- Email: [email protected]
A kinetic study of the effect of Co2+, Cd2+, Cu2+ and Ni2+ on yeast inorganic pyrophosphatase
208
millimolar range) to the enzyme in the absence of free metal ion, whereas the latter two show an enhancement in binding affinity in the presence of free metal ion [5,7]. Extensive kinetic studies of Mg 2+ activation of the yeast pyrophosphatase are consistent with a model in which both free Mg 2+ and the Mg-PE complex bind to the enzyme to produce the central catalytic complex [9-13]. Uncomplexed PPi is a competitive inhibitor with respect to the Mg-PP i substrate [9,10]. This type of model postulates two distinct roles for the metal ion: complexation with the enzyme t o produce an activated enzyme (activator role), and complexation of PPi to produce the substrate (substrate role). This dual-role model for metal ion effect has also been shown to provide a consistent explanation for Zn 2+ , Cd 2÷ , Mn 2+ and Co2+ activation [14-16] as well as Ca 2+ inhibition [17] kinetics. The pyrophosphatasecatalyzed hydrolysis of exchange inert Co3+ and Cr 3+ complexes of PPi in the presence of free Mg 2+, Mn 2+ or Zn 2+ has also clearly demonstrated two roles for the metal ion cofactor [6,12,18]. The purpose of this work is to determine kinetitally the effect that the metal ion which is complexed to the PPi exerts on the processes of metalPPi ligand binding and PPi hydrolysis. Results of an investigation of the inhibition of the Mg 2+activated inorganic pyrophosphatase reaction by CO 2+, Cd 2+, Cu 2+ and Ni 2+ are presented here. Inhibition constants for the binding of the PPi complexes of these metal ions to the Mg2+-en zyme, as well as relative rate constants for the hydrolysis of Co. PPi by the Co 2+- and Mg2+-en zyme are reported.
metal chloride salts used in this study were obtained from J.T. Baker. Tetramethylammonium chloride, 97%, was obtained from Aldrich. All chemicals were reagent grade and were used without further purification. Distilled water was passed through a Barnstead standard mixed bed resin deionizing column prior to use in solution preparation. Stock solutions of all metal ion salts were standardized by complexometric titration with standard ethylenediaminetetraacetic acid [19]. Murexide was used as an indicator for Co 2÷, Cu 2÷ and Ni 2÷ standardizations and Eriochrome black T was used for the standardization of Cd 2÷ and Mg 2+. Hydrochloric acid solutions were standardized with sodium carbonate [20]. Pyrophosphate stock solutions were standardized with standard HC1. Metal-pyrophosphate stability constants. Stability constants for the Cd 2+, Co2+, Cu 2+ and Ni 2+ complexes of pyrophosphate were determined, under the conditions of the kinetic assay, by potentiometric titration [21]. Titrations were carried out at 25.0°C under a water-saturated nitrogen atmosphere using a Coming model 12 pH meter with Fisher glass and calomel electrodes. Ionic strength was maintained at 0.20 with tetramethylammonium chloride. Evaluations of the stability constants for the metal-pyrophosphate complexes was accomplished by fitting the experimental titrations curves to the derived titration functions [22] using a nonlinear regression algorithm [23]. Definitions of the 1 : 1 stability constants are: KMpP = [MP2072- ]/[1)2074- ][M 2+ ] and
Experimentalprocedm'es KMm, v
Materials. Inorganic pyrophosphatase from bakers yeast was purchased from both Worthington (crystalline suspension, 600-800 units/mg) and Sigma (lyophilized powder, 500 units/mg). One unit of enzyme activity is defined as 1 micromole of inorganic phosphate produced per rain. Tetrasodium pyrophosphate, p-methylaminophenol sulfate and ammonium molybdate were obtained from Sigma. Murexide, Eriochrome black T, the disodium salt of EDTA and the hydrated
= [MHP2017 - ]/[HP203- ][M 2+ ]
The stability constants determined by this method are given in Table I, and are compared with available literature values [24-28] obtained under different experimental conditions. Stability constants for the Mg-PPi and K-PP i complexes were obtained directly from the literature: log KMsl,l, = 5.41 and log KMsHPP = 3.06 [29,30] and log KKpP = 0.80 [31]. Enzyme rate determination. Inorganic phosphate
209 TABLE I M E T A L - P Y R O P H O S P H A T E STABILITY C O N S T A N T S D E T E R M I N E D A T 25°C A N D IONIC S T R E N G T H 0.20 Metal ion
Constant
log K + S.D.
Literature values
Ni 2+
NNiPP KNiHPp
6.56+0.07 3.70 + 0.07
7.01 a, 6.22 b, 6.35 c 3.85 a, 3.50 b
Co 2+
Kcopr Kcoar r
6.64+0.07 3.88 + 0.06
7.36 a, 6.1 d 4.07 a, 5.7 d
Cu 2+
Kcupp KCuHPp
8.99 + 0.29 5.59 5:0.34
9.07 c 7.3 d 9.85 c 5.37 ¢, 5.4 d
C.d2+
Kcdpp KCdHP p
6.23+0.07 3.54+0.15
7.41 ': none
" b c d c
Ref. Ref. Ref. Ref. Ref.
24, 25, 26, 27, 28,
25°C, 15°C, 30°C, 25°C, 25°C,
I = 0.1, p H titration. I = 0.1, p H titration. PbO 2 electrode. I = 0.1, p H titration. I = 1.0 polarography.
production was determined as a function of time using a modification [32] of the Phosphomolybdenum blue method-of Fiske and SubbaRow [33]. The conditions for the kinetic determinations were: pH 7.0, 25.0°C, and an ionic strength of 0.20 (KC1). No pH buffer was used in these determinations because of the tendency of the transition metal ions to form strong complexes with the conjugate base forms of amines and oxyacids [34] which are commonly used in buffers. Initial rates were measured at less than 10% reaction and were strictly linear, indicating that any pH changes accompanying pyrophosphate hydrolysis were insignificant. Because of the use of different enzyme preparations of varying specific activity, a normalization procedure was used to compare experiments carried out over the course of the study. A standard reaction medium, containing 1.50 mM MgC12 and 1.00 mM Na4P2OT, was assayed daily and assigned an arbitrary value of 100%. Rates in all figures are therefore expressed as percent relative rate. Calculations. For each kinetic determination, the equilibrium concentrations of all ionic forms of free and metal-ion-complexed pyrophosphate, as well as the concentrations of free metal ions, were calculated from the total concentrations of pyrophosphate and metal ion using the experimen-
tally measured metal-pyrophosphate stability constants and literature values for the pyrophosphate acid association constants [22], as described previously [10]. Since this study was carried out at constant pH, the ratios of the various ionic forms of free and complexed pyrophosphate were constant. Thus, for convenience, the following concentration sums were defined and used in the kinetic analysis: uncomplexed pyrophosphate, [PP] -- ([H2P2O2- ] + [HP2073- ] + [P2074- ] + [KP2073-]); and metal-pyrophosphate complex, [MPP] = ([MP20 2-] + [MHP2017-]). This type of treatment results in the determination of conditional (composite) kinetic constants. R ~ Inhibition by Co 2 +, Cd e +, Cu 2 + and N i 2 +
The inhibitory effects of Co2+ , Cd 2+ and Ni 2+ on the Mg2+-activated pyrophosphatase reaction are shown in Figs. 1, 2 and 3, respectively. A similar study was carried out for Cu 2+ but is not
I
I
I
I
I
tOO i 8q
6( %REL,
RATE 4{
]
"
I 0
,1
I
I
.2 .3 COCI2, mlA
I
I
.4
.5
Fig. 1. C o 2 + inhibition of the Mg2+-activated yeast inorganic pyrophosphatase. The pyrophosphate concentrations were 1.00 m M and 0.246 m M In the upper and lower curves, respectively. The Mg 2+ concentration was 1.50 m M in both curves. Conditions were 250C, p H %0, and 0.20 ionic strength. Error bars represent standard deviations and the solid lines are calculated theoretical curves as described in the text.
