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35Cl-NMR studies on zinc-pyruvate kinase complexes
ARCHIVES
OF
BIOCHEMISTRY
35CI-NMR
AND
Studies
BIOPHYSICS
on Zinc-Pyruvate
GENE L. COTTAM Veterans
303-314 (1969)
132,
Kinase
AND RAYMOND
Complexes’
L. WARD3
Administration Hospital, Dallas, Texas 76216, and Lawrence Laboratory, Livermore, California 94660 Received
January
27, 1969; accepted
March
Radiation
25, 1969
%Cl NMR is used to study the interaction of zinc with pyruvate kinase. The number of zinc ions bound per molecule of pyruvate kinase is determined to be 4.17 f 0.2 and all four sites apparently have the same association constant of 0.81 f 0.07 X lo6 M-‘. Decreases in the WI-NMR line width are observed upon addition of HCOa-, ATP, or ADP to the zinc-pyruvate kinase binary complex. These effects could be explained by ternary complex formation or conformational changes effecting the environment of the bound zinc ion.
The recent technique of using chloride ions as a chemical probe for certain metal ions (Stengle and Baldeschwieler, 1966) had extended the use of nuclear magnetic resonance (NMR) to many interesting biological systems. This technique has been used to study antibody-hapten interactions (Haugland et al., 1967) and zinc-ADP interactions (Ward and Happe, 1968). Since those reports we have used the 35C1-NMR techniques to study enzymes which either contain zinc or require the addition of zinc ions for activity. This report on a zinc activated activity of pyruvate kinase will readily illustrate the usefulness of the 35Cl-NMR technique. Zinc-activated pyruvate kinase catalyzes a bicarbonate- and hydroxylamine-dependent cleavage of ATP (reaction below) which results in the formation of ADP and o-phosphorylhydroxylamine: 1 This work was performed under the auspices of the U. S. Atomic Energy Commission. The work was supported by the Division of Biology and Medicine of the Lawrence Radiation Laboratory and U.S.P.H.S. Grant AM 1131301 to the University of Texas Southwestern Medical School. 2 Veterans Administration Hospital and Department of Biochemistry, University of Texas Southwestern Medical School, Dallas, Texas. 3 Lawrence Radiation Laboratory, University of California, Livermore, California.
ATP
+
NHzOH ADP
p +
(HCOa-1 Zn2+ 7 K+
0)
H2N-O-PO,-.
The divalent metal Zn2+, cannot be replaced by Mg”+ and only partially by Mn2+ in the hydroxylamine phosphorylation. This order of divalent metal activation is reversed in the other two reactions catalyzed by the same of ADP by enzyme, the phosphorylation PEP and the phosphorylation of fluoride by ATP (Kupiecki and Coon, 1959). A monovalent cation such as K+ is also required to activate the enzyme (Boyer et al., 1943). The function of bicarbonate in the mechanism of the hydroxylamine phosphorylation was recently examined in a kinetic study and indicated that bicarbonate is a competitive inhibitor of the PEP in the Mg2+- or Mnz+-activated phosphorylation of ADP (Cottam et al., 1968). It is the purpose of this paper to demonstrate interaction between Zn2+ and pyruvate kinase using 35C1 nuclear magnetic resonance. This data is then used to calculate the number of Zn2+ binding sites per enzyme molecule and the Zn2+-pyruvate kinase association constant. 35Cl nuclear magnetic resonance was also used in attempts to detect ternary or higher complexes formed by
308
WI
NMR
ON ZINC-PYRUVATE
addition of bicarbonate, as well as other sub&rates, to the pyruvate kinase-ZIP+ binary complex. MATERIALS
AND
METHOD8
SMh! Measurements. The Wl spectra were obtained at 5.88 MHz/set with a Tarian V-4311 fixed-frequency RF unit. Baseline stabilization was achieved by field modulation at 175 cps and phase sensitive detection of the cent,er band with a PAR model HB-8 lock-in amplifier. The amplitude of the modulation was chosen to maximize the center band. A fixed magnetic field to frequency ratio was maintained by the use of an external proton lock signal. The spectra were calibrated using the first side bands of the field modulation system. All spectra were obtained at the probe temperature, approxima,tely 32”. The reported line widths are the average of at least five spectra and are reproducible to about 5% for the narrowest lines and to about 10% for the broadest lilies. The term “line width” refers to the full width of the NMR absorption measured at half maximiun amplitude. The concentration of chloride ion was 0.5 A~throughout all experiments. Chloride ioils possess electric quadrupole moments and as a result the line width of the single NMR absorption is very sensitive to departures from spherical charge distributions about the chlorine nucleus. Since chloride-metal ion complex formation readily leads to an asymmetric charge distribution, the resultant change in the WI-NMR line width can be used to study various metal ion systems. A description of the use of SW1 NMR for this type of study and the associated line width theory has been presented by Stengle and Baldeschwieler, (1966). It will s&ice t,o emphasize two points. First, the NMR observation of interest here depends on the ability of chloride ions to exchange rapidly among various sites so that the NMR line width is determined by a weighted average of these various sites; i.e., Av = ZAviPi . Av is the observed line width at half maximum amplitude, Aui is the width of the 35Cl resonance at site i, and Pi is the probability that the chloride ion is at site i. Second, the width of the NMR absorption, Avi , of a quadrupolar nucleus in a particular environment depends on the electric field gradient, q, at the nuclear site, and the correlation time, 7, for the quadrupolar nucleus, and is best described by the equation Av~ =
(const.)
