Studies on Adenosine Triphosphate Transphosphorylases

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 245, No. 13, Issue of July 10. PP. 3305-3314, 1970

Printed in U.S.A.

Studies on Adenosine Triphosphate Transphosphorylases

IX. KINETIC PROPERTIES OF THE CRYSTALLINE ADENOSINE TRIPHOSPHATE-CREATINE TRANSPHOSPHORYLASE FROM CALF BRAIN*

(Received for publication, December 3, 1969)

HANS K. JACOBS AND STEPHEN A. RUBY

From the Laboratory for the Study of Hereditary and Metabolic Disorders and the Departments of Biological Chemistry and Medicine, University of Utah, Xalt Lake City, Utah 84112

SUMMARY

The steady state kinetics of the calf brain ATP-creatine transphosphorylase seems to be adequately expressed by a random quasi-equilibrium type of mechanism with a rate- limiting step at the interconversion of the ternary complexes and for a case without independent binding of the substrates. Values for the kinetic parameters at pH 8.8, 30”, have been deduced. The over-all equilibrium constant, calculated kinetically from the Haldane relations, agreed satisfactorily with the thermodynamic value assigned previously (Kuby and Noltmann, The enzymes, Vol. 6, 1962, p. 515). Values for K,, versus &, (i.e. intrinsic dissociation constants of the substrate from the ternary and binary complexes, respectively) differed very_significantly (e.g. for the forward reaction: KMgaTP2- and K-MpATPz- were 1.35 x 10m4 and 0.93 x 10e3 M, Kcreatine and Kcrestine were 3.7 X 10m3 M and 2.9 X 1O-2 M, respectively). The possibility might then be entertained, that at pH 8.8, an enhancement in binding of the individual substrate in the ternary complex has occurred compared to the binary enzyme-substrate complex (cj. Morrison and James, Biochem. J., 97, 37 (1965)). Also, it has been tentatively concluded that ADP3- may compete with MgADP- for binding to the enzyme with a type of inhibition at high ADPo concentrations which has been evaluated in terms of an abortive and inactive ternary complex with creatine phosphate2-, and which forms also in a random manner.

The kinetic parameters have also been estimated for the calf muscle isoenzyme at pH 8.8 (and calculated for the above mechanism). Certain distinguishing kinetic features of each isoenzyme at pH 8.8 are then briefly outlined.

A further comparison of the calf brain enzyme’s kinetic data obtained at pH 8.8 with those obtained at pH 7.4 reveal several significant differences in the derived parameters, especially in VET (with a large increase), in ~cr,p~- (with a large decrease), and in comparatively smaller (or even insignificant) decreases between respective Es1 and K,, values (in which case, the brain-type enzyme seems to approach the muscle-type enzyme in its kinetic characteristics).

* This work was supported in part by grants from the National Science Foundation and the National Institutes of Health. The eighth paper of this series is Yue et al. (2).

An over-all evaluation of the data (physical, chemical, and kinetic) gathered for the calf brain ATP-creatine transphosphorylase seems to lead to the conclusion that it is susceptible to gross conformational changes as a result of environmental influences, in contrast to the more stable molecular unit to be found in the muscle-type ATP-creatine transphosphorylase.

Several molecular properties of the crystalline ATP-creatine transphosphorylase from calf brain (1) have been delineated (2)l to provide a future basis of comparison with a few crystalline skeletal muscle and brain ATP-creatine transphosphorylases (3) (and their hybrids) which have been isolated in this laboratory from calf (3), man (4), and rabbit (5). A final comparison and analysis will be presented later (except for a pertinent kinetic comparison between calf muscle and brain isoenzymes given here) on all of these enzymes and these may aid in the elucidation of the mechanism or mechanisms of action and the relation of structure to enzymatic function. Further, since the rabbit muscle ATP-creatine transphosphorylase, first isolated in crystalline form by Kuby, Noda, and Lardy (5), has been the subject of extensive investigations, kinetically (e.g. summarized in 6, and cf. 7 and 8), chemically (e.g. reviewed in 6, and cf. 9), physicochemically (e.g. lo), and since its mechanism has been examined by a wide variety of techniques and approaches (e.g. reviewed in 6, cf. 11, also 12-25,26,27, and 28), it thus forms the framework on which to present these future comparisons between the two-chain (e.g. 10 and 29), muscle-type, brain-type, and hybrid ATP-creatine transphosphorylases. It is the belief that from such critical comparative studies, especially on the isoenzymes from the same species, certain subtle differences and similarities will so manifest themselves as to facilitate this final development of unified concepts in regard to their mechanism or mechanisms of action. This report will be concerned specifically with a preliminary kinetic analysis of the calf brain enzyme’s catalyzed reaction which may have a bearing on its

1 K. Okabe, H. K. Jacobs, and S. A. Kuby, Reactivity and Anal- ysis of the Sulfhydryl Groups of the ATP-Creatine Transphos- phorylase from Calf Brain, submitted for publication.

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3306 Kinetics of Calf Brain ATP-Creatine Transphosphorylase Vol. 245, No. 13

native molecular structure and mechanism. A preliminary report of this work has been presented (3).

EXPERIMESJTAL PROCEDURE

dfaterials-Preparations of the crystalline calf brain enzyme were made according to the method of Keutel et al. (1). The sources of other materials have been indicated (1). The isola- tion procedure for the crystalline calf muscle isoenzyme (3) will be presented later.2 Wherever possible, the best analytical grade chemicals or biochemicals were employed and reagents were prepared in glass-distilled double deionized water (degassed by boiling).

ilfethods’rhe potentiometric (titrimetric pH-stat) procedure (30, 31) was employed for the velocity measurements, in either direction, and utilized the Radiometer TTTlaSBR2C, with pH A 630T scale expander and autoburette-type ABUlb. The use of an 0.25.ml autoburette (i.e. 0.25-ml delivery volume equivalent to full scale recorder deflection) permitted twice the sensitivity previously described (30).

The method of calculations and the values assigned for the chelation and dissociation constants were as listed earlier (6). Calculations employed a fixed concentration of free magnesium of 1 X lop3 M Mg++ (adjusted with magnesium acetate) for the kinetic studies on either forward or reverse direction, and where either MgATP*- or creatine were variable substrates, or MgADP- and Cr-P* for the reverse direction, with each substrate varied at fixed concentrations of the complementary substrate. All reaction mixtures contained, in addition to calculated concentrations of substrates, 1 mg per ml of albumin to stabilize the enzyme (30) and 10m6 M EDTA (to complex traces of copper to be found in commercial samples of albumin (33). This concentration of EDTA did not significantly affect the calculated values for magnesium complexes or Mg&

For titrant, the concentration of NaOH ranged from 3 X 1OV’ to 1 x 10-2 N, dependent on the sensitivity desired for the forward reaction: MgATP2- + creatine* --f MgADP + 0-P*- + H+.3 Equivalent concentrations of HCl were utilized as titrant for the reverse direction: i.e. MgADP- + Cr-P- + H+ --f MgATP” + creatine*.