210 t
I
kl
M g E + M g 2+ ~ Mg2E k2 k3
kmm
Mg2E+ MgPP ~ Mg2E(MgPP ) --, Products k4
so;
k~
kn,x
M g 2 E + X P P ~ MgeE(XPP ) ~ Products k6
60, ~. REL.
k7
M g 2 E + PP ~ Mg2E(PP ) ks
RATE 4020 Scheme I. X represents either Co 2+ , Cd 2+, Cu 2+ or N'i2+; MgPP and XPP represent the sum of all ionic forms of the Mg-PPi and inhibitory metal-PPi complexes; and PP represents all forms of the inhibitory uncomplexed PPi anion.
_ 0
20
I 40 CdCl2, uM
I 60
Fig. 2. Cd 2+ inhibition of the Mg2+-a~tivated yeast inorganic pyrophosphatase. Concentrations of pyrophosphate were 0.987 and 0.311 mM in the upper and lower curves, respectively. Mg 2+ concentrations were 1.49 and 0.992 mM in the upper and lower curves, respectively. Conditions were 25°C, pH 7.0, and 0.20 ionic strength. Error bars represent standard deviations and solid lines are calculated theoretical curves.
shown. In all cases, an initial steep decline in reaction rate is observed, followed by an asymptotic approach toward a limiting finite rate in the case of Co 2+ , and toward a limiting zero rate for Cd 2+ , Cu 2+ and Ni 2+. Precipitation of metal-PPi complexes occurred at metal ion concentrations higher than those shown in Figs. 1-3, limiting the concentration ranges of the inhibition studies. Calculations of the equilibrium concentrations of all free metal ion, metal-PP i and free PPi species were carried out for each of the inhibition experiments. In all cases, decreasing reaction rate was in parallel with the increasing ratio of the concentration of inhibitory metal-PPi complex to that of the Mg-PP i complex. The concentrations of free Mg 2+ were calculated to be two to five orders of magnitude greater than the concentrations of the free inhibitory metal ions. Scheme I, shown below, was used to analyze the kinetic data for the four inhibitory metal ions. Several points of clarification with regard to this scheme need to be made.
First, previous kinetic studies [9,10] have been consistent with a rate law containing a single Mg2+-enzyme dissociation constant and a firstorder dependence on the concentration of free
I
I
I
I
.2
.3
.4
.S
.o\
~REL. RATE 40
20
I .1
NiCl 2 , mM
Fig. 3. Ni 2+ inhibition of the Mg2+-activated yeast inorgamc pyrophosphatase. Concentrations of pyropbosphate were 0.947 and 0.255 mM in the upper and lower curves, respectively. The concentration of Mg 2+ was 1.49 mM in both curves. Conditons were 25°C, pH %0, and 0.20 ionic strength. Error bars represent standard deviations and the solid lines are calculated theoretical curves.
211
Mg 2+. Binding studies [8], however, have demonstrated the existence of two types of M g 2+ site per enzyme protomer: a tight site (K d = 38 pM) and a' weak site (K o = 150-280 pM). Since the kinetically determined values for the dissociation constant are in closest agreement with the values for the weak binding site [9], it is probable that the tight site was saturated under the conditions of the kinetic studies. Because the concentration of Mg 2+ is maintained in the millimolar range in these studies, the first step in Scheme I governs only Mg 2+ binding at the weak site, producing MgeE. Secondly, the above scheme is shown as an ordered binding sequence leading to the central complex, although other kinetically and thermodynamically equivalent paths do exist as possibilities (e.g., E + MgPP # E(MgPP) + Mg 2+ # MgE (MgPP) + Mg 2+ # Mg2E(MgPP)). The preference for the ordered sequence shown in Scheme I is based on binding studies showing that: (1) neither free PPi nor Ca. PPi binds to the enzyme in the absence of free metal ion [5]; (2) in the presence of free Ca2+, Ca-PPi binds very strongly to the enzyme (K d ffi 7 nM) [5]; and (3) complexes between the enzyme and free metal ions are strong and readily measured [7,8]. The same ordered sequence has been used in other studies of pyrophosphatase kinetics [13,15,16]. Thirdly, since the calculated concentrations of Mg 2+ are several orders of magnitude greater than those calculated for the free inhibitory metal ions in these experiments, and since the values for the dissociation constants for the complexes of the enzyme with Mg 2+ [8], Co2+ [7,8] and Cd 2+ [15] are all within a factor of 5-10 (the constants for Ni 2+ and Cu 2+ are not available), it is assumed that the formation of enzyme-metal ion complexes other than Mg2E are negligible. Fourthly, the terms kmm and kn~ are composite rate constants which include the microscopic rate constants for PPi hydrolysis and release of the P~ products. All of the latter rate constants have been shown to be partially rate-limiting in the direction of PPi hydrolysis [13]. The overall rate-determining step, however, is the reverse rate constant k4 in Scheme I, for the dissociation of Mg- PPi from the central complex [13]. The steady-state rate law for Scheme I, assuming that k4, k6, kin= and k m < all other con-
T A B L E II MICHAELIS, I N H I B I T I O N A N D R A T E C O N S T A N T S DET E R M I N E D F R O M K I N E T I C S O F Co l+ , Cd l+ , Cu 2+ A N D Ni l+ I N H I B I T I O N A N D Co~+ A C T I V A T I O N O F INORGANIC PYROPHOSPHATASE Metal ion
Krupp (pM)) ( + S.D.)
Co l+
3.2 ( + 0 . 3 )
km~/km~ = 0.14 ( + 0.01) k,=/kmm = 0.09 (+0.01)
Cd l+ Cu l+ Ni l+
0.22 (+0.05) 1.6 ( + 0 . 2 ) 1.2 ( + 0 . 1 )
-
M g 2+
Relative Rate Constants ( + S.D.)
kmm/kmm = 1.00
17 a 5.0 b
(0.2)c Zn2+
5.2 d 0.10 •
Ca 2+ " b c d •
Ref. Ref. Ref. Ref. Ref.