[e2q &I%,
(2)
where & is the electric quadrupole moment and e is the electric charge. For small molecules such as zinc chloride, 7 is the rotational correlation time, but for large macromolecules such as zinc-
309
KINASE
pyruvate kinase, 7 can depend on either the rotational correlation time or the chloride exchange rate or both. Cohn and co-workers have made extensive use of an enhancement parameter originally defined by Eisinger et al., (1962) to describe changes in the nuclear magnetic relaxation rates of water molecules interacting with manganous ions bound to macromolecules. Although the physical mechanisms responsible for paramagnetic relaxation and quadrupole relaxation are entirely different, we find it convenient to describe our line broadening in terms of a quadrupolar enhancement, i.e., v* - vo* cr(=--
(3)
Y - VII
where Y is the observed Wl line width in the presence of ZG+, ~0the observed Wl line width in the absence of Zn2+, and the terms with the asterisks represent the same parameters in the presence of a complexing agent, in this case pyruvate kinase. The observed quadrupolar enhancement, eq , of a solution of free zinc ions, ZKLF , and bound zinc, Znb , is thus a weighted average of all forms and given by: E,l = -FIf er + $ e,, + 1 T T
= L
C Zni E; ,
ZllT
(4)
where ZnT is the total zinc concentrat.ion; er , the enchancement of free zinc, is equal to one by definition and Q is the enhancement of zinc bound in the complex. For a binary system, Zrrr = Znr + 2111,, and (5A)
Znb =
(4
eq - 1
ell -
1
ZnT .
Thus, once ~b has been determined for a system, a measurement of Q will allow a calculation of the Znr and Znb concentrations. The enhancement parameter, eb , may be determined from titration data as noted in the section on results. Pyruvate kinase was prepared from fresh rabbit muscle as described by Tietz and Ochoa (1958). The enzyme preparations which had specific activities higher than 240 pmoles of pyruvate formed/min/mg of protein were combined and stored as an ammonium sulfate suspension at a protein concentration of 20 mg/ml. The enzymic activity was determined spectrophotometrically by coupling the pyruvate kinase reaction to lactic dehydrogenase and measuring the decrease in optical density at 340 mp due to the oxidation of
310
COTTAM
AND WARD
DPNH (Biicher and Pfleiderer, 1955). The reaction mixture contained 100 rmoles of imidazole-HCl buffer, pH 7.0, 109 pmoles of KCl, 4 pmoles of MgClz , 1 pmole of PEP, 5 pmoles of ADP, 0.15 rmole of DPNH, 5 pg of lactate dehydrogenase, and pyruvate kinase to start the reaction in a final volume of 1.0 ml. The rate of disappearance of DPNH was determined with a Beckman DU spectrophotometer equipped with a Gilford cuvet positioner and multiple sample absorbance recorder. The temperature was 30”. The protein concentration was estimated from the absorbance at 280 nn6, using an extinction coefficient of 0.54 ml/mg/cm (Bticher and Pfleiderer, 1955) and the molecular weight of 237,000 (Warner, 1958). To prepare protein solutions for the a5C1-NMR studies, the ammonium sulfate suspension of pyruvate kinase was centrifuged at 35,000g for 15 min and the protein pellet dissolved in 0.01 M Tris-HCl buffer, pH 7.4, containing 0.5 M KCl. RESULTS
The 35C1line width in a 0.5 M sodium or potassium chloride solution is approximately 12 cps. This narrow line width is due to the nearly symmetrical environment of the solvated chloride ion. If zinc ions are added to the solution, one observes that the 35C1NMR line width increases linearly with zinc concentration. This linear increase in 35C1 line width is pH independent to the point where zinc hydroxide precipitates and can be described for a 0.5 M sodium chloride solution by the equation (Ward and Happe, 1968) Av = vc1 + 2 X lo3 ‘5
[Zn’+],
(6)
where VCI is the 35C1line width in the absence of zinc. The observed line width is a weighted average of the free chloride ions in the bulk solution and those which form a complex with zinc. Since it appears reasonable that the effective correlation time for the zinc chloride complex is the rotational correlation time, it is likely that the molecular tumbling and the electric field gradient at 35C1nucleus due to chloride bound to zinc both contribute appreciably to the increase in the 35C1line width. The addition of a complexing agent such as citrate or EDTA will effectively chelate the zinc ions and the 35C1 line width will revert to that of aqueous chloride. The addi-
FIG. 1. Change in the 35Cl-NMR line width, Y - YCI, as a function of pyruvate kinase concentration in a solution 0.01 M Tris-HCl, pH 7.39, and 0.5 M KCl. Each point is the average of six line width measurements and the deviation from the average was 5% or less. The abscissa is the increase of the 3%1-NMR line width above the line width of an aqueous 0.5 M chloride standard.
tion of a molecule like adenosine diphosphate or the enzyme pyruvate kinase will, however, increase the 36C1line width over that due to Zn2+ alone. This enhanced broadening must be due to an increase in the effective correlation time for the zinc chloride bond or a change in q since Pi can only decrease upon the formation of a complex. A linear increase in the 35C1line width is also observed when pyruvate kinase alone is added to a 0.5 M potassium chloride solution (Fig. 1) This increase in line width is a result of either the non-specific binding of chloride ions by pyruvate kinase, metal ion contaminants, or viscosity effects. If one titrates a fixed amount of pyruvate kinase, 1.58 X UY5 M, with zinc ions using the 35C1 line width as the measured parameter, an increase in line width over that observed for the non-specific broadening is observed (Fig. 2). Equally as well, one can titrate a fixed amount of zinc, 1.43 X 10U5 M, with pyruvate kinase using the 36C1line width as the measured parameter (Fig. 3). We are interested in obtaining the number of pyruvate kinase zinc binding sites and the
“CL NMR ON ZINC-PYRUVATE
Once Znb can point of (5B). A
KINASE
311
eb has been determined, Znr and be calculated for each experimental Fig. 2 by using equations (5A) and Hughes and Klotz plot, i.e.,
(Pyruvate
Kinase)
1
+ jl-
= nK,(Zn,)
&b>
n
of these quantities will allow a determination of n, the number of zinc binding sites, and K, , the association constant for these sites. This plot appears in Fig. 5. The intercept on [Zinc]
M xI0” I
Change in the “Cl-NMR line width, Y - VC~, as a function of zinc concentration in a solution containing 0.01 M Tris-HCl, pH 7.39, 0.5 M KCl, and 1.58 X 10h5M pyruvate kinase. The residual broadening with no added zinc is due to pyruvate kinase. FIG. 2.
