Allosteric Interactions between Metal Ion and Phosphate at ...Allosteric Interactions between Metal...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 252, No. 20. Issue of October 26. pp. 1053-7061, 1977

Pm&d tn Cl S.A.

Allosteric Interactions between Metal Ion and Phosphate at the Active Sites of Alkaline Phosphatase as Determined by 31P NMR and l13Cd NMR*

(Received for publication, April 5, 1977)

JAN F. CHLEBOWSKI, IAN M. ARMITAGE, AND JOSEPH E. COLEMAN

From the Department of Molecular Biophysics and Biochemistry and the Section of Physical Sciences, Yale University School of Medicine, New Haven, Connecticut 06510

““Cd and :“P NMR have been used to determine the effect of variations in the nature and stoichiometry of bound metal ion and pH on the allosteric interactions (negative cooperativity) induced on association of phosphate with the dimeric zinc metalloenzyme Escherichia coli alkaline phosphatase. At pH 8.0, successive additions of an extra two Zn’+ ions and one Mg”+ ion to the Znzs+ enzyme result in structural alterations of the noncovalent phosphate complex (E. P) reflected in a progressive upfield chemical shift of the :“P resonance from 5.1 ppm (Zngz+ enzyme) to 4.2 and 3.5 ppm, respectively. Variation of metal ion content does not alter the stoichiometry of tight phosphate binding; in all cases a single phosphate ligand is bound per protein dimer. Phosphate binding to the CoL2+ enzyme at pH 8.0 (where tightly bound and free P, exist under slow exchange conditions) and pH 6.5 (where Pi is in fast exchange) are similarly consistent with the tight association of one Pi/ dimer. Observation of the NMR of both the ligand and metal ion nuclei in the covalent phosphate complex (E .P) of the Cd,?+ enzyme at pH 6.5 confirms these results. Two equivalents of Cd”+/dimer are required to generate the maximum of 1 eq of phosphoryl enzymeldimer, observed as a characteristic low field (-8.0 ppm) resonance in the n’P NMR spectrum. The “Wd NMR spectrum of the unliganded enzyme shows the Cd”+ ions to exist in identical environments, since a single resonance is present 170 ppm downfield from the standard, 0.1 M CdCIO,. Covalent phosphorylation at a single site results in appearance of two resonances of equal intensity at 142 and 55 ppm. The presence of 1 mol of excess phosphate does not alter the stoichiometry of phosphoryl enzyme formation or the altered environments of the metal ions. These results are consistent only with the existence of negative homotropic interactions between the subunits induced on ligand (phosphate) binding resulting in

* This work was supported by Grants AM 09070-13 and AM 18778-02 from the National Institutes of Health and by Grant PCM76-82231 from the National Science Foundation. Acknowledg- ment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C Seciton 1734 solely to indicate this fact.

conformational alterations at both active centers of the dimeric enzyme.

‘“P NMR has been shown to be a powerful means of exploring the chemical nature of the phosphate complexes of the Zr?+ metalloenzyme, Escherichia coli alkaline phosphatase (l-3). Initial studies have identified the phosphorus chemical shifts of the noncovalent complex of the enzyme, E. P, with the product, inorganic phosphate and the covalent intermediate, E-P, resulting from the phosphorylation of the hydroxyl group of serine 99 by phosphate or substrate.’ The low field position of the phosphorus resonance for E-P, -8 ppm, suggests that the enzyme phosphoserine is a phosphate ester of unusual geometry (1, 4, 5).

Since ‘r’P NMR can monitor simultaneously all forms of phosphate in the solution, it is potentially the best method for determining the stoichiometry of the various distinct forms of enzyme-bound phosphate. Phosphate binding to alkaline phosphatase has been shown to be metal ion-dependent (6). There- fore the stoichiometry of phosphate binding may be expected to depend on the metal ion stoichiometry. Conversely the presence of phosphate or the phosphoryl group might be expected to influence metal ion binding to the apoenzyme. The present paper explores in detail with 3’P NMR methods the chemical nature of enzyme-bound phosphate as functions of phosphate, metal ion (Zn’+, Cd’+, and Co’+), and magne- sium concentrations. Using ‘%d NMR, the chemical nature of the active center metal ion has been explored both in the absence and presence of phosphate ligand. Evidence is pre- sented consistent with the existence of stable structural isomers of enzyme. phosphate complexes as detected by differences in the chemical shifts of resonances assigned to tightly associated phosphate. The distribution of the enzyme in these conformational forms is controlled by the metal ion stoichiometry. Thus, the appearance of multiple E. P resonances can be related to the mode of preparation of the enzyme and

