see ref. 5), it may be estimated that one mole of Mg*÷ corresponds to a total of about 0.05 % magnesium oxide as a sh - - a reasonable figure. Thus it seems justifiable to assume that the additional 0.07 mM Mg ~+ required to give a better fit of the theoretical curve could have come from the added enzyme. In Fig. 3 the plots of observed percentage of activity and the drawn

oo

8O

>,

so

> / "~ 4 o

n,-

2 O

o I I I 0 0.5 1.0 1.5 g,O

Magnesium acetate (raM)

Fig. 3. E x p e r i m e n t a l and ca lcu la ted init ial ra tes of A T P a s e reac t ion as effected by m a g n e s i u m ace ta te a t o . i M glycine buffer (pH 9.0) a t 3 o°. S moo th curves were d r a w n t h r o u g h ca lcu la ted values. E x p e r i m e n t a l po in t s : ©, i m M A T P ; O, 0.3 m M ATP . Values ob ta ined a t m a g n e s i u m ace ta te concen t ra t ion less t h a n 0. 5 m M were increased b y o.o 7 m M to allow for Mg 2+ a s s u m e d

b o u n d to e n z y m e (see text) .

Biochim. Biophys. Acta, 8i (I964) 27o-279

ATPAsE ACTIVITY OF CREATINE KINASE 277

curve of theoretically calculated activity for increasing magnesium acetate concentra- tion again show good agreement.

Because of the agreement of theoretical and experimental values for ATPase activity (Figs. 2 and 3), as in the case of the creatine kinase analysis, it seems justifiable to conclude that MgATP ~- is also the true substrate for ATPase activity.

As previously reported, creatine kinase is inhibited by HATP 3- present in a re- action mixture at low magnesium ion concentration and low pH's and furthermore it was found that observed and calculated reaction velocities are in agreement for a Ki value for HATP 3- of 0.3 mM (see ref. 3). Quite comparably, the Ki value for HATP 3- for ATPase activity has been found to be 0. 4 mM.

The similarity of the effect of pH on ATPase and creatine kinase activities is shown in Fig. 4 in which the rate of reaction at pH 9 was taken as IOO°/o . The pK of ATPase activity is about pH 6.5, the same value as that previously reported for creatine kinase activity 3.

t t O I

8 0

6c

o >

._>

© n.-

2 ¢

I 0 0 r:l I ~ - -

o I I ] I 5 6 7 8 9

pH

Fig. 4- Initial rates of reaction a~s effected by pH at3o ° and I mM ATP; 0 - - 0 , ATPase and [2]--[~, creative kinase activities measured as in Table I.

The determination of Km values for MgATP 2- at various pH's under conditions of excess magnesium acetate showed the Km for MgATP 2- to be essentially constant over the range pH 5.5-9 and recalls the constancy of Km with pH for creatine kinase over the same pH range. However, there was a quantitative difference in that Km for MgATP 2- for ATPase activity was found to be 0. 7 mM, compared to the previously reported value of 0. 4 mM for creatine kinase activity.

Anion inhibition of ATPase activity was observed to be essentially like that for creatine kinase activity. Kl values are shown in Table IV, calculated by the method of HUNTER AND DOWNS 16, together with the values for creatine kinase activity 3 for purposes of comparison.

Biochim. Biophys. Acta, 81 (I964) 270-279

278 T. SASA, L. N O D A

T A B L E I V

COMPETITIVE INHIBITION BY ANIONS

( kinase ) ( A T Pase ) (M) (M)

S o d i u m a c e t a t e 0. 4 o. 4 NaC1 o.12 o.14 KC1 o.12 o . i ,

N a N O 3 0.022 0 .03 N a 2 S O 4 0 . o o 6 o . o i

N a 3 P O 4 O.Ol 3 O.Ol 4 Na.zP~O 7 O.OI l O.OI A D P 0.2 7 • IO -a 0 .2 • i o -a H A T P 3- o.2 8 • i o - S o. 4 • i o - 3 " "

* P r e v i o u s l y r e p o r t e d v a l u e s 8. ** B y t h e m e t h o d o f H U N T E R AND DOWNS 16.

*** E s t i m a t e d b y t h e m e t h o d u s e d f o r k i n a s O .

Determination of the energy of activation by Arrhenius plots gave the same value of 9.6 kcal for the two activities in this study. This is quite in agreement with the value of i i . i ± 2 kcal previously reported 9.

D I S C U S S I O N

The constancy of the ratio of ATPase activity to creatine kinase activity during the latter stages of purification and repeated precipitations, on chromatography, and during a variety of enzyme inactivating procedures all point to the ATPase activity as being inherently a part of the ATP-creatine phosphotransferase molecule rather than being a trace of contaminating ATPase activity in the enzyme preparation. Close similarity of kinetic properties for the two enzymic activities gives further evidence of the purity of the enzyme preparation and strongly suggests that the inter- action of the common substrate, MgATP ~-, with enzyme molecule as observed in studies of the two activities must be essentially the same.