As will be seen, the steady state kinetics conformed to first degree velocity expressions (34) for two substrate reactions. For the quasi-equilibrium random mechanism similar to the one proposed for the rabbit muscle enzyme (6, 8), but differing in that independent binding is not invoked (7), the following defined intrinsic constants may be given as:

SCHEME 1

To determine the purity and concentrations of the nucleotides employed, spectrophotometric coupled enzymatic analyses were utilized (e.g. the hexokinase-glucose g-phosphate dehydrogenase system for ,4TP, and when coupled to ATP-creatine transphosphorylase with ADP replacing ATP, for Cr-P; and the pyruvate kinase-lactic dehydrogenase system for ADP). For the best nucleotide samples, these often agreed with spectrophotometric det,erminations (E2” = 15.4 X lo3 at pH 7.0 and 259 rnp (32)). To convert to absolute concentration units, the value for the stoichiometric ratio vn+ (6), [(H+) formed]/[(ATP)o disappeared] at pH 8.8 was taken as 1.00 as previously measured (30) and found to be equal to 0.95 f 0.02 at pH 7.4 (30”) under conditions of measurement (see below). Subscript 0 implies total concentrations and appropriate valences are assigned to each of the ionizable or complex species.

Kl MA @

E.MA+ B

+ Kv E.MC + D K

Et "rkxf Et= k+fr \'MC

E.MA.B = E*MC.D +E

+B \ 4 KZ E.0 t MA 4

V;,/E,=ke5 -b &I

// D+ EDtMc 'G

where MA = MgATPz-; B = creatine*; MC = MgADP; D = Cr -P*; and where it is presumed that the interconversions of the ternary complexes (EMAB F’ EMCD) represent the rate-limiting steps. For the limiting cases, where the concentrations of products may be set to zero initially (see text), then for the forward and reverse initial velocities, respectively:

vnf = ” InSI

K3 &KS ’ + (ii& + (B) + (MA) (B)

(1)

Calculations of MgATP*- were adequately made, over the range of concentrations explored, by employing the set of ap- prosimate conservation equations:

ATPo S MgATPs- + ATF + HATPa- + MgHATP + NaATP”-

Mg, Y Mg2’ + MgATP” + MgHATP-

Kl.Ka = KyK, (2)

Ymax vg =

KS K9 KTKO l+ (MC) + 0 + (MC) (a

(3)

K,.Ks = K,.K, (4

where v,,~ and vO” denote initial velocities for forward and reverse Nao s Na+ + NaATP”

For the reverse direction, for calculation of MgADP- and reactions’ Cr-I’*-, the set of conservation equations could be adequately For variable MgATP*- (MA) and fixed creatine (B), e.g. a

approximated by primary plot of 1 /vo versus 1 /(flfA) would yield

ADPo z MgADP- + HADPz- + ADP3- + MgHADP

+ NaADP* (5)

Cr-PO s CrNPz- + HCr-P + MgCr-P + NaCr-P

Mg, s Mg2+ + MgADP- + MgHADP + MgCrmP

Nao s Na+ + NaADP2- + NaCr-P

and from the appropriate secondary plots of slopes and ordinate- intercepts, and Relation 2, values for K,-Kd may be estimated for the forward reaction. Thus,

2 H. J. Keutel, H. K. Jacobs, K. Okabe, T. Allison, F. Ziter, R. H. Yue, and S. A. Kuby, in preparation.

Slope 0’3

3 The abbreviation used is: Cr-P, creatine phosphate. and


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Issue of July 10, 1970 H. K. Jacobs and S. A. Kuby 3307

Y-intercept = +- + $- k max mzix 0

(7)

Also, for variable (B) and fixed (M-4); again all four constants (and VL,,) may be estimated for the forward reaction. Within the experimental errors and calculation uncertainties, the values for the kinetic parameters proved to be the same and average values will be presented together with their ranges of uncertainty. Similarly, appropriate secondary plots and Relation 4 yield values for all four constants for the reverse direction (cf. anal- ogous graphical treatments of data by Dalziel (35) and Florini and Vestling (36)).

As a measure of self-consistency, estimation of a defined over-all equilibrium constant (6) of the system, i.e.

K = (Mc) (D) . (H+) eq (MA) 03

may be obtained from any of four Haldane relations (see text, Equations 16 to 19), and compared with the calculated thermodynamic value of 2.81 x lO-‘O given (6). It must be stressed as indicated before (6), that the absolute values of the kinetic parameters hinge to some degree on the values selected for intrinsic dissociation and metal complexation constants, moreover, since all values for kinetic parameters, in principle represent derived values, the numerical estimations contain within themselves some inherent uncertainties.

For the kinetic studies, aliquots of the dissolved crystalline enzyme were passed through columns, 10 x 210 mm, of Sephadex G-75 to remove (NH4)zSOc and to equilibrate against 0.01 M

mercaptoethanol-0.001 M EDTA-0.05 M Tris, pH 7.8. Aliquots were then distributed into stoppered polycarbonate tubes and frozen in liquid nitrogen; when required, tubes were removed from the liquid nitrogen and the contents slowly thawed (O’), and kept at ice bath temperature for the day’s work. With repeated freezing and thawing, activity slowly decreased, but all velocity measurements were corrected to a reference value of 200 peq min+ . mg-I; samples, not repeatedly frozen and thawed, seemed to retain a constant specific activity for many months. Samples of three preparations (No. 8, 9, and 10) had been utilized for the kinetic studies reported here, but since some activity loss in Preparation 8 (from approximately 243 units per mg to approximately 190 to 200) had taken place prior to the introduction of the liquid nitrogen procedure, and since some loss in activity usually seemed to occur4 within a period of several hours after passage through Sephadex G-75, all velocity measurements were normalized to a value of 200 units per mg, by repeated pH-stat analyses under standard conditions (1, 31) with each set of velocity determinations (see text). With the introduction of the normalization procedure, and with the use of the graphical analysis procedure described above, values for the derived intrinsic constants (Kl-Kg) appeared to be reproduci- ble within the estimated uncertainties to be described below.