10, p H 7.40, T ffi 30°C. 9, p H 7.20, T ffi 25°C. 13, p H 7.00, T ffi 25°C (calculated dissociation constant). 14, p H 7.00, T ffi 25°C. 17, p H 7.40, T = 30°C.
stants, is given in Eqn. 1: v -E0 - =
kmm[MgPP]/K~, + km[XPP]/K,~p
1+
K m
[Mg l÷ ] In
this
equation,
K~pp
[XPPI
[PP]
Kxpp
K~p
K m == k 2 / k l ,
(1)
Kmppm _~ ( k 4 +
k m m ) / k 3, Kxpp -- (k 6 + k m ~ ) / k 5, K~p = k s / k ~,
and E 0 is the total enzyme concentration. In fitting the above rate law to the inhibition data, previously determined fiterature values for several parameters were utilized as known constants: Kmpp--15 pM [9,10]; K mffi 150 pM [9]; and K~p = 7.9 pM [10]. The value of km~ was extrapolated to zero (i.e., less than 1% of kmm) for Cd 2+, Cu 2+ and Ni 2+, but the value for Co2+, expressed as the normalized ratio, k m J k m m , was determined in the data analysis. Best-fit values for all unknown parameters were determined by nonlinear regression analysis [10,23] and these values are presented in Table II. The best-fit parameter values were used with the known literature values in Eqn. 1 to calculate theoretical curves, which are shown in the figures as solid lines.
212
Activation by Co2 + The parameter, kmJkmm , for Co~ + (see Table II) is a relative rate constant for the hydrolysis of the Co-PPi complex by Mg2E. Evaluation of the relative rate constant (symbolized by kcc/kmm) for the hydrolysis of the Co-PPi complex by C02E would allow comparison of two different metal ions in the activator role (i.e., Mg2E and C02E ) catalyzing the hydrolysis of the same substrate. To determine the koJkmm value, the reaction rate was measured as a function of added Co2+ at a constant PPi concentration. This produced a saturation in both Co-PPi substrate and free Co2+ (see Fig. 4). Solubility problems precluded a wider concentration range, but the data was sufficient for a good estimate of the rate constant. The kinetic model used to analyze the Co2+ activation data was identical to Scheme I, but omitting the third step and substituting Co2+ for
I
I
'
it
.2 .3 COCI2, mM
.4
6
~, REL. RAT[ 4
0
.1
.S
Fig. 4. The percent relative rate of yeast inorgamc pyrophosphatase is plotted versus the concentration of CoC12. The pyrophosphate concentration was 0.197 mM at pH 7.0, 25°C, and 0.20 ionic strength. Error bars represent average deviation values. The solid line through the points is a calculated theoretical curve as described in the te~xt.
Mg 2+ . The rate law in this case is given below: o --E0 =
k~[covp]/r~ __g ~
[CoPP] + [pp]
(2)
The dissociation constant, K c, was taken to be 25 /~M from equilibrium binding studies [8], and bestfit values were determined for the other parameters. The value determined for k~/kmm is given in Table II. The theoretical curve in Fig. 4 was calculated from Eqn. 2 and the best-fit parameters. Discussion An unambiguous determination of the catalytic role of the divalent metal ion cofactor in the yeast inorganic pyrophosphatase reaction is complicated by the fact that the metal ion independently binds to the free enzyme (activator role) and to the pyrophosphate species (substrate role). Several approaches have been utilized to experimentaUy isolate the metal ion effect in the two distinct roles. One approach, placing Co3+ and Cr 3+ in exchange inert complexes with PPi (producing both substrates and inhibitors), while using Mg 2+ or Zn2+ in the activator role [12,18], has provided strong evidence that a bridged, P1,p2-bidentate metal-PPi complex is the active form of the substrate. In another type of experiment, the interaction of Pi with the Cd2+-complexed enzyme was studied using NMR techniques, demonstrating inner-sphere binding between Pi and at least one enzyme-bound Cd 2+ [15]. The approach taken in this work has been to carry out inhibition studies under conditions such that the inhibitory metal ion binds preferentially to the pyrophosphate anion rather than to the enzyme. This is experimentally possible because the formation constants for the Cd 2+ , Co2+ , Cu 2+ and Ni 2+ complexes of PPi are very large (Table I), greater by one to three orders of magnitude than the corresponding constants for Mg 2+ [29,30]. Equilibrium calculations show that the majority of the total inhibitory metal ion is complexed with PPi rather than existing in free form, even in the presence of the excess Mg 2+ used in these experiments. A similar approach has been used to study the hydrolysis of trivalent metal-PPi complexes by
213 the Mg2+-enzyme [35]. The predominant effect of the inhibitory metal ions in these experiments, then, is the binding of their PPi complexes (X. PP) to Mg2E, in competition with Mg. PP, to form nonreactive, or slowly reactive, central complexes. This type of kinetic model has given an excellent fit to the inhibition data (see theoretical curves in Figs. 1-3). The Kxpp values determined in this study are compared in Table II with literature values for Mg 2+, Zn 2+ and Ca2+. The Kx~v parameters are Michaelis constants for the substrates Mg. PPi, Co. PPi and Zn. PPi, and are inhibition (dissociation) constants for Ca. PPi, Cd. PPi, Cu. PPi and Ni. PPi. Since the rate constant for the dissociation of the metal-PPi substrate from the central complex is slow in comparison to the catalytic constant [13], the true dissociation constants for Mg. PPi, Co. PPi and Zn. PPi from their central complexes must be less than their measured Michaelis constants (e.g., Kx~p = (k 6 + kmx)/k 5 in Scheme I; if k 6 < kmx, then Kxn~p> K d = k6/k5). Thus, for Mg. PPi, the Michaelis constant has been measured to be 5-17 #M [9,10,13], while its dissociation constant has been determined to be 0.2 ~tM [13] (Table II). Although the Michaelis and dissociation constant values in Table II cannot be directly compared, several points can nonetheless be made. It is evident from Table II that the nature of the metal ion complexed to PPi does exert a considerable effect on the interaction of the metal-PPi complex with the Mg2+-enzyme. There is a general trend in K~p values toward tighter binding as the ionic radius of the central metal ion increases. The strongest inhibitor binding is exhibited by Ca. PPi and Cd. PPi in which the central metal ions have filled outer shell electron orbitals and relatively large ionic radii with respect to Mg 2+ (Goldschmidt ionic radius values in ~-agstroms are: Mg 2+ = 0.65, Ni 2+ = 0.68, Zn2+ = 0.69, Coz+ = 0.70, Cu 2+ = 0.92, Cd 2+ = 0.92' and Ca2+ = 0.94) [361. The true dissociation constant for Mg. PPi, 0.2 #M [13], places its binding affinity in the same range as Cd. PPi and Ca. PPi, which is somewhat surprising. Strong experimental evidence has been obtained for the involvement of two types of dec_ trophilic group, Arg-77 [37] and at least one of the two activator role metal ions [15,18], in the hind-