association constants for these sites. Since we have only one measured parameter, E,it is necessary to relate this parameter to the pyruvate kinase and zinc concentration. This is accomplished, following the guide of Rlildvan and Cohn (1963). The definition of parameters ZIlb , in terms of enhancement (5B), is substituted into the definition of K A , the association constant for zinc and pyruvate kinase (7). Znt is the free zinc concentration, Znb is the bound zinc concentration, and Pr is the free protein concentration. KAY = (Znf>(Pf) (znb)
’
I
I
I
I
I
I
r -l
16 14 IZ-
[Pyruvaie
Kinose]
M ~10”
FIG. 3. Change in the WI-NMR line width, Y - YCI, as a function of the pyruvate kinase concentration at a fixed ZnClz concentration, 1.43 X 1OWM. The solution contains 0.01 M Tris-HCI, pH 7.39 and 0.5 M KCI.
(7)
From these two equations one can obtain the relationship expressed in (8) : (8) Under the condition Pr >> KD/tt,, which holds for Zn-pyruvate kinase, we can substitute the total pyruvate kinase concentration (P,) for the free protein concentration in the calculations. A plot of l/e4 vs. l/Pt, using the experimental points of Fig. 3, yields the curve of Fig. 4. Extrapolation of f/eq to infinite protein concentration then yields a value of eb = 196 and from the slope K, = 0.81 f 0.07 x lo5 M-l.
I.0
20 [Pyruvate
30
4.0
Kinose]e’
M-’
~10~’
FIG. 4. Double reciprocal plot of observed quadrupolar enhancement vs. pyruvate kinase concentration.
312
COTTAM
FIG. 5. Hughes and Klotz type plot of the results of a titration of pyruvate kinase with zinc. The concentration of free and bound zinc was calculated from the observed 3U-NMR line width and q = 196 as described in the text. In this type of graph the intercept on the ordinate gives l/n where n (4.17 f 0.2) is the number of binding sites and on the abscissa gives --KA , where KA (0.81 f 0.07 X lo5 M-I) is the association constant.
the ordinate yields a value of n = 4.17 f 0.2 and the intercept on the abscissa yields the negative of the association constant, KA = 0.81 f 0.07 x lo5 M-l. It is interesting to compare the binding of manganese ion to pyruvate kinase as studied by Mildvan and Cohn (1964, 1965) using pulsed nuclear magnetic resonance techniques. They observe n = 2.06 f 0.03 and K, = 1.33 X lo4 ~-l when potassium was included in the reaction mixture. Yet, if the nonactivating tetramethylammonium ion is substituted for potassium, 4 moles of manganese are bound per mole of pyruvate kinase. Since the potassium concentration used in the 35C1-NMR studies is higher than that used by Mildvan and Cohn, we repeated their pulsed NMR and ESR experiments on manganese binding to pyruvate kinase under our conditions of 0.5 M potassium chloride and obtained two Mn’J+ binding sites per mole of pyruvate kinase. The proposed mechanisms of the three reactions catalyzed by pyruvate kinase assume that the same binding site exists for Mg2+, MrP+, and Zn2+; we find, however,
AND
WARD
tha,t n = 4 for Zn2+ and n = 2 for Mn2+. We have used electron spin resonance techniques to follow the concentration of free Mn2+ in the presence of pyruvate kinase under conditions similar to those of our chloride-NMR studies. Upon addition of equimolar Zn2+ to the MrP+-pyruvate kinase system, the concentration of free Mn2+ increases. This observation, however, does not allow one to distinguish between two Zn2+ ions at the same Mn2+ site or two separate sets of binding sites. 35C1line width studies of pyruvate kinase in the presence of both Zn2+ and lug2+ exhibit a narrower line width than when compared to Zn2+ alone. However, we have not been able to displace all of the bound Zn2f with Mg2+ due to the larger KA for Zn2+. Efects of added substrates on enhancements of zinc-pyruvate kinase. We have titrated zinc-pyruvate kinase with KHC03 at pH 8.0, Fig. 6. This decrease in line width must be due to the effect of HC03- on the environment of the zinc ion. Either HC03- occupies a coordination site of the bound zinc preventing access of chloride ion or HCO3binds to the enzyme producing a conformational change at the zinc site which affects either q, 7, or even Pi . The association constant for HC03- estimated from these
“t
66
64
.
c 60 a n
$I/ \I 0
0.033
0.066 KHC03 M
0.10
FIG. 6. Titration of the zinc-pyruvate kinase binary complex with potassium bicarbonate. The reaction mixtures contained 14.8 mg of pyruvate kinase, 0.5 M KCl, 0.0167 M Tris-HCl, pH 8.0, 8.33 X 1OP M ZnCln and the noted concentration of KHCOs in a total volume of 0.60 ml of water. The %l-NMR line width in the absence of ZnCL was 40.2 cps.