RESULTS AND DISCUSSION

Kinetic Analysis-The kinetic analysis employed the random, quasi-equilibrium mechanism (see above), and additional con- fidence in the validity of such a mechanism could be obtained,

4 The unique-SH group reactivity of this brain enzyme towards molecular O2 with attendant losses in activity, in contrast to that of the muscle-type enzyme (37), will be described later (see Foot- note 1).

for example, by studies of product inhibition or by isotope exchange studies at equilibrium (38). I f the analogy to the rabbit muscle enzyme were to hold, where such studies (e.g. 28), especially product inhibition studies (6, 7, 37) had been carried out, and after considerations of possible dead end complexes (6, 7), the pattern of product inhibition tended to eliminate considerations of an ordered mechanism, then the random mechanism as presented above would appear to be most probable (provided the former restriction (6) as to independent substrate binding (cf. 7) were omitted in this case, see below). Further- more, with the demonstration for the calf brain enzyme of binary enzyme-substrate complexes for the reverse direction5 and of enzyme-nucleotide complexes for forward or reverse reaction,5 the possibility of any ordered mechanism could again be considered unlikely, if it presumed that binary enzyme-substrate complexes became insignificant in the steady state. Thus, the kinetic analysis which therefore evolved was that of the quasi- equilibrium random mechanism as presented. Additional justification for this mechanism for the case of the calf brain enzyme may be postponed when, it is hoped, both product inhibition and substrate-binding data may be presented. There- fore with the assumption as to mechanism made, it may now be shown from over-all velocity expressions that t.he methods of graphical analysis employed above will yield valid measures of the basic kinetic parameters, even independent of the assumption as to dead end complexes (6, 7). However, it appears necessary in the case of the calf brain enzyme to invoke additional considerations as to ADP3- inhibition at high ADPo concentrations, and in this case analyzed also in a manner other than that of the independent binding restriction made earlier (6) for the rabbit muscle enzyme case.

Independent of whether dead end complexes (6, 7) are formed or not, from the over-all velocity expressions for either case (i.e. with or without dead end complexes) the limiting expressions when both products approach “zero” (i.e. by initial velocity studies), for the forward or reverse reaction, reduce to the equations given; but of course, the product inhibition patterns will be altered. Thus, consider the mechanism as given above (see “Experimental Procedure” 6), and if either or both dead end

5 E. A. Noltmann and S. A. Kuby, unpublished observations. The calf brain enzyme molecule, in contrast to the muscle-type enzyme, is apparently more susceptible to environmental influences, such that interaction with CrwPz- or ADPa- (to form binary enzyme complexes) may be qualitatively followed by the technique of fluorescence quenching of its tryptophan fluorescence. Fur- thermore, in the case of the calf brain enzyme, to which the fluorescent probe, fluorescein mercuric acetate, had been attached to its two essential sulfhydryl groups (or one per subunit, see Footnote l), binding of ADP3- or ATP4- may be measured by fluorescence enhancement of the probe.

6 For the mechanism as given above (under “Experimental Pro- cedure”), and without dead end complexes, then :

VL (MA) (B) _ (MC) K&i,

"' = KIKS + Ka(MA) + Kd(B) + (MA) (B) 1

(14)

+ KlK,(MC) (D) + KlKdMC? + KIKsK.dD)

K&9 K7 IGIG and

JK~K~ = KzK4 (2) [K~K~ = KyKg (4)

where K'equil is the apparent equilibrium constant at a pH, i.e.


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3308 Kinetics of Calf Brain ATP-C~eatine Transphosphorylase Vol. 245, No. 13

vgf = (13)

KIK, + K,(MA) + K,(B) + (MA) (B) + &K&(D)

&& +

KlKdMC) (D) + K,(D) (MA)

&i-L KY

complexes, E. B’MC or E .MA. D, could form as they had been deduced for the ATP-creatine transphosphorylase from rabbit muscle (6, 7), then in addition to the reactions involving RI-K, (inclusive) described above, one may add :

if one assumes that an ADP3- (C) competes for binding wit,h MgADP- (i%fC) and not with CrmPz- (D), and that an abortive and inactive complex E.C. D forms in a random manner as for the basic mechanism (leading to another value for KCr,.+- for the abortive ternary complex with ADP3-, uiz. K’,) i.e. for the reverse direction :

K, 0-m + (MC) I

KP E.B.MC ( E.MC + B (9)

E.MA + D KY KS

( E-MA.D e ED + MA (10)

and the Equalities 2 and 4 are extended6 to include

i

K,K2 = KsK7 (11)

K,KI = KaK6 02)

which leads to the over-all velocity expression shown in Equa- tion 13 and which reduces (as it does also for the above mechanism without dead end complexes”) in the case where (MC) = (D) + 0, to the limiting forward velocity expression (1) given above.

Thus, the limiting velocity expressions for the mechanism presented above under “Experimental Procedure” will permit an evaluation of K&g values by suitable measurements of Al,,’ and vi as a function of either the metal-nucleotide concentration at various fixed values of the nonnucleotide, or the converse, provided of course that other species of the several possible ionizable and metal complexes of the substrates in solution, do not seriously interfere by inhibition. (This complex set of equilibria has been discussed earlier in the case of the rabbit muscle enzyme-catalyzed reactions (6) .) To facilitate this first set of kinetic analyses, a fixed concentration of free Mg++ = 1 X 1O-3 M, by introduction of magnesium acetate, was employed throughout. In the case of the forward reaction (i.e. MgATP2- + creatine* --f MgADP + CrmPz- + H+), this reduces the concentrations, at pH 8.8, of all nucleotide species (including ATPa-) other than MgATP” to a negligible concentration over the range of concentrations of ATPo employed. In the reverse direction, at pH 8.8, the ADP3- concentration thereby rises to one-third the values of MgADP- at a fixed value of free Mg++ = 1 X lo+ M, for the approximate conservation equations employed, and therefore ADP3- can approach very significant values at the highest concentrations of ADPo employed. It will be shown, however, that over most of the concentration range employed, ADPa- does not appear to interfere, but appears to cause a slight inhibition at values of ADPo 2 1 to 2 X 10m3 at pH 8.8. ‘However, at pH 7.4, the inhibition by ADPo appears at a somewhat lower concentration range than for pH 8.8 and is larger in its extent. the Mdi&,

By fixing also a systematic quantitative evaluation of the inhibition

by varying ADP& concentrations is made difficult and this will make the subject of further study, where ADPa- may in turn be systematically fixed. However, a very approximate idea of the dissociation constants for ADP+ may be obtained,

[(MC)(D)]/[(MA)(B)]. In the limits of (MC) = (D) + 0, the equation reduces to Equation 1 for v ,f (cf. also Equation 13). The four Haldane relations (as presented in the text Equations 16 to 19) follow from the definition of the selected eauilibrium const,ant (Equation 8) (6), and the Equalities 3 and 4. *

and which equilibria (designated by primed values) should be added to the basic mechanism (for K1-Kg) given above. (Note that Iis (or RCrupz-) remains the same.) Thus, K17Klg = KeK’, (in addition to K7Kg = K,K,) and the limiting velocity expression for reverse reaction is amended to :

Accurate values for K’?, K’,, and K’, are difficult to assess under these conditions and by successive approximations and curve fitting may yield only rough measures of these constants.