’ The phosphorylated serine is residue 99 from the NH,-terminal threonine residue (100 from the NH,-terminal arginine in the arginine isozyme) in the preliminary numbering of the sequence as it is presently available from the work of R. A. Bradshaw, P. A. Neumann, F. Cancedda, K. Schrifla, J. D. Hecht, and M. J. Schlesinger (personal communication from R. A. Bradshaw).

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7054 ‘Td NMR and :“P NMR Studies of Alkaline Phosphatase

its consequent metal ion content. Under all conditions the enzyme-bound phosphate stoichiometry remains constant at 1 molimol of enzyme dimer. Thus binding of ligand at one site of the symmetrical dimer must prevent ligand binding at the second site. This negative cooperativity is unambiguously confirmed by “:‘Cd NMR which shows the two Cd” + ions to occupy identical chemical environments in the unliganded enzyme, but two different environments when a single phosphate anion is covalently bound to the dimer.

MATERIALS AND METHODS

Enzymes and Chemicals -Crystalline alkaline phosphatase was prepared from Escherichia cd (strain CW 3747) as described by Applebury et al. (6). Enzyme concentrations were determined spec- trophotometrically at 278 nm with E:$ = 0.72 (71. For molar calculations a molecular weight of 86,000 was used.’ Buffer solutions, HCl, and NH,OH were prepared metal-free (81. Apo- and Me”+ phosphatases were prepared as previously described (6, 8). For the NMR studies, the Co” and Cd” enzymes were prepared by the addition of slightly less than 2 eq of Me”+/apoenzyme dimer. In the case of the cadmium enzyme 96 atom % of ““Cd(H) salts (Oak Ridge Laboratories) were added to solutions of the apoenzyme, followed by concentration to a volume of -1 ml. Titration of apoalkaline phosphatase with Me” ions as followed by several spectroscopic techniques shows that the first two metal ions added are tightly bound at the sites occupied by the catalytically active Zn’+ ions of the native enzyme (6, 9-12). All NMR samples and equipment were prepared metal-free following procedures previously described (1).

NMR Techniques -Fourier transform :“P and ““Cd NMR spectra were obtained on an extensively modified Bruker HFX-90 MHz spectrometer operating at 36.44 and 19.96 MHz, respectively, at 25 ? 2” (13, 14). D,O in a 3-mm co-axial capillary insert was used as an external field frequency lock. All :“P NMR spectra were obtained under conditions of proton noise decoupling. ““Cd NMR spectra were obtained in the absence of proton noise decoupling because of the negative value of the nuclear magnetic moment for ““Cd and the predicted dependence of the negative nuclear Overhauser en- hancement on correlation time. A spectral width of 5000 Hz was used throughout to maximize the S/N improvement from a 5000 Hz bandwidth crystal filter. Typical parameters for J’P NMR spectra include an acquisition time of 0.2 s, a pulse delay of 0.2 s, and a pulse width of 60”. For ‘13Cd NMR spectra they were as follows: acquisition time, 0.1 s; pulse delay, 0.3 s; pulse width, 70”. For all spectra shown an interpolation expansion routine was employed providing a resolution of 1.2 Hz/point for 31P and 5.0 Hz/point for ‘13Cd (15). Measurements were made on -l.O-ml samples contained in lo-mm sample tubes fitted with Vortex plugs to confine the solution within the transmitter coil. Typical spectra required from 40,000 to 250,000 transients (4 to 24 h) depending on nucleus and protein concentration. “‘P chemical shifts were determined relative to external 85% H,PO,. ‘I:’ Cd chemical shifts are expressed relative to the resonance position of 0.1 M CdClO,. Signs for chemical shift values reflect the IUPAC standard nomenclature, increasing num- bers to low field.