=OL NMR
ON ZINC-PYRUVATE
studies is similar in magnitude to the constant calculated from kinetic studies. The addition of ATP or ADP to a solution of Zn2f and pyruvate kinase decreases the observed chloride line width. Factors such as competition between pyruvate kinase and ATP or ADP for Zn*+ or ternary complex formation could cause the reduction in line width. Further work is required to interpret the observed results on this ternary system. We have used the chloride-NMR technique to investigate possible effects of other substrates or products on the pyruvate kinasezinc binary complex and as yet have failed to observe other measurable effects on the chloride line width. DISCUSSION
Reynard et al. (1961) reported that pyruvate kinase has at least two binding sites for PEP. The conclusion was based on the equilibrium binding of PEP to pyruvate kinase as measured with an ultracentrifugal technique. Mildvan and Cohn (1964, 1965) have reported data from pulsed NMR and ESR techniques and have calculated that 2 Mn2+ are bound per molecule of pyruvate kinase when the reaction mixture contains 0.1 M K+, an activating cation. However, when tetramethylammonium ion, a nonactivating cation, was substituted for K+, 4 Mn2+ are bound per molecule of pyruvate kinase. They also demonstrate 2 K+ bound per molecule of enzyme. From their data they suggest that there is one divalent and one monovalent cation activator at each active site on pyruvate kinase. Ultraviolet difference spectrophotometry data from Suelter s laboratory are also consistent with these observations (Suelter et al., 1966). In the experiments reported here we have demonstrated that 35C1NMR allows us to detect interaction of Zn2+ with pyruvate kinase. This data is used to calculate the number of Zn2f bound per enzyme molecule and the association constant for the pyruvate kinase-Zn2f interaction. We find that 4 moles of Zn2+ are bound per mole of pyruvate kinase in the presence of 0.5 M K+. The association constant, 0.81 f 0.07 X lo5 iv+ is apparently the same for all four of the Zn2f binding sites. When identical conditions
KINASE
313
to those in the 35C1-NMR experiments are used with the pulsed NMR and ESR techniques to detect Mn2+ binding, we can determine only 2 Mn2+ bound per molecule of enzyme. Our results could mean that there are 2 Zn2+ ions binding at each active site, possibly by preventing the activating monovalent cation from binding to the enzyme. Another possible explanation could be that 2 Zn2+ ions bind to other sites on the enzyme which have the same association constant but are not identical to the two catalytic sites. It seems unlikely that the Zn2+ ions are binding to the protein at a completely different site than the Mg2+ or J!In2f because of the demonstration with ESR that the Zn2f and Mn2+ apparently compete for the same binding site on the enzyme. Since bicarbonate is required for hydroxylamine phosphorylation, yet no function for bicarbonate in this reaction has been demonstrated, we have attempted to detect interaction of bicarbonate with the pyruvate kinase-Zn2+ complex. When the binary Zn2fpyruvate kinase complex is titrated with KHCO, , pH 8.0, a decrease in the 35Cl-NhIR line width is observed. Perhaps if the bicarbonate anion occupies the same site on the enzyme as the carboxylate group of pyruvate, as suggested by Boyer (1962) and supported by kinetic data (Cottam et al., 1968), this may cause a conformational change similar to that observed with pyruvate (Suelter et al., 1966), which sufficiently changes the environment around the enzyme bound zinc ion to obtain the observed change in the 35C1-NMR spectra. Further experiments must be completed to distinguish between the possibilities of bicarbonate either causing a conformational change in pyruvate kinase or occupying a coordination site of the enzyme bound zinc. REFERENCES BACHHAWAT, B. K., AND COON, M. J., J. Bid. Chem. 231, 625 (1958). BOYER, P. D., in “The Enzymes” (P. D. Boyer, H. A. Lardy, and K. Myrbiick Eds.), Vol. 6, p. 95. Academic Press, New York (1962). BOYER, P. D., LARDY, H. A., AND PHILLIPS, P.H., J. Bid. Chem. 149, 529 (1943). BUTCHER,T., ANDPFLEIDERER, G., Methods Enzymol. 1, 435 (1955).
314
COTTAM
COTTAM, G. L., KUPIECKI, F. P., AND COON, M. J., J. Biol. Chem. 243,163O (1968). EISINOER, J., SHULMAN, R. G., AND SZYMANSKI, B. M., J. Chem. Phys. %,1721 (1962). HAUGLAND, R. P., STRYER, L., STENGLE, T. R., AND BALDESCHWIELER, J. C., Biochemistry 6, 498 (1967). HUGHES, T. R., AND KLOTZ, I. M., “Methods of Biochemical Analysis,” Vol. III, p. 265. (Wiley Interscience) New York (1956). KUPIECKI, F. P., AND COON, M. J., J. Biol. Chem. 234, 2428 (1959). MILDVAN, A. S., AND COHN, M., Biochemistry 2, 910 (1963). MILDVAN, A. S., AND COHN, M., J. Biol. Chem. 240, 238 (1965). MILDVAN, A. S., AND COHN M., Abstracts Sixth
AND
WARD
International Congress of Biochemistry, I.U.B., 32. p. 322, IV-111 (1964). REYNARD, A. M., HASS, L. F., JACOBSEN, D. D., AND BOYER, P. D., J. Biol. Chem. 236, 2277 (1961). STENGLE, T. R. AND BALDESCHWIELER, J. D., Proc. Natl. Acad. Sci. U. S. 65,102O (1966). SUELTER, C. H., SINQLETON, R., JR., KAYNE, F. J., ARRINGTON, S., GLASS, J., AND MILDVAN, A. S., Biochemistry 6,131 (1!%6). TIETZ, A., AND OCHOA, S., Arch. Biochem. Biophys. 78,477 (1958). WARD, R. L., AND HAPPE, J. A., Biochem. Biophys. Res. Common. 28, 785 (1968). WARNER, R. C., Arch. Biochem. Biophys. 78, 494 (1958).