Kinetics at pH 8.8-Systematic measurements of the initial velocities as a function of one substrate concentration (with its paired substrate held fixed, and MgLL fixed at 1 x 10m3 M)

yield at pH 8.8 and 30” essentially linear double reciprocal plots, and therefore approximately first degree (34) velocity expressions. The results (in terms of double reciprocal plots of l/v0 versus l/substrate) for the forward reactions are given in Fig. 1, A and B, for variable MgL4TP2- at several fixed values of creatine* (1B) and for creatine*, at several fixed values of MgATPz- (1A). Insets A’ and B’ in Fig. 1 contain the secondary plots derived from the slopes and ordinate-intercepts of the primary plots. Similarly, Fig. 1, C and D, contains the data obtained for the reverse direction at pH 8.8, in terms of plots for the variable substrate MgADP and Cr=P*, respectively. The primary plots of Fig. 1, -4, B, and D, are essentially a set of linear curves which intersect at or near the second quadrant; and in the case of variable CrmP2- (Fig. lD), the common intersection is almost at the ordinate axis. In Fig. 1C (for variable MgADP, varied over almost a loo-fold range of concentrations) at the highest calculated values of MgADP (and therefore highest values of ADP3-), or lowest reciprocal value, a slight deviation from linearity may be observed (which becomes very noticeable at pH 7.4, see Fig. 2C) implying that ADP+ may cause inhibition. The common intersection for Fig. 1C (variable MgADP-), extrapolated from the linear portions, extends far removed into the second quadrant compared to the case of Fig. 1B for variable MgATP2- or for variable creatine* (Fig. IA). Results of the final analyses are given in Table I in terms of the calculated values for the derived kinetic parameters at pH 8.8. Accepting the analyses, it is of interest that


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n’ A TABLE I

B' B [Creolinc’]

“0 20~20 mi 200,

/

2 x IOJM

W~M~ATP2-lb10-3 holer-‘x lhlerl~10-3

[cr- P2-l

05rl0-4M /

ll/[MgAOP-Il~10-3 knoler-1 x lIlerl~l03

$dqAOP-:

5 110’5M

I x 10-4

2 x 10-4 3 I IO.4

f 8 x 10-4

W~MpAOP-lIW3 5 5.

-I +Y’ 0 5 IO I5 20

fI/[Cr-P*-It do-2 Imoler-’ x Mer).lO -2

FIG. 1. Kinetics, at pH 8.8 and 30”, of forward reaction, MgATP2- + creatine* -+ MgADP- + Cr~pz- + Hf, and of reverse reaction, MgADP + CrwPz- + H+ -+ MgATPz- + creatine*, catalyzed by ATP-creatine transphosphorylase from calf brain. Measurements conducted by potentiometric pH-stat procedure. Primary plots: A, l/vJ versus l/(creatine*) at several fixed values of (MgATPt-); (Mg;:,) fixed at 1 X 10-a M. (Mgo adjusted with magnesium acetate, see text). Secondary plots: A’ of slopes (0-O) and of ordinate-intercepts (O-0) of primary plots (A) versus l/(MgATPz-) ; secondary plots fitted to the expressions :

Slope = K3 KIK, 1

V f + z (MA) In&X V

Values for derived kinetic parameters at pH 8.8 (SO”) for ATP- creatine transphosphorulase from calf brain

Derived kinetic

parameters Defined equilibrium

Kl (zt 0.74) X 10-a K2 EB F? E + B (A 1.1) X lo-’

K3 EMAB * EMA + B (Koreatine 1 3.7 (f 0.4) x 10-s

K4 EMAB is

EB + MA (KM~ATPP-)~ 1.3~ (=t 0.45) x 10-4

Kt3 ED 8 E f D (&+p2-) 2.0 (h 0.6) x lo-2 K7 EMC * E + MC (IfMeADP-) 1.2 (zt 0.1) X 10-d

K3 EMCD F?

ED + MC (KGADP-) 1.0 (4~ 0.3) X 10e5

K9 EMCDG

EMc + D &r.,+-) 2.0 (AZ 0.1) x lo+

V fn:yd/Et EMAB + EMCD 2.0” (f 0.1) X 102 umoles min-1 rncl

V ::y/~, IEMCD 4 EMAB j 1 7.b” (h 0.2) X 10;

I I fimoles min-’ mg-l

a i?, denotes an intrinsic dissociation constant of the particular substrates from the binary complex. K, denotes a dissociation constant of the particular substrate from the ternary complex.

b If a correction is applied for an initial 20% loss in activity (see text). Vi,JEl = 2.5 X lo2 and V&,/E, = 8.8 X 1O1.

1 K4 1 Y-intercept = Al,, + c (MA)

B, l/z$ versus l/(MgATPz-) at several fixed values of (creatine*) ; (Mg:Ae)-_fixed at 1 X 1OF M. Secondary plots: B’ of slopes (O----O) and of Y-intercepts (O--O) from primary plots (B) versus I/(creatinef); secondary plots fitted to the expressions:

KzKa 1 Slope = -$- + - -

nlax v;,, (B)

& 1 Y-intercept = $- + - -

msx v;., (B)

C, l/vor versus l/(MgADP-) at several fixed values of (CrwPz-); (Mg::,) fixed at 1 X 1OW M. Secondary plots: C’ of slopes (O---O) and Y-intercepts (O---O) from linear, extrapolated, primary plots (C) versus l/(Cr-P”); secondary plots fitted to the expressions :

K8 KS& 1 Slope = vz + c (0)

Y-intercept = --+- + v+ -!- msx rnSX CD)

D, l/vO’ versus l/(Cr-P”) at several fixed values of (MgADP); (Mg:&) fixed at 1 X 1OW M. Secondary p1ot.s: D’ of slopes (O--O) and Y-intercepts (O--O) from primary plots (D) versus l/(MgADP); secondary splots fitted t,o the expressions:

K9 Slope = -

K7K9 1

KiX +zm

Y-intercept = KS 1

-L+-- V msx Cl,* (MC’)


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3310 Kinetics of Calf Brain ATP-Creatine Transphosphorylase Vol. 245, r\To. 13

K,, (for ES, F) E + Si) values are consistently much larger (7- to 12-fold larger) than the corresponding K,, (for ES,& F!

ES, + X,) values for MgATP2-, for creatinef, or in the reverse direction, for MgADP- and for CrmPz- (e.g. compare K, and

KS for MgADP-; Kl and Kd for MgATP+; K2 and Ii3 for creatine*; Kg and Kg for CrwPz-). A plausible explanation might involve a substrate pair-induced conformational change occurring within the ternary complexes (E. MA . B or E .MC. D)

to permit tighter binding of the magnesium nucleotide complex or its nonnucleotide substrate to the enzyme, than in the case where only a single member of each substrate pair is associated with the enzyme. Morrison and James (7) have invoked this hypothesis for the rabbit muscle ATP-creatine transphosphorylase and it is of interest, that the relative differences between &I and &, values (i.e. the dissociation constant for a particular substrate from the ternary versus binary complex, Table I) are comparatively large for each substrate, compared to the rabbit muscle enzyme, where the comparable situation was even considered to be negligible or relatively small in former analyses on the rabbit muscle enzyme (6, 8).