ing of Pi and PPi ligands, presumably through interactions with the negative phosphate oxyanions. Since the cyclic p1, p2_bidentate form of the metal-PPi complex has been shown to be the most probable form of the substrate (or competitive inhibitor) [18], it would be reasonable to expect that a change in the central metal ion from Mg 2+ to the much larger Ca2+ or Cd 2+ would have a significant effect on the conformation of the pyrophosphate moiety in the complex, and therefore on its pattern of binding interactions with active site groups. This suggests the possibility that Cd. PPi and Ca. PPi may bind to the active site with a different set of binding interactions than for Mg. PPi, which are equally strong, but catalytically nonproductive. Ting and Dunaway-Mariano [35] have shown, in a study of the hydrolysis of trivalent metal-PPi complexes by the Mg2+-enzyme, that a series of weaker binding complexes (K m = 150-1000/tM) had appreciable catalytic activity, but that tight-binding Sc. PPi ( K i = 8 ~M) was inactive [35]. The possibility exists, then, that some metal-PPi complexes may be catalytically inactive and strongly inhibitory due to very tight 'wrongway' binding at the active site. Also compared in Table II are kmc/kmm and k~/kmm, which are the relative rate constants for the hydrolysis of the Co-PPi substrate by Mg2E and Co2E, respectively. The value for k~/kmm (0.09) compares favorably with a value of 0.05 ([Co2+ ] = 0.25 mM) obtained by Welsh et al. [16] under similar conditions. The value for kmJkmm (0.14) is surprisingly very close to kcJkmm which indicates, at least in the case of the Co-PPi complex, that the metal ion bound to the enzyme (activator role) has only a small influence on the observed rate of substrate hydrolysis. A similar finding for the hydrolysis of the Zn-PPi complex by Mg2E and Zn2E has been reported [14]. However, by comparing kcJkmm (0.09) with kmm/kmm (1.00), it can be seen that changing the metal ion in both substrate and activator roles from Co2+ to Mg 2+ causes an ll-fold increase in the hydrolytic rate constant. These two comparisons, taken together, argue that the metal ion complexed with the PPi in the substrate role is the more influential in affecting the rate of PPi hydrolysis. Welsh et al. [16] have shown that the difference
214
in rate constants for the pyrophosphatase-catalyzed hydrolysis of PPi for Mg 2+ and Co2+ can be explained by a difference in the rate of dissociation of the first reaction product a metal-Pi complex, from the enzyme. A reasonable assumption, then, is that both kmc and k~ are primarily controlled by the rate of release of the Co-Pi complex, the Co2÷ therein coming from the Co-PPi substrate. In the case of the kmc rate constant, there is a second possible mechanistic interpretation which cannot be ruled out by this kinetic data and which should be mentioned. It is conceivable that interchange of activator and substrate metal ions could occur on the enzyme surface in the central catalytic complex. If very rapid, this exchange could be complete prior to PPi hydrolysis and product release, which would mean that kmc would be a complex measure of the catalytic breakdown of the two possible forms of MR. CoE(MgPP) (two possible sites for activator metal ions) as well as of Mg2E(CoPP). While there is no direct experimental evidence for this type of rapid interchange in the pyrophosphate literature, there is also no way at this time to definitely exclude it as a possibility.
Acknowledgement The authors acknowledge and appreciate the support that was provided for this work by Research Corporation through the Cottrell College Science Grants Program.
References 1 Cohen, S.A., Sterner, R., Keim, P.S. and Heim-ickson, R.L. (1978) J. Biol. Chem. 253, 889-897 2 Butler, L.G. (1971) in The Enzymes (Boyer, P.D., ed.), 3rd. FAn., Vol. 4, p. 535, Academic Press, New York 3 Butler, L.G. and Sperow, J.W. (1977) Bioinorg, Chem. 7, 141-150 4 Cooperman, B.S. and Chin, N.Y. (1973) Biochemistry 12, 1670-1676 5 Ridlington, J.W. and Butler, L.G. (1972) J. Biol. Chem. 247, 7303-7307 6 Knight, W.B., Dunaway-Mariano, D., Ransom, S.C. and Villafranca, J.J. (1984) J. Biol. Chem. 259, 2886-2895 7 Cooperman, B.S., Panackal, A., Springs, B. and Harem, D.J. (1981) Biochemistry 20, 6051-6060 8 Rapoport, T.A., Hohne, W.E., HeRman, P. and Rapoport, S. (1973) Eur. J. Biocham. 33, 341-347
9 Rapoport, T.A., Hohme, W.E., Reich, J., HeRman, P. and Rapoport, S.M. (1972) Eur. J. Biochem. 26, 237-246 10 Moe, O.A. and Butler, L.G. (1972) J. Biol. Chem. 247, 7308-7315 11 Shafransldi, Y.A., Baikov, A.A., Andrinkovich, P.F. and Aveava, S.M. (1977) Biokhimiya 42, 1244-1250 12 'Knight W.B., Fitts, S.W. and Dunaway-Mariano, D. (1981) Biochemistry 20, 4079-4086 13 Springs, B., Welch, K.M. and Cooperman, B.S. (1981) Biochemistry 20, 6384-6391 14 Moe, O.A., Jr., Pham, S., Dang, T. and Stryer, L. (1979) Arch. Biochem. Biophys. 196, 73-78 15 Welsh, K.M., Armitage, I.M. and Cooperman, B.S. (1983) Biochemistry 22, 1046-1054 16 Welsh, K.M., Jacobyansky, A., Springs, B. and Cooperman, B.S. (1983) Biochemistry 22, 2243-2248 17 Moe, O.A. and Butler, L.G. (1972) J. Biol. Chem. 247, 7315-7319 18 Knight, W.B., Ting, S., Chuang, S., Dunaway-Mariano, D., Haromy, T. and Sundaralingam, M. (1983) Arch. Biochem. Biophys. 227, 302-309 19 Schwartzenbach, G. and Flascha, J. (1969) Complexometric Titrations, Methuen, London 20 Vogel, A.J. (1961) Quantitative Inorganic Analysis, 3rd Edn., p. 433, Wiley, New York 21 Rossotti, F.J.C. and gossotti, H. (1961) The Determination of Stability Constants, Chapter 7, McGraw-Hill, New York 22 Moe, O.A., Jr. and Wiest, S.A. (1977) Anal. Biochem. 77, 73-781 23 Kim, H. (1970) J. Chem. Educ. 47, 120-122 24 Hammes, G.G. and Morrell, M.L. (1964) J. Am. Chem. Soc. 86, 1497-1502 25 Frey, C.H. and Stuehr, J.E. (1972) J. Am. Chem. Soc. 94, 8898-8903 26 Karweik, D.H. and Huber, C.O. (1978) Anal. Chem. 50, 1209 27 Johansson, A. and Wanninen, E. (1963) Talanta 10, 769-777 28 Schupp, O.E., Sturrock, P.E. and Watters, J.I. (1962) Inorg. Chem. 2, 106-112 29 Lambert, S.M. and Watters, J.I. (1957) J. Am. Chem. Soc. 79, 5606-5608 30 Josse, J. (1966) J. Biol. Chem. 241, 1948-1955 31 Lambert, S.M. and Watters, J.I. (1957) J. Am. Chem. Soc. 79, 4262-4265 32 Ridlington, J.W., Yang, Y. and Butler, L.G. (1972) Arch. Biochem. Biophys. 153, 714-725 33 Fiske, C.H. and SubbaRow, Y. (1925) J. Biol. Chem. 66, 375 34 Sillen, L.G. and Martel, A.E. (1964) Stability Constants of Metal-Ion Complexes, Special Publication, No. 17, The Chemical Society, London 35 Ting, S. and Dunaway-Mariano, D. (1984) FEBS Lett. 165, 251-253 36 Cotton, F.A. and Wilkinson, G. (1966) Advanced Inorganic Chemistry, 2nd Edn., p. 45, Interscienee, New York 37 Cooperman, B.S. (1982) Methods Enzymol. 87, 526-548
millimolar range) to the enzyme in the absence of free metal ion, whereas the latter two show an enhancement in binding affinity in the presence of free metal ion [5,7]. Extensive kinetic studies of Mg 2+ activation of the yeast pyrophosphatase are consistent with a model in which both free Mg 2+ and the Mg-PE complex bind to the enzyme to produce the central catalytic complex [9-13]. Uncomplexed PPi is a competitive inhibitor with respect to the Mg-PP i substrate [9,10]. This type of model postulates two distinct roles for the metal ion: complexation with the enzyme t o produce an activated enzyme (activator role), and complexation of PPi to produce the substrate (substrate role). This dual-role model for metal ion effect has also been shown to provide a consistent explanation for Zn 2+ , Cd 2÷ , Mn 2+ and Co2+ activation [14-16] as well as Ca 2+ inhibition [17] kinetics. The pyrophosphatasecatalyzed hydrolysis of exchange inert Co3+ and Cr 3+ complexes of PPi in the presence of free Mg 2+, Mn 2+ or Zn 2+ has also clearly demonstrated two roles for the metal ion cofactor [6,12,18]. The purpose of this work is to determine kinetitally the effect that the metal ion which is complexed to the PPi exerts on the processes of metalPPi ligand binding and PPi hydrolysis. Results of an investigation of the inhibition of the Mg 2+activated inorganic pyrophosphatase reaction by CO 2+, Cd 2+, Cu 2+ and Ni 2+ are presented here. Inhibition constants for the binding of the PPi complexes of these metal ions to the Mg2+-en zyme, as well as relative rate constants for the hydrolysis of Co. PPi by the Co 2+- and Mg2+-en zyme are reported.