OF
BIOCHEMISTRY
35CI-NMR
AND
Studies
BIOPHYSICS
on Zinc-Pyruvate
GENE L. COTTAM Veterans
303-314 (1969)
132,
Kinase
AND RAYMOND
Complexes’
L. WARD3
Administration Hospital, Dallas, Texas 76216, and Lawrence Laboratory, Livermore, California 94660 Received
January
27, 1969; accepted
March
Radiation
25, 1969
%Cl NMR is used to study the interaction of zinc with pyruvate kinase. The number of zinc ions bound per molecule of pyruvate kinase is determined to be 4.17 f 0.2 and all four sites apparently have the same association constant of 0.81 f 0.07 X lo6 M-‘. Decreases in the WI-NMR line width are observed upon addition of HCOa-, ATP, or ADP to the zinc-pyruvate kinase binary complex. These effects could be explained by ternary complex formation or conformational changes effecting the environment of the bound zinc ion.
The recent technique of using chloride ions as a chemical probe for certain metal ions (Stengle and Baldeschwieler, 1966) had extended the use of nuclear magnetic resonance (NMR) to many interesting biological systems. This technique has been used to study antibody-hapten interactions (Haugland et al., 1967) and zinc-ADP interactions (Ward and Happe, 1968). Since those reports we have used the 35C1-NMR techniques to study enzymes which either contain zinc or require the addition of zinc ions for activity. This report on a zinc activated activity of pyruvate kinase will readily illustrate the usefulness of the 35Cl-NMR technique. Zinc-activated pyruvate kinase catalyzes a bicarbonate- and hydroxylamine-dependent cleavage of ATP (reaction below) which results in the formation of ADP and o-phosphorylhydroxylamine: 1 This work was performed under the auspices of the U. S. Atomic Energy Commission. The work was supported by the Division of Biology and Medicine of the Lawrence Radiation Laboratory and U.S.P.H.S. Grant AM 1131301 to the University of Texas Southwestern Medical School. 2 Veterans Administration Hospital and Department of Biochemistry, University of Texas Southwestern Medical School, Dallas, Texas. 3 Lawrence Radiation Laboratory, University of California, Livermore, California.
ATP
+
NHzOH ADP
p +
(HCOa-1 Zn2+ 7 K+
0)
H2N-O-PO,-.
The divalent metal Zn2+, cannot be replaced by Mg”+ and only partially by Mn2+ in the hydroxylamine phosphorylation. This order of divalent metal activation is reversed in the other two reactions catalyzed by the same of ADP by enzyme, the phosphorylation PEP and the phosphorylation of fluoride by ATP (Kupiecki and Coon, 1959). A monovalent cation such as K+ is also required to activate the enzyme (Boyer et al., 1943). The function of bicarbonate in the mechanism of the hydroxylamine phosphorylation was recently examined in a kinetic study and indicated that bicarbonate is a competitive inhibitor of the PEP in the Mg2+- or Mnz+-activated phosphorylation of ADP (Cottam et al., 1968). It is the purpose of this paper to demonstrate interaction between Zn2+ and pyruvate kinase using 35C1 nuclear magnetic resonance. This data is then used to calculate the number of Zn2+ binding sites per enzyme molecule and the Zn2+-pyruvate kinase association constant. 35Cl nuclear magnetic resonance was also used in attempts to detect ternary or higher complexes formed by
308
WI
NMR
ON ZINC-PYRUVATE
addition of bicarbonate, as well as other sub&rates, to the pyruvate kinase-ZIP+ binary complex. MATERIALS
AND
METHOD8
SMh! Measurements. The Wl spectra were obtained at 5.88 MHz/set with a Tarian V-4311 fixed-frequency RF unit. Baseline stabilization was achieved by field modulation at 175 cps and phase sensitive detection of the cent,er band with a PAR model HB-8 lock-in amplifier. The amplitude of the modulation was chosen to maximize the center band. A fixed magnetic field to frequency ratio was maintained by the use of an external proton lock signal. The spectra were calibrated using the first side bands of the field modulation system. All spectra were obtained at the probe temperature, approxima,tely 32”. The reported line widths are the average of at least five spectra and are reproducible to about 5% for the narrowest lines and to about 10% for the broadest lilies. The term “line width” refers to the full width of the NMR absorption measured at half maximiun amplitude. The concentration of chloride ion was 0.5 A~throughout all experiments. Chloride ioils possess electric quadrupole moments and as a result the line width of the single NMR absorption is very sensitive to departures from spherical charge distributions about the chlorine nucleus. Since chloride-metal ion complex formation readily leads to an asymmetric charge distribution, the resultant change in the WI-NMR line width can be used to study various metal ion systems. A description of the use of SW1 NMR for this type of study and the associated line width theory has been presented by Stengle and Baldeschwieler, (1966). It will s&ice t,o emphasize two points. First, the NMR observation of interest here depends on the ability of chloride ions to exchange rapidly among various sites so that the NMR line width is determined by a weighted average of these various sites; i.e., Av = ZAviPi . Av is the observed line width at half maximum amplitude, Aui is the width of the 35Cl resonance at site i, and Pi is the probability that the chloride ion is at site i. Second, the width of the NMR absorption, Avi , of a quadrupolar nucleus in a particular environment depends on the electric field gradient, q, at the nuclear site, and the correlation time, 7, for the quadrupolar nucleus, and is best described by the equation Av~ =
(const.)