As in the case of the rabbit muscle enzyme (6, 11) (and shown by equilibrium-binding measurements), it appears that the uncomplexed species, ADP+, can compete for binding with MgADP- but only very tentative values may be assigned for the calf brain enzyme at pH 8.8, with relatively high uncertainties; only the order of magnitude is considered significant for the present, viz. I<‘, s 0.6 X 1OW M; K’s S 1.5 X 10s4 M;

K’g s 5 x 10v3 M (refer to the assigned equilibria involved). I f these values prove approximately correct, it would appear that at pH 8.8 the binding coefficients of ADP3- to the free enzyme or in the ternary complex are approximately one order of magnitude less than for the case of MgADP- (KT = 1.2 x 10-d M; Z& s 1.0 X lop5 M) with the value for KICrNP for the abortive complex (K’,) increasing only slightly and probably not significantly over the reactive ternary complex (1c9 = 2 x lo-+ M).

Although very large attendant errors are involved in such analyses of derived kinetic parameters (Table I), which hinge on the values for magnesium complexation and dissociation constants selected (6), nevertheless since identical values were employed here as utilized earlier in an evaluation of the thermo-

TABLE II

Values for derived kinetic parameters at pH 8.8 (SO’) for ATP- creatine transphosphorylase from calf muscle

Derived kinetic parameters

Defined intrinsic issociation constant

RXg*TP2- Zcreatine* K oreatine* K MgATPP-

&-P2-

KM,ADP-

K YgADP- K Cr-Pz-

Value

M

9.7 (zk 1.8) X 10-4 5.3 (* 3.8) x IO-2 2.1 (A 0.4) x IO-2 7.8 (zt 5.6) x 10-d 4.5 (31 0.42) x 1O-2 1.7 (* 0.22) x 10-4 9.4 (Yk 0.44) x 10-s 2.3 (zk 0.63) x lo+

2.2 (& 0.025) X 102pmoles min-1 mg-l

1.3 (i 0.42) X lo2 ,umoles min-’ mg-l

dynamic equilibrium value, e.g. MgATP2- + creatine* , Kequil

) MgADP- + CrNP2 + (H+) (viz. 2.81 (& 0.41) & lo-lo, for =I= 1~) a self-consistent mechanism would require that the estimate of Kequil from kinetic analyses (i.e. from its four Haldane relations) at least yield a similar value, within the uncertainties, although the attendant errors might yield fortui- tous agreement. From over-all velocity Equations 13 or 14 and Equalities 2 and 4 as given above, the four Haldane expressions follow from the definition of this assigned equilibrium constant (Equation 8). Thus at pH 8.8,

K = KLK,K~H+) 09”lI

Vk&,K3 = 3.16 x lo-10 (16)

K = ~LK~K,(H+) WUll

J&&K4 = 2.31 X lo-10 (17)

K = FLK,K,(H+) W”ll

VLJGK, = 2.78 x lo-'0 (18)

K = V',axK6K8(H+) = 2.61 x lo-‘0 esd VkdGK,

(19)

or an average value of Kequil = 2.72 x lo-lo (& 0.35) x lO+O). The agreement at pH 8.8 is surprisingly close to the assigned

value of 2.81 x lo-lo and testifies possibly to the precision of the pH-stat procedure for velocity determinations.

It is of interest at this point to present, at least briefly, a comparison of kinetic data obtained for its muscle enzyme counterpart, tiz. for the calf muscle isoenzyme, ATP-creatine transphosphorylase. More complete data will be given later.’ However, for the present, if it may be assumed that the kinetic analysis, as given for the brain isoenzyme, holds also for the muscle isoenzyme, then the kinetic data may be evaluated and the kinetic parameters summarized in Table II, for the calf muscle enzyme, at the same pH of 8.8 (at 30”) and under similar conditions (see “Experimental Procedure”) as for the calf brain enzyme (Table I). A comparison of Table II (for muscle-type), therefore with Table I (for brain-type) reveals the following significant differences, or similarities, in the derived kinetic parameters. (a) For the forward reaction constants, the muscle- type possesses much larger dissociation constants for dissociation of the substrates from ternary complexes, e.g. larger values for Ka (K ereatd and K4 (K MpATpz-); but similar values for K, and Kz (for dissociation from the binary complexes) are observa- bl;Orfa;dboth isoenzymes; and surprisingly, they possess similar V max values. (b) However, the differences between RX1 and

KS, values for the forward reaction catalyzed by the muscle enzyme are either insignificant (cf. i!?MgATp2- and &gATPz-) or are decreased by only 2.5-fold from Rcreatine to Kereatine (K2 versus KS), in contrast to the very much larger differences, viz. approximately one order of magnitude, observed for the brain enzyme (see above). (c) For the reverse direction, KCr++-

is almost 12 times larger for the muscle-type than the brain-type and Khlg~~p-- almost 10 times larger, whereas the equivalent values for &,, are again not too dissimilar for the two isoenzymes, which again reduces the differences between K,, and Es1 to about a-fold or less. The Vk,, for the reverse direction for the brain-type is surprisingly low compared to its VziFd (and apparently a characteristic of all brain-type enzymes studied thus

7 H. K. Jacobs, K. Okabe, and S. A. Kuby, in preparation.


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Issue of July lo,1970 H. K. Jacobs and S. A. Kuby 3311

[M~ATP*~ .,-4..

-5 0 5

(I/[Creotine’1l~l0~* holes~l x liler)~lO~*

B' B

[Creatlne’]

2r~~-3~

4x10-3 W[Creotine’lblO~*

I x 10-Z

4 x 10-2

,; 2’0

ll/[MgATP2~1)~10~3 holes~‘x liter),10-3

(moles-t x liter I .10m3

(I/[Cr-P*‘ll 10-2 holeil x liter1 ‘IO‘*

FIG. 2. Steady-state kinetics at pH 7.4, 30’ of reactions catalyzed by calf brain ATP-creatine transphosphorylase. Primary plots: A, l/vJ versus l/(creatine*) at several fixed values of (MgATPz-). (Mg:LJ fixed at 1 X 10m3 M; Mg, adjusted with magnesium acetate (see text), Secondary plots: A’ of slopes ( O-O) and of ordinate-intercepts (O--O) of primary plots (A) versus l/(MgATP3-). B, l/vJ versus l/(MgATPz-) at several fixed values of (creatine’); (MgTr:,) fixed at 1 X lo-) M. Secondary plots: B’ of slope (O--O) and Y-intercepts (O--O) from primary plots uersus (B) l/(creatine*). C, l/vor uersus l/ (MgADP) at several fixed values of (CrwPz-); and Mg:ze fixed at 1 X W3 M. Secondary plots: C’ of slopes (O--O) and Y-intercepts (O-O) from linear, extrapolated, primary plots

far) and within a factor of two of the Vgly for the muscle-type. These characteristic points will be briefly dealt with again; suffice it to say here that, interestingly, certain very noticeable differences and similarities are made apparent between the two isoenzymes (and parenthetically it would appear that, surprisingly, both enzymes appear to be nicely adapted to their respective internal environments, provided correlations may be drawn between rates determined by both KS, and & and the physiological substrate concentrations under resting or certain steady state conditions).