metal chloride salts used in this study were obtained from J.T. Baker. Tetramethylammonium chloride, 97%, was obtained from Aldrich. All chemicals were reagent grade and were used without further purification. Distilled water was passed through a Barnstead standard mixed bed resin deionizing column prior to use in solution preparation. Stock solutions of all metal ion salts were standardized by complexometric titration with standard ethylenediaminetetraacetic acid [19]. Murexide was used as an indicator for Co 2÷, Cu 2÷ and Ni 2÷ standardizations and Eriochrome black T was used for the standardization of Cd 2÷ and Mg 2+. Hydrochloric acid solutions were standardized with sodium carbonate [20]. Pyrophosphate stock solutions were standardized with standard HC1. Metal-pyrophosphate stability constants. Stability constants for the Cd 2+, Co2+, Cu 2+ and Ni 2+ complexes of pyrophosphate were determined, under the conditions of the kinetic assay, by potentiometric titration [21]. Titrations were carried out at 25.0°C under a water-saturated nitrogen atmosphere using a Coming model 12 pH meter with Fisher glass and calomel electrodes. Ionic strength was maintained at 0.20 with tetramethylammonium chloride. Evaluations of the stability constants for the metal-pyrophosphate complexes was accomplished by fitting the experimental titrations curves to the derived titration functions [22] using a nonlinear regression algorithm [23]. Definitions of the 1 : 1 stability constants are: KMpP = [MP2072- ]/[1)2074- ][M 2+ ] and
Experimentalprocedm'es KMm, v
Materials. Inorganic pyrophosphatase from bakers yeast was purchased from both Worthington (crystalline suspension, 600-800 units/mg) and Sigma (lyophilized powder, 500 units/mg). One unit of enzyme activity is defined as 1 micromole of inorganic phosphate produced per rain. Tetrasodium pyrophosphate, p-methylaminophenol sulfate and ammonium molybdate were obtained from Sigma. Murexide, Eriochrome black T, the disodium salt of EDTA and the hydrated
= [MHP2017 - ]/[HP203- ][M 2+ ]
The stability constants determined by this method are given in Table I, and are compared with available literature values [24-28] obtained under different experimental conditions. Stability constants for the Mg-PPi and K-PP i complexes were obtained directly from the literature: log KMsl,l, = 5.41 and log KMsHPP = 3.06 [29,30] and log KKpP = 0.80 [31]. Enzyme rate determination. Inorganic phosphate
209 TABLE I M E T A L - P Y R O P H O S P H A T E STABILITY C O N S T A N T S D E T E R M I N E D A T 25°C A N D IONIC S T R E N G T H 0.20 Metal ion
Constant
log K + S.D.
Literature values
Ni 2+
NNiPP KNiHPp
6.56+0.07 3.70 + 0.07
7.01 a, 6.22 b, 6.35 c 3.85 a, 3.50 b
Co 2+
Kcopr Kcoar r
6.64+0.07 3.88 + 0.06
7.36 a, 6.1 d 4.07 a, 5.7 d
Cu 2+
Kcupp KCuHPp
8.99 + 0.29 5.59 5:0.34
9.07 c 7.3 d 9.85 c 5.37 ¢, 5.4 d
C.d2+
Kcdpp KCdHP p
6.23+0.07 3.54+0.15
7.41 ': none
" b c d c
Ref. Ref. Ref. Ref. Ref.
24, 25, 26, 27, 28,
25°C, 15°C, 30°C, 25°C, 25°C,
I = 0.1, p H titration. I = 0.1, p H titration. PbO 2 electrode. I = 0.1, p H titration. I = 1.0 polarography.
production was determined as a function of time using a modification [32] of the Phosphomolybdenum blue method-of Fiske and SubbaRow [33]. The conditions for the kinetic determinations were: pH 7.0, 25.0°C, and an ionic strength of 0.20 (KC1). No pH buffer was used in these determinations because of the tendency of the transition metal ions to form strong complexes with the conjugate base forms of amines and oxyacids [34] which are commonly used in buffers. Initial rates were measured at less than 10% reaction and were strictly linear, indicating that any pH changes accompanying pyrophosphate hydrolysis were insignificant. Because of the use of different enzyme preparations of varying specific activity, a normalization procedure was used to compare experiments carried out over the course of the study. A standard reaction medium, containing 1.50 mM MgC12 and 1.00 mM Na4P2OT, was assayed daily and assigned an arbitrary value of 100%. Rates in all figures are therefore expressed as percent relative rate. Calculations. For each kinetic determination, the equilibrium concentrations of all ionic forms of free and metal-ion-complexed pyrophosphate, as well as the concentrations of free metal ions, were calculated from the total concentrations of pyrophosphate and metal ion using the experimen-
tally measured metal-pyrophosphate stability constants and literature values for the pyrophosphate acid association constants [22], as described previously [10]. Since this study was carried out at constant pH, the ratios of the various ionic forms of free and complexed pyrophosphate were constant. Thus, for convenience, the following concentration sums were defined and used in the kinetic analysis: uncomplexed pyrophosphate, [PP] -- ([H2P2O2- ] + [HP2073- ] + [P2074- ] + [KP2073-]); and metal-pyrophosphate complex, [MPP] = ([MP20 2-] + [MHP2017-]). This type of treatment results in the determination of conditional (composite) kinetic constants. R ~ Inhibition by Co 2 +, Cd e +, Cu 2 + and N i 2 +
The inhibitory effects of Co2+ , Cd 2+ and Ni 2+ on the Mg2+-activated pyrophosphatase reaction are shown in Figs. 1, 2 and 3, respectively. A similar study was carried out for Cu 2+ but is not
I
I
I
I
I
tOO i 8q
6( %REL,
RATE 4{
]
"
I 0
,1
I
I
.2 .3 COCI2, mlA
I
I
.4
.5
Fig. 1. C o 2 + inhibition of the Mg2+-activated yeast inorganic pyrophosphatase. The pyrophosphate concentrations were 1.00 m M and 0.246 m M In the upper and lower curves, respectively. The Mg 2+ concentration was 1.50 m M in both curves. Conditions were 250C, p H %0, and 0.20 ionic strength. Error bars represent standard deviations and the solid lines are calculated theoretical curves as described in the text.