[e2q &I%,
(2)
where & is the electric quadrupole moment and e is the electric charge. For small molecules such as zinc chloride, 7 is the rotational correlation time, but for large macromolecules such as zinc-
309
KINASE
pyruvate kinase, 7 can depend on either the rotational correlation time or the chloride exchange rate or both. Cohn and co-workers have made extensive use of an enhancement parameter originally defined by Eisinger et al., (1962) to describe changes in the nuclear magnetic relaxation rates of water molecules interacting with manganous ions bound to macromolecules. Although the physical mechanisms responsible for paramagnetic relaxation and quadrupole relaxation are entirely different, we find it convenient to describe our line broadening in terms of a quadrupolar enhancement, i.e., v* - vo* cr(=--
(3)
Y - VII
where Y is the observed Wl line width in the presence of ZG+, ~0the observed Wl line width in the absence of Zn2+, and the terms with the asterisks represent the same parameters in the presence of a complexing agent, in this case pyruvate kinase. The observed quadrupolar enhancement, eq , of a solution of free zinc ions, ZKLF , and bound zinc, Znb , is thus a weighted average of all forms and given by: E,l = -FIf er + $ e,, + 1 T T
= L
C Zni E; ,
ZllT
(4)
where ZnT is the total zinc concentrat.ion; er , the enchancement of free zinc, is equal to one by definition and Q is the enhancement of zinc bound in the complex. For a binary system, Zrrr = Znr + 2111,, and (5A)
Znb =
(4
eq - 1
ell -
1
ZnT .
Thus, once ~b has been determined for a system, a measurement of Q will allow a calculation of the Znr and Znb concentrations. The enhancement parameter, eb , may be determined from titration data as noted in the section on results. Pyruvate kinase was prepared from fresh rabbit muscle as described by Tietz and Ochoa (1958). The enzyme preparations which had specific activities higher than 240 pmoles of pyruvate formed/min/mg of protein were combined and stored as an ammonium sulfate suspension at a protein concentration of 20 mg/ml. The enzymic activity was determined spectrophotometrically by coupling the pyruvate kinase reaction to lactic dehydrogenase and measuring the decrease in optical density at 340 mp due to the oxidation of
310
COTTAM
AND WARD
DPNH (Biicher and Pfleiderer, 1955). The reaction mixture contained 100 rmoles of imidazole-HCl buffer, pH 7.0, 109 pmoles of KCl, 4 pmoles of MgClz , 1 pmole of PEP, 5 pmoles of ADP, 0.15 rmole of DPNH, 5 pg of lactate dehydrogenase, and pyruvate kinase to start the reaction in a final volume of 1.0 ml. The rate of disappearance of DPNH was determined with a Beckman DU spectrophotometer equipped with a Gilford cuvet positioner and multiple sample absorbance recorder. The temperature was 30”. The protein concentration was estimated from the absorbance at 280 nn6, using an extinction coefficient of 0.54 ml/mg/cm (Bticher and Pfleiderer, 1955) and the molecular weight of 237,000 (Warner, 1958). To prepare protein solutions for the a5C1-NMR studies, the ammonium sulfate suspension of pyruvate kinase was centrifuged at 35,000g for 15 min and the protein pellet dissolved in 0.01 M Tris-HCl buffer, pH 7.4, containing 0.5 M KCl. RESULTS
The 35C1line width in a 0.5 M sodium or potassium chloride solution is approximately 12 cps. This narrow line width is due to the nearly symmetrical environment of the solvated chloride ion. If zinc ions are added to the solution, one observes that the 35C1NMR line width increases linearly with zinc concentration. This linear increase in 35C1 line width is pH independent to the point where zinc hydroxide precipitates and can be described for a 0.5 M sodium chloride solution by the equation (Ward and Happe, 1968) Av = vc1 + 2 X lo3 ‘5
[Zn’+],
(6)
where VCI is the 35C1line width in the absence of zinc. The observed line width is a weighted average of the free chloride ions in the bulk solution and those which form a complex with zinc. Since it appears reasonable that the effective correlation time for the zinc chloride complex is the rotational correlation time, it is likely that the molecular tumbling and the electric field gradient at 35C1nucleus due to chloride bound to zinc both contribute appreciably to the increase in the 35C1line width. The addition of a complexing agent such as citrate or EDTA will effectively chelate the zinc ions and the 35C1 line width will revert to that of aqueous chloride. The addi-
FIG. 1. Change in the 35Cl-NMR line width, Y - YCI, as a function of pyruvate kinase concentration in a solution 0.01 M Tris-HCl, pH 7.39, and 0.5 M KCl. Each point is the average of six line width measurements and the deviation from the average was 5% or less. The abscissa is the increase of the 3%1-NMR line width above the line width of an aqueous 0.5 M chloride standard.
tion of a molecule like adenosine diphosphate or the enzyme pyruvate kinase will, however, increase the 36C1line width over that due to Zn2+ alone. This enhanced broadening must be due to an increase in the effective correlation time for the zinc chloride bond or a change in q since Pi can only decrease upon the formation of a complex. A linear increase in the 35C1line width is also observed when pyruvate kinase alone is added to a 0.5 M potassium chloride solution (Fig. 1) This increase in line width is a result of either the non-specific binding of chloride ions by pyruvate kinase, metal ion contaminants, or viscosity effects. If one titrates a fixed amount of pyruvate kinase, 1.58 X UY5 M, with zinc ions using the 35C1 line width as the measured parameter, an increase in line width over that observed for the non-specific broadening is observed (Fig. 2). Equally as well, one can titrate a fixed amount of zinc, 1.43 X 10U5 M, with pyruvate kinase using the 36C1line width as the measured parameter (Fig. 3). We are interested in obtaining the number of pyruvate kinase zinc binding sites and the
“CL NMR ON ZINC-PYRUVATE
Once Znb can point of (5B). A
KINASE
311
eb has been determined, Znr and be calculated for each experimental Fig. 2 by using equations (5A) and Hughes and Klotz plot, i.e.,
(Pyruvate
Kinase)
1
+ jl-
= nK,(Zn,)
&b>
n
of these quantities will allow a determination of n, the number of zinc binding sites, and K, , the association constant for these sites. This plot appears in Fig. 5. The intercept on [Zinc]
M xI0” I
Change in the “Cl-NMR line width, Y - VC~, as a function of zinc concentration in a solution containing 0.01 M Tris-HCl, pH 7.39, 0.5 M KCl, and 1.58 X 10h5M pyruvate kinase. The residual broadening with no added zinc is due to pyruvate kinase. FIG. 2.