In addition to hexokinase and creatine kinase having nearly the same ratio of ATPase to kinase activity, the trace hydrolytic property of sucrose phosphorylase reported by WEIMBERG AND D O U D O R O F F 17 and of levansucrose reported by HESTRIN, FEINGOLD AND AVlGAD TM should be mentioned. If phosphatases be thought of as catalyzing transfer of the phosphoryl group to water, then the observed transfer of the phosphoryl group to alcohol can be thought to be a very closely related enzymic property as has been pointed out by AXELROD 19. In the presence of added alcohols of lower molecular weight, citric phosphatase more rapidly catalyzes the liberation of nitrophenol from nitrophenyl phosphate than in the presence of water alone as solvent S°. HEPPEL AND WHITFIELD 21 reported that ribonuclease can catalyze the formation of cytidine-3'-methyl phosphate from the 2',3'-phosphate ester in the presence of methanol. FINDLAY, MATHIAS AND RABIN 2z have studied the hydrolysis and alcoholysis of cytidine 2',3'-phosphate by RNAase to yield cytidine 3'-phosphate and the ester of cytidine 3'-phosphate respectively. Alcohols inhibit the hydrolysis of the sub-

Bioch im. B i o p h y s . Acta, 81 ( I 9 6 4 ) 2 7 0 - 2 7 9

A T P A s E ACTIVITY OF CREATINE KINASE 279

s t r a t e n o n - c o m p e t i t i v e l y w i t h r e spec t to t h e n u c l e o t i d e b u t c o m p e t i t i v e l y w i t h

respec t to wa te r . T h e effect of a d d i t i o n of f o r m a m i d e or d i o x a n e on t h e r e l a t i v e r a t e s

was m e a s u r e d a n d t h e a u t h o r s sugges t t h a t w a t e r or a l coho l is b o u n d to a specif ic s i te on t h e e n z y m e . F o r R N A a s e t h e r a t i o o f hyd ro ly s i s a c t i v i t y to a lcoholys is is a b o u t

o . I c o m p a r e d to lO -5 for t h e r a t i o o f A T P a s e to k inase a c t i v i t y for c r ea t i ne e n z y m e .

Th is g r e a t d i f ference in r a t i o does n o t s eem to c o n t r a d i c t t h e n o t i o n t h a t w i t h R N A a s e

b o t h a c c e p t o r molecu les a re b o u n d on t h e e n z y m e whi le w i t h c rea t ine k inase t h e g r e a t

d i f ference in r a t e s m i g h t be a t t r i b u t e d to t h e l ack o f b i n d i n g of w a t e r on t h e e n z y m e

i f i t be a s s u m e d t h a t t h e o x y g e n of w a t e r a n d t h e n i t r o g e n of c r ea t i ne a re of t h e s a m e

o rde r o f m a g n i t u d e in nuc leoph i l i c a t t a c k i n g ab i l i ty . T h e o b s e r v a t i o n t h a t c r e a t i n e k inase has a b o u t 5 % or less o f A D P a s e a c t i v i t y

c o m p a r e d to t h e A T P a s e a c t i v i t y (all m e a s u r e d in g lyc ine buffer (pH 9)), does n o t s e e m surpr i s ing since i t m i g h t be e x p e c t e d t h a t th is e n z y m e wh ich is e q u i p p e d to

f a c i l i t a t e t r ans f e r o f t h e ? - p h o s p h a t e o f A T P w o u l d n o t i n t e r a c t in t h e s a m e m a n n e r

w i t h t h e E - p h o s p h a t e o f A D P as w i t h t h e t e r m i n a l p h o s p h a t e o f A T P . P rope r t i e s o f t h e e n z y m e as an A T P a s e a n d as c r ea t i ne k inase a re u n i f o r m l y

s im i l a r for v a r i o u s k inds of t r e a t m e n t s . T h e s ingle d i f ference o b s e r v e d (Table III) is

t h a t tw ice m o l a r c o n c e n t r a t i o n s o f P C M B a t p H 9 i n a c t i v a t e A T P a s e b u t n o t t h e

k inase a c t i v i t y . Since A T P a n d c r ea t i ne b i n d i n d e p e n d e n t l y on t h e enzyme3, s, t h e

o b s e r v a t i o n m i g h t be i n t e r p r e t e d to i n d i c a t e t h a t e n z y m e - S H groups are n o t d i r e c t l y

i n v o l v e d in t h e b i n d i n g of subs t r a t e , b u t r a t h e r m a y be i n d i r e c t l y i n v o l v e d in m a i n t a i n -

ing or i n d u c i n g n e c e s s a r y changes o f e n z y m e c o n f o r m a t i o n .

ACKNOWLEDGEMENT

G r a t e f u l a c k n o w l e d g e m e n t is m a d e for s u p p o r t o f th is i n v e s t i g a t i o n b y R e s e a r c h

G r a n t 1-1-3599 f r o m t h e N a t i o n a l H e a r t I n s t i t u t e , N a t i o n a l I n s t i t u t e s of H e a l t h , U n i t e d S t a t e s P u b l i c H e a l t h Serv ice .

R E F E R E N C E S

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l0 L. NODA, S. A. KUBY AND I-I. A. L.kRDY, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 2, Academic Press, New York, 1953, p. 6o5.

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Biochim. Biophys. Acta, 81 (1964) 270-279