210 t
I
kl
M g E + M g 2+ ~ Mg2E k2 k3
kmm
Mg2E+ MgPP ~ Mg2E(MgPP ) --, Products k4
so;
k~
kn,x
M g 2 E + X P P ~ MgeE(XPP ) ~ Products k6
60, ~. REL.
k7
M g 2 E + PP ~ Mg2E(PP ) ks
RATE 4020 Scheme I. X represents either Co 2+ , Cd 2+, Cu 2+ or N'i2+; MgPP and XPP represent the sum of all ionic forms of the Mg-PPi and inhibitory metal-PPi complexes; and PP represents all forms of the inhibitory uncomplexed PPi anion.
_ 0
20
I 40 CdCl2, uM
I 60
Fig. 2. Cd 2+ inhibition of the Mg2+-a~tivated yeast inorganic pyrophosphatase. Concentrations of pyrophosphate were 0.987 and 0.311 mM in the upper and lower curves, respectively. Mg 2+ concentrations were 1.49 and 0.992 mM in the upper and lower curves, respectively. Conditions were 25°C, pH 7.0, and 0.20 ionic strength. Error bars represent standard deviations and solid lines are calculated theoretical curves.
shown. In all cases, an initial steep decline in reaction rate is observed, followed by an asymptotic approach toward a limiting finite rate in the case of Co 2+ , and toward a limiting zero rate for Cd 2+ , Cu 2+ and Ni 2+. Precipitation of metal-PPi complexes occurred at metal ion concentrations higher than those shown in Figs. 1-3, limiting the concentration ranges of the inhibition studies. Calculations of the equilibrium concentrations of all free metal ion, metal-PP i and free PPi species were carried out for each of the inhibition experiments. In all cases, decreasing reaction rate was in parallel with the increasing ratio of the concentration of inhibitory metal-PPi complex to that of the Mg-PP i complex. The concentrations of free Mg 2+ were calculated to be two to five orders of magnitude greater than the concentrations of the free inhibitory metal ions. Scheme I, shown below, was used to analyze the kinetic data for the four inhibitory metal ions. Several points of clarification with regard to this scheme need to be made.
First, previous kinetic studies [9,10] have been consistent with a rate law containing a single Mg2+-enzyme dissociation constant and a firstorder dependence on the concentration of free
I
I
I
I
.2
.3
.4
.S
.o\
~REL. RATE 40
20
I .1
NiCl 2 , mM
Fig. 3. Ni 2+ inhibition of the Mg2+-activated yeast inorgamc pyrophosphatase. Concentrations of pyropbosphate were 0.947 and 0.255 mM in the upper and lower curves, respectively. The concentration of Mg 2+ was 1.49 mM in both curves. Conditons were 25°C, pH %0, and 0.20 ionic strength. Error bars represent standard deviations and the solid lines are calculated theoretical curves.
211
Mg 2+. Binding studies [8], however, have demonstrated the existence of two types of M g 2+ site per enzyme protomer: a tight site (K d = 38 pM) and a' weak site (K o = 150-280 pM). Since the kinetically determined values for the dissociation constant are in closest agreement with the values for the weak binding site [9], it is probable that the tight site was saturated under the conditions of the kinetic studies. Because the concentration of Mg 2+ is maintained in the millimolar range in these studies, the first step in Scheme I governs only Mg 2+ binding at the weak site, producing MgeE. Secondly, the above scheme is shown as an ordered binding sequence leading to the central complex, although other kinetically and thermodynamically equivalent paths do exist as possibilities (e.g., E + MgPP # E(MgPP) + Mg 2+ # MgE (MgPP) + Mg 2+ # Mg2E(MgPP)). The preference for the ordered sequence shown in Scheme I is based on binding studies showing that: (1) neither free PPi nor Ca. PPi binds to the enzyme in the absence of free metal ion [5]; (2) in the presence of free Ca2+, Ca-PPi binds very strongly to the enzyme (K d ffi 7 nM) [5]; and (3) complexes between the enzyme and free metal ions are strong and readily measured [7,8]. The same ordered sequence has been used in other studies of pyrophosphatase kinetics [13,15,16]. Thirdly, since the calculated concentrations of Mg 2+ are several orders of magnitude greater than those calculated for the free inhibitory metal ions in these experiments, and since the values for the dissociation constants for the complexes of the enzyme with Mg 2+ [8], Co2+ [7,8] and Cd 2+ [15] are all within a factor of 5-10 (the constants for Ni 2+ and Cu 2+ are not available), it is assumed that the formation of enzyme-metal ion complexes other than Mg2E are negligible. Fourthly, the terms kmm and kn~ are composite rate constants which include the microscopic rate constants for PPi hydrolysis and release of the P~ products. All of the latter rate constants have been shown to be partially rate-limiting in the direction of PPi hydrolysis [13]. The overall rate-determining step, however, is the reverse rate constant k4 in Scheme I, for the dissociation of Mg- PPi from the central complex [13]. The steady-state rate law for Scheme I, assuming that k4, k6, kin= and k m < all other con-
T A B L E II MICHAELIS, I N H I B I T I O N A N D R A T E C O N S T A N T S DET E R M I N E D F R O M K I N E T I C S O F Co l+ , Cd l+ , Cu 2+ A N D Ni l+ I N H I B I T I O N A N D Co~+ A C T I V A T I O N O F INORGANIC PYROPHOSPHATASE Metal ion
Krupp (pM)) ( + S.D.)
Co l+
3.2 ( + 0 . 3 )
km~/km~ = 0.14 ( + 0.01) k,=/kmm = 0.09 (+0.01)
Cd l+ Cu l+ Ni l+
0.22 (+0.05) 1.6 ( + 0 . 2 ) 1.2 ( + 0 . 1 )
-
M g 2+
Relative Rate Constants ( + S.D.)
kmm/kmm = 1.00
17 a 5.0 b
(0.2)c Zn2+
5.2 d 0.10 •
Ca 2+ " b c d •
Ref. Ref. Ref. Ref. Ref.