Kinetics at pH 7...$-In addition to the results presented above for pH 8.8, kinetic measurements on the calf brain enzyme were also conducted at pH 7.4 and evaluated with the use of a stoichiometric ratio, Y=+, experimentally determined for this pH value (see “Experimental Procedure,” by removal of aliquots from the pH-stat vessel followed by nucleotide analysis). These results at pH 7.4, as will be seen, indicate some quantitative differences in the intrinsic constants as well as a relatively large increase (almost one order of magnitude) in VLax for the reverse direction, for example, as compared to pH 8.8. Thus, kinetically operable ionization constant or constants or pH-related conformational changes (or both) in the ternary complex, may have to be entertained in the final analysis of its kinetic mechanism. The gathering of these data at pH 7.4 as well as at pH 8.8 had been prompted by two reasons. (a) The brain enzyme displayed a surprisingly low value of Vii,, at pH 8.8 compared to VL,, (Table I) and which might therefore be the result of an ionizable function kinetically operable in the ternary complex (i.e. H+.E. MC.D). (Parenthetically, one may note again that this feature of a relatively low value of Vi’%? at pH 8.8 seems to be characteristic of the brain type of enzyme in contrast to the muscle type of enzyme as indicated above and as will be revealed in a future communication which will deal with an interesting kinetic comparison of the calf muscle, calf brain, and hybrid ATP- creatine transphosphorylase.7) (b) The general lability of the brain enzyme (1, 2) might be reflected in pH-dependent conformational changes which in turn might lead to secondary changes in the kinetic parameters.

The results at pH 7.4 are presented graphically in Fig. 2. Qualitatively, one may discern significant differences between Fig. 1 for pH 8.8 and Fig. 2 for pH 7.4, e.g. in the primary plots for MgATP” and for MgADP- as the variable substrate (cf. Fig. 2, B and C and Fig. 1, B and C) and especially in the case of CrmPz- as variable substrate, where the common intersection of the last case (Fig. 20) is far removed into the third quadrant in contrast to the case for pH 8.8 (Fig. 1D) in which case it is almost at the Y-ordinate. Similarly, but less pronounced, the common intersection for MgADP-, extrapolated from the straight line portions, lies in the third quadrant (Fig. 2C) in contrast to the second quadrant for the pH 8.8 data (Fig. 1C). Further, the inhibition at relatively high values of ADPo is very discernible in Fig. 2C (depicted by the dotted concave-upward curves) ; and there is an almost one order of magnitude difference in the ordinate values for l/v0 at pH 7.4 (in Fig. 2, C and D) compared to pH 8.8 (Fig. 1, C and D) at comparable values for l/S1 and fixed SZ. These qualitative differences lead, in turn,

versus (C) l/(Cr-P2-). D, l/v? versus l/(CrmPZ-) at several fixed values of (MgADP); (and Mgtrt, fixed at 1 X lo+ M). Secondaryplots: D’of slopes (O--O) and Y-intercept (O--O) from primary plots (0) versus l/(MgADP).


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Kinetics of Calf Brain ATP-Creatine Transphosphorylase Vol. 245, No. 13

TABLE III

Values for derived kinetic parameters at pH 7.4 (SO”) for ATP- creatine transphosphorylase from calf brain

Defined intrinsic dissociation

constant

KM~ATPP-

Roreatine

LCltilX

KMgAW- gcr-z- KY~ADP-

K MgADP-

&-P2-

Value

M

1.8 (zt 0.3) x 10-4 1.3 (zt 0.5) x 10-z 3.0 (zk 0.4) x 10-s 4.7 (A 1.5) x 10-S 6.7 (+ 1.4) X 1OP 3.9 (32 0.3) x 10-s 3.0 (zk 0.3) x 10-S 5.2 (h 1.2) X 1O-4

1.2a (* 0.1) X 102pmoles min-1 mg-l

5.18 (+ 0.5) X 102~moles mine1 mg-1

Q Correcting for a 20% loss in activity (see text). V&/Et = 1.5 X lo2 and V&,/E, = 6.4 X 10%.

to quantitative differences in the derived values for the intrinsic constants at pH 7.4 as well as a relatively huge increase (almost one order of magnitude) in I%,, for the reverse direction, as compared to the values for pH 8.8. Thus, although it now appears likely that there exist kinetically operable ionization constants, or pH-related conformational changes in the ternary complex, t,hese kinetic data alone would not provide an un- ambiguous assignment of either of these two factors. However, in a following communication,1 data on the chemical reactivity of its -SH groups will provide additional support for pH-dependent conformational changes. The data at pH 7.4 are summarized in Table III, in terms of calculated values for the derived kinetic parameters, where it may be noted, firstly, that whereas VL,, is within 60% of the value at pH 8.8 (cf. Table I) V' max has increased at pH 7.4, over ‘i-fold to a corrected value of 640 pmoles (min)-l (mg)+ or to 27,250 moles (min)-l mole (catalytic sites)+ (assuming that the two polypeptide chains in the dimer of molecular weight 85,200 (2) contain two catalytic sites). It may be remarked parenthetically that this velocity would be more than sufficient to account for its physiological role in the central nervous system, via. that of a rapid resynthesis of ATPo from ADPo and CrwPo (39, 40), and that the kinetic parameters as given in Table III make the brain enzyme, in contrast to the muscle enzyme: fairly well adapted to the brain milieu and attendant steady state substrate concentrations. This is especially striking in the lower values for KCr,+z- and Kcreatine in the case of the brain enzyme and note the surprisingly large change (decrease) of almost 30-fold in the values of RCrNP (Ks) at pH 7.4 (Table III) versus pH 8.8 (Table I). It is of further interest to note here that the relatively large differences in the values for K,, versus Es, at pH 8.8 (ranging from 7- t,o 1Sfold increases, see Table I, for all four substrates) have decreased at pH 7.4 (see Table III) to much smaller differences which range only from an insignificant 1.3-fold change in values for the reverse reaction constants to about 4-fold change in the difference between the forward reaction constants. If the above interpretation at pH 8.8 has merit, i.e. involving substrate pair-induced conformational changes occurring within the ternary complexes (so as to enhance the binding of each substrate in the

