Studies on Adenosine Triphosphate Transphosphorylases

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  • 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, respec- tively) differed very_significantly (e.g. for the forward reac- tion: 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 enter- tained, 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 com- plex 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 enzymes 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 in- significant) 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 transphos- phorylase 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 transphosphoryl- ase.

    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 mecha- nism or mechanisms of action. This report will be concerned specifically with a preliminary kinetic analysis of the calf brain enzymes 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.

    3305

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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).

    ilfethodsrhe 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 de- fined 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 transphos- phorylase 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 concen- trations 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 concen- trations 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 03

    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 thermo- dynamic 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, more- over, since all values for kinetic parameters, in principle represent derived values, the numerical estimations contain within them- selves 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 days 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 mecha- nism 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 con- sidered 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. Addit...

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