10, p H 7.40, T ffi 30°C. 9, p H 7.20, T ffi 25°C. 13, p H 7.00, T ffi 25°C (calculated dissociation constant). 14, p H 7.00, T ffi 25°C. 17, p H 7.40, T = 30°C.
stants, is given in Eqn. 1: v -E0 - =
kmm[MgPP]/K~, + km[XPP]/K,~p
1+
K m
[Mg l÷ ] In
this
equation,
K~pp
[XPPI
[PP]
Kxpp
K~p
K m == k 2 / k l ,
(1)
Kmppm _~ ( k 4 +
k m m ) / k 3, Kxpp -- (k 6 + k m ~ ) / k 5, K~p = k s / k ~,
and E 0 is the total enzyme concentration. In fitting the above rate law to the inhibition data, previously determined fiterature values for several parameters were utilized as known constants: Kmpp--15 pM [9,10]; K mffi 150 pM [9]; and K~p = 7.9 pM [10]. The value of km~ was extrapolated to zero (i.e., less than 1% of kmm) for Cd 2+, Cu 2+ and Ni 2+, but the value for Co2+, expressed as the normalized ratio, k m J k m m , was determined in the data analysis. Best-fit values for all unknown parameters were determined by nonlinear regression analysis [10,23] and these values are presented in Table II. The best-fit parameter values were used with the known literature values in Eqn. 1 to calculate theoretical curves, which are shown in the figures as solid lines.
212
Activation by Co2 + The parameter, kmJkmm , for Co~ + (see Table II) is a relative rate constant for the hydrolysis of the Co-PPi complex by Mg2E. Evaluation of the relative rate constant (symbolized by kcc/kmm) for the hydrolysis of the Co-PPi complex by C02E would allow comparison of two different metal ions in the activator role (i.e., Mg2E and C02E ) catalyzing the hydrolysis of the same substrate. To determine the koJkmm value, the reaction rate was measured as a function of added Co2+ at a constant PPi concentration. This produced a saturation in both Co-PPi substrate and free Co2+ (see Fig. 4). Solubility problems precluded a wider concentration range, but the data was sufficient for a good estimate of the rate constant. The kinetic model used to analyze the Co2+ activation data was identical to Scheme I, but omitting the third step and substituting Co2+ for
I
I
'
it
.2 .3 COCI2, mM
.4
6
~, REL. RAT[ 4
0
.1
.S
Fig. 4. The percent relative rate of yeast inorgamc pyrophosphatase is plotted versus the concentration of CoC12. The pyrophosphate concentration was 0.197 mM at pH 7.0, 25°C, and 0.20 ionic strength. Error bars represent average deviation values. The solid line through the points is a calculated theoretical curve as described in the te~xt.
Mg 2+ . The rate law in this case is given below: o --E0 =
k~[covp]/r~ __g ~
[CoPP] + [pp]
(2)
The dissociation constant, K c, was taken to be 25 /~M from equilibrium binding studies [8], and bestfit values were determined for the other parameters. The value determined for k~/kmm is given in Table II. The theoretical curve in Fig. 4 was calculated from Eqn. 2 and the best-fit parameters. Discussion An unambiguous determination of the catalytic role of the divalent metal ion cofactor in the yeast inorganic pyrophosphatase reaction is complicated by the fact that the metal ion independently binds to the free enzyme (activator role) and to the pyrophosphate species (substrate role). Several approaches have been utilized to experimentaUy isolate the metal ion effect in the two distinct roles. One approach, placing Co3+ and Cr 3+ in exchange inert complexes with PPi (producing both substrates and inhibitors), while using Mg 2+ or Zn2+ in the activator role [12,18], has provided strong evidence that a bridged, P1,p2-bidentate metal-PPi complex is the active form of the substrate. In another type of experiment, the interaction of Pi with the Cd2+-complexed enzyme was studied using NMR techniques, demonstrating inner-sphere binding between Pi and at least one enzyme-bound Cd 2+ [15]. The approach taken in this work has been to carry out inhibition studies under conditions such that the inhibitory metal ion binds preferentially to the pyrophosphate anion rather than to the enzyme. This is experimentally possible because the formation constants for the Cd 2+ , Co2+ , Cu 2+ and Ni 2+ complexes of PPi are very large (Table I), greater by one to three orders of magnitude than the corresponding constants for Mg 2+ [29,30]. Equilibrium calculations show that the majority of the total inhibitory metal ion is complexed with PPi rather than existing in free form, even in the presence of the excess Mg 2+ used in these experiments. A similar approach has been used to study the hydrolysis of trivalent metal-PPi complexes by
213 the Mg2+-enzyme [35]. The predominant effect of the inhibitory metal ions in these experiments, then, is the binding of their PPi complexes (X. PP) to Mg2E, in competition with Mg. PP, to form nonreactive, or slowly reactive, central complexes. This type of kinetic model has given an excellent fit to the inhibition data (see theoretical curves in Figs. 1-3). The Kxpp values determined in this study are compared in Table II with literature values for Mg 2+, Zn 2+ and Ca2+. The Kx~v parameters are Michaelis constants for the substrates Mg. PPi, Co. PPi and Zn. PPi, and are inhibition (dissociation) constants for Ca. PPi, Cd. PPi, Cu. PPi and Ni. PPi. Since the rate constant for the dissociation of the metal-PPi substrate from the central complex is slow in comparison to the catalytic constant [13], the true dissociation constants for Mg. PPi, Co. PPi and Zn. PPi from their central complexes must be less than their measured Michaelis constants (e.g., Kx~p = (k 6 + kmx)/k 5 in Scheme I; if k 6 < kmx, then Kxn~p> K d = k6/k5). Thus, for Mg. PPi, the Michaelis constant has been measured to be 5-17 #M [9,10,13], while its dissociation constant has been determined to be 0.2 ~tM [13] (Table II). Although the Michaelis and dissociation constant values in Table II cannot be directly compared, several points can nonetheless be made. It is evident from Table II that the nature of the metal ion complexed to PPi does exert a considerable effect on the interaction of the metal-PPi complex with the Mg2+-enzyme. There is a general trend in K~p values toward tighter binding as the ionic radius of the central metal ion increases. The strongest inhibitor binding is exhibited by Ca. PPi and Cd. PPi in which the central metal ions have filled outer shell electron orbitals and relatively large ionic radii with respect to Mg 2+ (Goldschmidt ionic radius values in ~-agstroms are: Mg 2+ = 0.65, Ni 2+ = 0.68, Zn2+ = 0.69, Coz+ = 0.70, Cu 2+ = 0.92, Cd 2+ = 0.92' and Ca2+ = 0.94) [361. The true dissociation constant for Mg. PPi, 0.2 #M [13], places its binding affinity in the same range as Cd. PPi and Ca. PPi, which is somewhat surprising. Strong experimental evidence has been obtained for the involvement of two types of dec_ trophilic group, Arg-77 [37] and at least one of the two activator role metal ions [15,18], in the hind-