ternary complex compared to the binary complex), then the argument would likely be extended to another pH-dependent conformational change of the enzyme itself, so as to permit a tightening of the protein structure at pH 7.4 versus pH 8.8 and near the substrate-binding sites, i.e. to a more rigid structure at pH 7.4 which would better resist environmental influences, and which would retain a structure similar to the final kinetically active ternary complex. Thus, the pH-dependent conformational change would lead therefore, at pH 7.4, to smaller or nonexistent cooperative substrate effects, by having the substrate-induced conformational changes overshadowed by this pH-dependent conformational change; and, as will be shown more clearly later,7 (see Table II and compare Table I) this facet of the brain enzyme kinetics at pH 7.4 (viz. K,, values approach- ing &&) therefore approaches muscle enzyme kinetics. One final argument to support this point of view may be seen in the ADP3- inhibition constants. As indicated above, the inhibition by ADPo is much more pronounced at pH 7.4 compared to pH 8.8 (cf. Figs. 2C versus 1C). If analyses by curve fitting and successive approximations are conducted in the same manner as above, for K'g, K',, and K',, highly tentative values of 0.3 x 10W4, 0.8 X 10e4, and 1.6 X 1O-3 M, respectively, are obtained. In contrast to the case for pH 8.8, at pH 7.4, the values of K *npa- (K',) for the abortive ternary complex and KMgAnP (3 x 10m5) for the active ternary complex do not differ by at least the same order of magnitude to be found at pH 8.8; moreover, the dissociation of CrwPz- from the abortive E.ADP+. CrNPZ- ternary complex, i.e. K',, and Kg (KCr,+-2 = 5.2 x

10m4) also differ only by a factor of 3-fold, as well as the binding of ADP3- and MgADP+ to the enzyme (KfADPa and ffMMpADP3-) which approach similar values within a factor of 3-fold (KIT versus KT) at pH 7.4, provided these very highly tentative values derived by curve-fitting may be taken as significant. In any case, the enhanced inhibition by ADPo is explainable qualitatively by the decreased values for K’?, K'*, K',, at pH 7.4 versus pH 8.8, relative to the values of K6-Kg; such enhancement in binding perhaps could be the result of the pH-dependent conformational change in the protein so as to permit tighter binding at pH 7.4 versus pH 8.8 of the uncomplexed ADP3- (versus MgADP) to the enzyme and also within the abortive ternary complex containing CrmPz-. Finally, and in confirmation of the consistency of the derived parameters obtained for the proposed mechanism at pH 7.4, estimates of the four Haldane constants are again presented as was done for pH 8.8 (see above, Equations 16 to 19), viz., 3.02 X lo-lo, 3.09 x lo-lo, 3.1 x lo-lo, 3.49 x lo-lo; with an average value of Kequil at pH 7.4 = 3.3 x lo-lo f 0.23 X lo-lo (for f la); which is in reasonable agreement with the assigned thermodynamic value (6), viz. 2.81 (rt 0.41) x lO+O, after consideration of the combined uncertainties.

FINAL DISCUSSION

In summary, then, it is highly probable that both kinetically operable ionization constants within the pH region of 7 to 8 and superimposed pH-related conformational changes in the ternary complex may have to be considered in the final analysis of the kinetic mechanism of the calf brain enzyme (but kinetic data obtained at only pH values of 8.8 and 7.4, and for such a complex system are insufficient to make a final assignment of these ionization constants). Moreover, cooperative substrate-binding effects at pH 8.8 are likely the result of paired substrate-induced


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Issue of July 10, 1970 H. K. Jacobs and X. A. Kuby 3313

conformational changes in the ternary complex containing the magnesium nucleotide and guanidine base substrate. But much smaller effects are to be observed at pH 7.4, which is very likely the result of a general tightening of the tertiary structure of this unusual protein which appears easily altered in its properties by environmental influences (and to be discussed and summarized later). That the native two-chain brain enzyme (2) contains a “looser” or less restrained geometric structure than the calf muscle enzyme (the subject also of a future communication) seems evident from the kinetics of -SH titrations under denaturing conditions,l and from its susceptibility to oxidative conditions1 (1, 2), from the possibility of the relatively large conformational changes discussed above which may occur during kinetic formation of the ternary complexes and relatively smaller ones to be found in the calf muscle enzyme7 (also see Table II), and from the general lability of the enzyme, e.g. under treatments at acidic and alkaline pH values (1, 2), especially under elec- trophoretic conditions (2). This brain enzyme may then contain within it the direct means of studying conformationally dependent processes (and hinted at from these kinetic analyses), and perhaps interactions between catalytic sites, presumably one per subunit for the noncovalently linked two polypeptide chain structure (2), whereas such studies seem difficult indeed in the case of the more “tightly knit” and stable rabbit muscle enzyme structure (10). Also, in the case of the more refractory rabbit muscle enzyme, efforts to observe directly by several physical means conformational changes accompanying substrate- binding or substrate-dependent conformational changes, have not been too successful or revealed only relatively small or minor changes, e.g. in sedimentation coefficients, hydrogen-deuterium exchange, intrinsic viscosity, optical rotatory dispersion (e.g. 12-14), or in the lack of spectrophotometric changes accompanying binding of creatine (e.g. 15). Such postulated conformational changes for the rabbit muscle enzyme, accompanying substrate binding and suggested from the steady-state kinetics (7, 16, 17), have been deduced only indirectly, e.g. from alterations in reactivity of protein functional groups or effects on the kinetics of reaction with protein derivatization reagents when in the presence of substrates or equilibrium mixtures of substrates (12,14, N-20, 23), from susceptibility to tryptic digestions (12, 25), from anti- enzyme inhibition studies (22), or from solvent proton nuclear magnetic resonance relaxation rates in presence of paramagnetic ions such as Mnf2 (19, 27). It is of interest in this regard, that in a kinetic study on the rabbit muscle enzyme (24) by temperature jump means with a pH indicator to monitor rapid pH changes in the approach to equilibrium after temperature per- turbation, Hammes and Hurst (24) had interpreted their results in terms of conformational changes following the binding of either ADPa- or ATP4- or their metal complexes to the enzyme; whereas, no conformational change could be assigned to the binding of creatine or creatine phosphate. Any conformational changes (or “isomerizations” (24)) in the ternary complex or complexes could not be precisely defined by (24) because of the complexity of the relaxation processes involved when in the presence of metal nucleotides. Whether there are at least two types of isomerizations or conformational changes, one affecting the reactivity of certain reactive groups (e.g. -SH groups,8 and

8 As a result of an unusual oxidation-reduction reactivity of its exnosed sulfhvdrvl EVOUZ) (see Footnote 4). one Dossibilitv which may be enter&&d& tgat‘of an oxidative’reaction lead& to disulfide formation which in turn would lead to alterations in con-

a point which will be dealt wit’h later’) and the other affecting the pK values of certain ionizable residues possibly involved in the cooperative substrate effects, is equally tenable as discussed by Hammes and Hurst (24). It is of interest to note in passing, that at pH 7.6 and ll”, Hammes and Hurst (24) could detect no difference between rates of isomerization of the ADPa--enzyme complex and the metal nucleotide-enzyme complex (as revealed by pH changes) and is thus not in disagreement with the deductions reached here for the calf brain enzyme at pH 7.4. But it should be remarked that such rapid isomerizations would, in the steady state, not affect the steady state analysis based here on a rate-limiting step at the interconversion of the ternary complexes, and would thus not permit any unique assignments of isomerization other than those at the rate-limiting steps.