association constants for these sites. Since we have only one measured parameter, E,it is necessary to relate this parameter to the pyruvate kinase and zinc concentration. This is accomplished, following the guide of Rlildvan and Cohn (1963). The definition of parameters ZIlb , in terms of enhancement (5B), is substituted into the definition of K A , the association constant for zinc and pyruvate kinase (7). Znt is the free zinc concentration, Znb is the bound zinc concentration, and Pr is the free protein concentration. KAY = (Znf>(Pf) (znb)
’
I
I
I
I
I
I
r -l
16 14 IZ-
[Pyruvaie
Kinose]
M ~10”
FIG. 3. Change in the WI-NMR line width, Y - YCI, as a function of the pyruvate kinase concentration at a fixed ZnClz concentration, 1.43 X 1OWM. The solution contains 0.01 M Tris-HCI, pH 7.39 and 0.5 M KCI.
(7)
From these two equations one can obtain the relationship expressed in (8) : (8) Under the condition Pr >> KD/tt,, which holds for Zn-pyruvate kinase, we can substitute the total pyruvate kinase concentration (P,) for the free protein concentration in the calculations. A plot of l/e4 vs. l/Pt, using the experimental points of Fig. 3, yields the curve of Fig. 4. Extrapolation of f/eq to infinite protein concentration then yields a value of eb = 196 and from the slope K, = 0.81 f 0.07 x lo5 M-l.
I.0
20 [Pyruvate
30
4.0
Kinose]e’
M-’
~10~’
FIG. 4. Double reciprocal plot of observed quadrupolar enhancement vs. pyruvate kinase concentration.
312
COTTAM
FIG. 5. Hughes and Klotz type plot of the results of a titration of pyruvate kinase with zinc. The concentration of free and bound zinc was calculated from the observed 3U-NMR line width and q = 196 as described in the text. In this type of graph the intercept on the ordinate gives l/n where n (4.17 f 0.2) is the number of binding sites and on the abscissa gives --KA , where KA (0.81 f 0.07 X lo5 M-I) is the association constant.
the ordinate yields a value of n = 4.17 f 0.2 and the intercept on the abscissa yields the negative of the association constant, KA = 0.81 f 0.07 x lo5 M-l. It is interesting to compare the binding of manganese ion to pyruvate kinase as studied by Mildvan and Cohn (1964, 1965) using pulsed nuclear magnetic resonance techniques. They observe n = 2.06 f 0.03 and K, = 1.33 X lo4 ~-l when potassium was included in the reaction mixture. Yet, if the nonactivating tetramethylammonium ion is substituted for potassium, 4 moles of manganese are bound per mole of pyruvate kinase. Since the potassium concentration used in the 35C1-NMR studies is higher than that used by Mildvan and Cohn, we repeated their pulsed NMR and ESR experiments on manganese binding to pyruvate kinase under our conditions of 0.5 M potassium chloride and obtained two Mn’J+ binding sites per mole of pyruvate kinase. The proposed mechanisms of the three reactions catalyzed by pyruvate kinase assume that the same binding site exists for Mg2+, MrP+, and Zn2+; we find, however,
AND
WARD
tha,t n = 4 for Zn2+ and n = 2 for Mn2+. We have used electron spin resonance techniques to follow the concentration of free Mn2+ in the presence of pyruvate kinase under conditions similar to those of our chloride-NMR studies. Upon addition of equimolar Zn2+ to the MrP+-pyruvate kinase system, the concentration of free Mn2+ increases. This observation, however, does not allow one to distinguish between two Zn2+ ions at the same Mn2+ site or two separate sets of binding sites. 35C1line width studies of pyruvate kinase in the presence of both Zn2+ and lug2+ exhibit a narrower line width than when compared to Zn2+ alone. However, we have not been able to displace all of the bound Zn2f with Mg2+ due to the larger KA for Zn2+. Efects of added substrates on enhancements of zinc-pyruvate kinase. We have titrated zinc-pyruvate kinase with KHC03 at pH 8.0, Fig. 6. This decrease in line width must be due to the effect of HC03- on the environment of the zinc ion. Either HC03- occupies a coordination site of the bound zinc preventing access of chloride ion or HCO3binds to the enzyme producing a conformational change at the zinc site which affects either q, 7, or even Pi . The association constant for HC03- estimated from these
“t
66
64
.
c 60 a n
$I/ \I 0
0.033
0.066 KHC03 M
0.10
FIG. 6. Titration of the zinc-pyruvate kinase binary complex with potassium bicarbonate. The reaction mixtures contained 14.8 mg of pyruvate kinase, 0.5 M KCl, 0.0167 M Tris-HCl, pH 8.0, 8.33 X 1OP M ZnCln and the noted concentration of KHCOs in a total volume of 0.60 ml of water. The %l-NMR line width in the absence of ZnCL was 40.2 cps.