ing of Pi and PPi ligands, presumably through interactions with the negative phosphate oxyanions. Since the cyclic p1, p2_bidentate form of the metal-PPi complex has been shown to be the most probable form of the substrate (or competitive inhibitor) [18], it would be reasonable to expect that a change in the central metal ion from Mg 2+ to the much larger Ca2+ or Cd 2+ would have a significant effect on the conformation of the pyrophosphate moiety in the complex, and therefore on its pattern of binding interactions with active site groups. This suggests the possibility that Cd. PPi and Ca. PPi may bind to the active site with a different set of binding interactions than for Mg. PPi, which are equally strong, but catalytically nonproductive. Ting and Dunaway-Mariano [35] have shown, in a study of the hydrolysis of trivalent metal-PPi complexes by the Mg2+-enzyme, that a series of weaker binding complexes (K m = 150-1000/tM) had appreciable catalytic activity, but that tight-binding Sc. PPi ( K i = 8 ~M) was inactive [35]. The possibility exists, then, that some metal-PPi complexes may be catalytically inactive and strongly inhibitory due to very tight 'wrongway' binding at the active site. Also compared in Table II are kmc/kmm and k~/kmm, which are the relative rate constants for the hydrolysis of the Co-PPi substrate by Mg2E and Co2E, respectively. The value for k~/kmm (0.09) compares favorably with a value of 0.05 ([Co2+ ] = 0.25 mM) obtained by Welsh et al. [16] under similar conditions. The value for kmJkmm (0.14) is surprisingly very close to kcJkmm which indicates, at least in the case of the Co-PPi complex, that the metal ion bound to the enzyme (activator role) has only a small influence on the observed rate of substrate hydrolysis. A similar finding for the hydrolysis of the Zn-PPi complex by Mg2E and Zn2E has been reported [14]. However, by comparing kcJkmm (0.09) with kmm/kmm (1.00), it can be seen that changing the metal ion in both substrate and activator roles from Co2+ to Mg 2+ causes an ll-fold increase in the hydrolytic rate constant. These two comparisons, taken together, argue that the metal ion complexed with the PPi in the substrate role is the more influential in affecting the rate of PPi hydrolysis. Welsh et al. [16] have shown that the difference
214
in rate constants for the pyrophosphatase-catalyzed hydrolysis of PPi for Mg 2+ and Co2+ can be explained by a difference in the rate of dissociation of the first reaction product a metal-Pi complex, from the enzyme. A reasonable assumption, then, is that both kmc and k~ are primarily controlled by the rate of release of the Co-Pi complex, the Co2÷ therein coming from the Co-PPi substrate. In the case of the kmc rate constant, there is a second possible mechanistic interpretation which cannot be ruled out by this kinetic data and which should be mentioned. It is conceivable that interchange of activator and substrate metal ions could occur on the enzyme surface in the central catalytic complex. If very rapid, this exchange could be complete prior to PPi hydrolysis and product release, which would mean that kmc would be a complex measure of the catalytic breakdown of the two possible forms of MR. CoE(MgPP) (two possible sites for activator metal ions) as well as of Mg2E(CoPP). While there is no direct experimental evidence for this type of rapid interchange in the pyrophosphate literature, there is also no way at this time to definitely exclude it as a possibility.
Acknowledgement The authors acknowledge and appreciate the support that was provided for this work by Research Corporation through the Cottrell College Science Grants Program.
References 1 Cohen, S.A., Sterner, R., Keim, P.S. and Heim-ickson, R.L. (1978) J. Biol. Chem. 253, 889-897 2 Butler, L.G. (1971) in The Enzymes (Boyer, P.D., ed.), 3rd. FAn., Vol. 4, p. 535, Academic Press, New York 3 Butler, L.G. and Sperow, J.W. (1977) Bioinorg, Chem. 7, 141-150 4 Cooperman, B.S. and Chin, N.Y. (1973) Biochemistry 12, 1670-1676 5 Ridlington, J.W. and Butler, L.G. (1972) J. Biol. Chem. 247, 7303-7307 6 Knight, W.B., Dunaway-Mariano, D., Ransom, S.C. and Villafranca, J.J. (1984) J. Biol. Chem. 259, 2886-2895 7 Cooperman, B.S., Panackal, A., Springs, B. and Harem, D.J. (1981) Biochemistry 20, 6051-6060 8 Rapoport, T.A., Hohne, W.E., HeRman, P. and Rapoport, S. (1973) Eur. J. Biocham. 33, 341-347
9 Rapoport, T.A., Hohme, W.E., Reich, J., HeRman, P. and Rapoport, S.M. (1972) Eur. J. Biochem. 26, 237-246 10 Moe, O.A. and Butler, L.G. (1972) J. Biol. Chem. 247, 7308-7315 11 Shafransldi, Y.A., Baikov, A.A., Andrinkovich, P.F. and Aveava, S.M. (1977) Biokhimiya 42, 1244-1250 12 'Knight W.B., Fitts, S.W. and Dunaway-Mariano, D. (1981) Biochemistry 20, 4079-4086 13 Springs, B., Welch, K.M. and Cooperman, B.S. (1981) Biochemistry 20, 6384-6391 14 Moe, O.A., Jr., Pham, S., Dang, T. and Stryer, L. (1979) Arch. Biochem. Biophys. 196, 73-78 15 Welsh, K.M., Armitage, I.M. and Cooperman, B.S. (1983) Biochemistry 22, 1046-1054 16 Welsh, K.M., Jacobyansky, A., Springs, B. and Cooperman, B.S. (1983) Biochemistry 22, 2243-2248 17 Moe, O.A. and Butler, L.G. (1972) J. Biol. Chem. 247, 7315-7319 18 Knight, W.B., Ting, S., Chuang, S., Dunaway-Mariano, D., Haromy, T. and Sundaralingam, M. (1983) Arch. Biochem. Biophys. 227, 302-309 19 Schwartzenbach, G. and Flascha, J. (1969) Complexometric Titrations, Methuen, London 20 Vogel, A.J. (1961) Quantitative Inorganic Analysis, 3rd Edn., p. 433, Wiley, New York 21 Rossotti, F.J.C. and gossotti, H. (1961) The Determination of Stability Constants, Chapter 7, McGraw-Hill, New York 22 Moe, O.A., Jr. and Wiest, S.A. (1977) Anal. Biochem. 77, 73-781 23 Kim, H. (1970) J. Chem. Educ. 47, 120-122 24 Hammes, G.G. and Morrell, M.L. (1964) J. Am. Chem. Soc. 86, 1497-1502 25 Frey, C.H. and Stuehr, J.E. (1972) J. Am. Chem. Soc. 94, 8898-8903 26 Karweik, D.H. and Huber, C.O. (1978) Anal. Chem. 50, 1209 27 Johansson, A. and Wanninen, E. (1963) Talanta 10, 769-777 28 Schupp, O.E., Sturrock, P.E. and Watters, J.I. (1962) Inorg. Chem. 2, 106-112 29 Lambert, S.M. and Watters, J.I. (1957) J. Am. Chem. Soc. 79, 5606-5608 30 Josse, J. (1966) J. Biol. Chem. 241, 1948-1955 31 Lambert, S.M. and Watters, J.I. (1957) J. Am. Chem. Soc. 79, 4262-4265 32 Ridlington, J.W., Yang, Y. and Butler, L.G. (1972) Arch. Biochem. Biophys. 153, 714-725 33 Fiske, C.H. and SubbaRow, Y. (1925) J. Biol. Chem. 66, 375 34 Sillen, L.G. and Martel, A.E. (1964) Stability Constants of Metal-Ion Complexes, Special Publication, No. 17, The Chemical Society, London 35 Ting, S. and Dunaway-Mariano, D. (1984) FEBS Lett. 165, 251-253 36 Cotton, F.A. and Wilkinson, G. (1966) Advanced Inorganic Chemistry, 2nd Edn., p. 45, Interscienee, New York 37 Cooperman, B.S. (1982) Methods Enzymol. 87, 526-548