Finally, a future communication7 will deal with a kinetic comparison of the brain, the muscle, and hybrid isoenzymes (3,4) all of which display subtle differences in their gross physical and chemical properties and which seem to be reflected in their kinetics, and whose kinetic parameters, for the most part, may be correlated with their steady state intracellular environment. From such a study, it is hoped, that further insight may be gained into the problem of interactions between like and unlike catalytic sites (as manifestly reflected in the hybrid enzyme whose kinetic parameters do not lie precisely intermediate between muscle and brain-type? (4)) ; and some additional and general understanding permitted of the over-all mechanism of t,he ATP-creatine transphosphorylases.

Acknowledgments-We are grateful for the participation of Doctors H. Keutel, K. Okabe, and F. Ziter, and Mr. T. Allison in the preparations of the enzyme used in this study.

1.

2.

3.

4.

5.

6.

7. 8.

9.

10.

11.

12.

13. 14.

REFERENCES

KEUTEL, H. J., JACOBS, H. K., OI~ABE, K., YUE, R. H., AND KUBY, S. A., Biochemistry, ‘7, 4283 (1968).

YUE, It. H., JACOBS, H. K., OICABE, K., KEUTEL, H. J., AND KUBY, S. A., Biochemistry, 7,429l (1968).

JACOBS, H. K., KEUTEL, H. J., YUE, R. H., OKABE, K., AND KUBY, S. A., Fed. Proc., 27,640 (1968).

JACOBS, II., OKADE, K., YUE, R., KEUTEL, H., ZITER, F., PALMIERI, R., TYLER, F., AND KUBY, S. A., Fed. Proc., 28, 346 (1969).

KUBY, S. A., NODA, L., AND LARDY, H. A., J. Biol. Chem., 209, 191 (1954).

KUBY, S. A., AND NOLTMANN, E. A., in P. D. BOYER, H. LARDY, AND K. MYRBXCK (Editors), The enzymes, Vol. 6, Academic Press, New York, 1962, p. 515.

MORRISON, J. F., AND JAMES, E., Biochem. J., 97,37 (1965). NIHEI, T., NODA, L., AND MORALES, M. F., J. Biol. Chem.,

236, 3203 (1961). NOLTMANN, E. A., MAHOWALD, T. A., AND KUBY, S. A., J.

Biol. Chem., 237, 1146 (1962). YUE, R. H., PALMIERI, R. H., OLSON, 0. E., MALAND, L. J.,

AND KUBY, S. A., Biochemistry, 6,3204 (1967). KUBY, S. A., MAHOWALD, T. A., AND NOLTMANN, E. A., Bio-

chemistry, 1, 748 (1962). LUI, N. S. T., AND CUNNINGHAM, L. W., Biochemistry, 6, 144

(1966). KHGI, J. H., AND LI, T. K., Fed. Proc., 24, 285 (1965). SAMUELS, A. J., NIHEI, T., AND NODA, L., Proc. Nat. Acad.

Sci. U.S. A., 42, 1992 (1961).

formational integrity and attendant losses in enzymatic activity. I f such a mechanism prevailed within the nerve cell environment, an unique regulatory mechanism for this enzyme might play a role, which would depend on a reversible interconversion of its reactive sulfhydryl groups to disulfide bonds and re-reduction via, for example, glutathione.


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3314 Kinetics of Calf Brain ATP-Creatine Transphosphorylase Vol. 245, No. 13

15.

16.

17.

18.

19.

20. 21. 22. 23.

24. 25.

26.

27.

28.

ROUSTAN, C., KASSAB, R., PRADEL, L. A., AND THOAI, N. V., Biochim. Biophys. Acta, 167, 326 (1968).

MORRISON, J. F., AND UHR, M. L., Biochim. Biophys. Acta, 122, 57 (1966).

JAMES, E., AND MORRISON, J. F., J. Biol. Chem., 241, 4758 (1966).

O’SULLIVAN, W. J., DIEFENBACH, H., AND COHN, M., Biochem- istry, 6, 2666 (1966).

O’SULLIVAN, W. J., AND COHN, M., J. Biol. Chem., 241, 3116 (1966).

WATTS, D. C., Biochem. J., 89,220 (1963). RABIN, B. R., AND WATTS, D. C., Nature, 188, 1163 (1960). SAMUELS, A. J., Biophys. J., 1,437 (1961). PRADEL, L. A., AND KASSAB, R., Biochim. Biophys. Acta, 16’7,

317 (1968). HAMMES, G. G., AND HURST, J. K., Biochemistry, 8,1083 (1969). JACOBS, G., AND CUNNINGHAM, L. W., Biochemistry, 7, 143

(1968). THOMSON. A. R., EVELEIGH, J. W., AND MILES, B. J., Nature,

203, 267 (1964). TAYLOR, J. S., LEIGH, J. S., AND COHN, M., Proc. Nat. Acad.

Sci. U. S. A., 64, 219 (1969). MORRISON, J. F., AND CLELAND, W. W., J. Biol. Chem., 241,

673 (1966).

29.

30.

31.

32.

33.

34.

35. 36.

37.

38. 39.

40.

DAWSON, D. M., EPPENBERGER, H. M., AND KAPLAN, N. O., J. Biol. Chem., 242, 210 (1967).

MAHOWALD, T. A., NOLTMANN, E. A., AND KUBY, S. A., J. Biol. Chem., 237, 1535 (1962).

KUBY, S. A., Nat. Acad. Sci. Nat. Res. Count. Publ., 1344, 231 (1967).

BOCK, It. M., LING, N. S., MORELL, S. A., AND LIPTON, S. H., Arch. Biochem. Biophys., 62, 253 (1956).

YUE, R. H., NOLTMANN, E. A., AND KUBY, S. A., J. Biol. Chem., 244, 1353 (1969).

WONG, J. T. F., AND HANES, C. S., Can. J. Biochem. Physiol., 40, 763 (1962).

DALZIEL, K., Acta Chem. Scund., 11, 1706 (1957). FLORINI, J. R., AND VESTLING, C. S., Biochim. Biophys. Acta,

26, 575 (1957). KUBY, S. A., NODA, L., AND LARDY, H. A. L., J. Biol. Chem.,

210, 65 (1954). CLELAND, W. W., Annu. Rev. Biochem., 36,77 (1967). MCILWAIN, H., Biochemistry and the central nervous system,

Ed. 3, J. and A. Churchill, London, 1966. NACHMANSOHN, D., AND WILSON, I. B., Advan. Enzymol., 12,

259 (1951).


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Hans K. Jacobs and Stephen A. KubyBRAIN

TRIPHOSPHATE-CREATINE TRANSPHOSPHORYLASE FROM CALFPROPERTIES OF THE CRYSTALLINE ADENOSINE

Studies on Adenosine Triphosphate Transphosphorylases: IX. KINETIC

1970, 245:3305-3314.J. Biol. Chem.

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