=OL NMR
ON ZINC-PYRUVATE
studies is similar in magnitude to the constant calculated from kinetic studies. The addition of ATP or ADP to a solution of Zn2f and pyruvate kinase decreases the observed chloride line width. Factors such as competition between pyruvate kinase and ATP or ADP for Zn*+ or ternary complex formation could cause the reduction in line width. Further work is required to interpret the observed results on this ternary system. We have used the chloride-NMR technique to investigate possible effects of other substrates or products on the pyruvate kinasezinc binary complex and as yet have failed to observe other measurable effects on the chloride line width. DISCUSSION
Reynard et al. (1961) reported that pyruvate kinase has at least two binding sites for PEP. The conclusion was based on the equilibrium binding of PEP to pyruvate kinase as measured with an ultracentrifugal technique. Mildvan and Cohn (1964, 1965) have reported data from pulsed NMR and ESR techniques and have calculated that 2 Mn2+ are bound per molecule of pyruvate kinase when the reaction mixture contains 0.1 M K+, an activating cation. However, when tetramethylammonium ion, a nonactivating cation, was substituted for K+, 4 Mn2+ are bound per molecule of pyruvate kinase. They also demonstrate 2 K+ bound per molecule of enzyme. From their data they suggest that there is one divalent and one monovalent cation activator at each active site on pyruvate kinase. Ultraviolet difference spectrophotometry data from Suelter s laboratory are also consistent with these observations (Suelter et al., 1966). In the experiments reported here we have demonstrated that 35C1NMR allows us to detect interaction of Zn2+ with pyruvate kinase. This data is used to calculate the number of Zn2f bound per enzyme molecule and the association constant for the pyruvate kinase-Zn2f interaction. We find that 4 moles of Zn2+ are bound per mole of pyruvate kinase in the presence of 0.5 M K+. The association constant, 0.81 f 0.07 X lo5 iv+ is apparently the same for all four of the Zn2f binding sites. When identical conditions
KINASE
313
to those in the 35C1-NMR experiments are used with the pulsed NMR and ESR techniques to detect Mn2+ binding, we can determine only 2 Mn2+ bound per molecule of enzyme. Our results could mean that there are 2 Zn2+ ions binding at each active site, possibly by preventing the activating monovalent cation from binding to the enzyme. Another possible explanation could be that 2 Zn2+ ions bind to other sites on the enzyme which have the same association constant but are not identical to the two catalytic sites. It seems unlikely that the Zn2+ ions are binding to the protein at a completely different site than the Mg2+ or J!In2f because of the demonstration with ESR that the Zn2f and Mn2+ apparently compete for the same binding site on the enzyme. Since bicarbonate is required for hydroxylamine phosphorylation, yet no function for bicarbonate in this reaction has been demonstrated, we have attempted to detect interaction of bicarbonate with the pyruvate kinase-Zn2+ complex. When the binary Zn2fpyruvate kinase complex is titrated with KHCO, , pH 8.0, a decrease in the 35Cl-NhIR line width is observed. Perhaps if the bicarbonate anion occupies the same site on the enzyme as the carboxylate group of pyruvate, as suggested by Boyer (1962) and supported by kinetic data (Cottam et al., 1968), this may cause a conformational change similar to that observed with pyruvate (Suelter et al., 1966), which sufficiently changes the environment around the enzyme bound zinc ion to obtain the observed change in the 35C1-NMR spectra. Further experiments must be completed to distinguish between the possibilities of bicarbonate either causing a conformational change in pyruvate kinase or occupying a coordination site of the enzyme bound zinc. REFERENCES BACHHAWAT, B. K., AND COON, M. J., J. Bid. Chem. 231, 625 (1958). BOYER, P. D., in “The Enzymes” (P. D. Boyer, H. A. Lardy, and K. Myrbiick Eds.), Vol. 6, p. 95. Academic Press, New York (1962). BOYER, P. D., LARDY, H. A., AND PHILLIPS, P.H., J. Bid. Chem. 149, 529 (1943). BUTCHER,T., ANDPFLEIDERER, G., Methods Enzymol. 1, 435 (1955).
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COTTAM
COTTAM, G. L., KUPIECKI, F. P., AND COON, M. J., J. Biol. Chem. 243,163O (1968). EISINOER, J., SHULMAN, R. G., AND SZYMANSKI, B. M., J. Chem. Phys. %,1721 (1962). HAUGLAND, R. P., STRYER, L., STENGLE, T. R., AND BALDESCHWIELER, J. C., Biochemistry 6, 498 (1967). HUGHES, T. R., AND KLOTZ, I. M., “Methods of Biochemical Analysis,” Vol. III, p. 265. (Wiley Interscience) New York (1956). KUPIECKI, F. P., AND COON, M. J., J. Biol. Chem. 234, 2428 (1959). MILDVAN, A. S., AND COHN, M., Biochemistry 2, 910 (1963). MILDVAN, A. S., AND COHN, M., J. Biol. Chem. 240, 238 (1965). MILDVAN, A. S., AND COHN M., Abstracts Sixth
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WARD
International Congress of Biochemistry, I.U.B., 32. p. 322, IV-111 (1964). REYNARD, A. M., HASS, L. F., JACOBSEN, D. D., AND BOYER, P. D., J. Biol. Chem. 236, 2277 (1961). STENGLE, T. R. AND BALDESCHWIELER, J. D., Proc. Natl. Acad. Sci. U. S. 65,102O (1966). SUELTER, C. H., SINQLETON, R., JR., KAYNE, F. J., ARRINGTON, S., GLASS, J., AND MILDVAN, A. S., Biochemistry 6,131 (1!%6). TIETZ, A., AND OCHOA, S., Arch. Biochem. Biophys. 78,477 (1958). WARD, R. L., AND HAPPE, J. A., Biochem. Biophys. Res. Common. 28, 785 (1968). WARNER, R. C., Arch. Biochem. Biophys. 78, 494 (1958).