RESULTS AND DISCUSSION

Stoichiometry of Zny+ and Bound Phosphate -Despite in- vestigation by a variety of experimental methods, the stoichiometry of ligand (i.e. substrate, inhibitor, or product) binding to alkaline phosphatase has remained controversial. Under conditions of moderate ionic strength, /1 = 0.15, and low concentrations (10 mM) of Tris buffer, tight binding of one ligand (K,, f= lo-‘; Ml/enzyme dimer is observed (6, 9, 16-19). Interaction of enzyme dimer with a second equivalent of ligand was observed to occur with a greatly reduced affinity (K,, > 1Om:1 M) (6, 17). The methodology employed in these studies did not allow a distinction between different forms of

2 The value for the molecular weight was calculated from the tentative sequence of 425 amino residues in each monomer (R. A. Bradshaw, personal communication).

enzyme-bound phosphate and thus did not reveal whether binding of a second ligand is different from the first. All of the above studies have suggested that negative homotropic interactions exist between the two identical active sites on the dimer of alkaline phosphatase, interactions presumably mediated by conformational changes induced (or selected for) by the interaction of substrate or product with the enzyme and propagated across the monomer-monomer interface.

Several recent reports have suggested that 2 eq of phosphate bind to the enzyme and that binding is characterized by a single dissociation constant (3, 20, 21). While there is consid- erable variation in concentration in the several studies, the suggestion of phosphate contamination as a comprehensive explanation for apparent negative cooperativity (20) cannot apply to experiments in which the apoenzyme is an intermediate since the apoenzyme has negligible affinity for phosphate (6).

Phosphorus chemical shifts of the various forms of enzyme- bound phosphate span -6 ppm, hence :“P NMR is a particu- larly good method to evaluate the subtle differences in the interactions of phosphate with this enzyme. An equimolar mixture of apoalkaline phosphatase and inorganic phosphate at pH 8 shows a single sharp ‘“P resonance at the chemical shift position of P, in the absence of protein at the pH and ionic strength used (Fig. lA), confirming that there is negligible specific interaction between the apoenzyme and inorganic phosphate. Addition of 1 eq of Zn’* to the mixture of apoenzyme and inorganic phosphate results in the appearance of at least two additional broadened lines at lower field (Fig. 1B). Under these conditions the observed line broadening

A D ApcAP+leqP, APO AP + 1 cq P,

+ 4 eq znm

FIG. 1. :“P NMR spectra of phosphate binding to ZnY+ alkaline phosphatase. Variation of phosphate, Zn’+, and Mg’+ stoichiometry. Conditions: 0.01 M Tris, 0.01 M NaOAc, 0.1 M NaCl, pH 8.0. A, 1.37 x 10mx M apoalkaline phosphatase (APO AP), 1.45 x 10m9 M K,HPO,;

B, 1.33 x lo-” M apoalkaline phcsphatase, 1.41 x lo-” M K,HPO,, 1.42 x lo-:’ M Zn’+; C, 1.29 x lo-” M apoalkaline phosphatase, 1.37 x 10 :I M K,HPO,, 2.77 x lo-” M Zn”; D, 1.22 x 10m9 M apoalkaline phosphatase, 1.29 x 10m3 M K,HPO,, 5.26 x lo-:’ M Zn”+; E, 1.16 x lo-” M apoalkaline phosphatase, 2.78 x 10m3 M K,HPOI, 5.00 x lo-:’ M Zn’+; F, 1.13 x lo-” M apoalkaline phosphatase, 2.71 x 10~” M K,HPO,, 4.87 x lo-” M Zn’+, 1.21 x lo-” M Mg’+. Metal ion was added as Me’+ Cl,.


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“:‘Cd NMR and :“P NMR Studies of Alkaline Phosphatase

requires that enzyme species containing one Zn” ion have at least a transitory existence. Modulation of bound P, resonances from these species may result from direct transfer of P, between proteins of variable metal ion content or may be governed by the rate of metal ion exchange between protein forms. These alternatives, both plausible, cannot be distin- guished from the present results. The line width and chemical shift of the peak for the remaining free P, indicate that it is not an intermediate in this exchange process.

Addition of a second equivalent of Zn’+ converts most of the ‘“P intensity into a narrowed line at the resonance position expected forE .P (1) (Fig. 1C). The resonance of low amplitude at higher field reflects a slight excess of P, beyond a stoichiometry of one P,/reconstituted dimer. A content of two Zn”+ ionsidimer has been shown to be the minimum metal content required for activity (6, 10, 22). The above sequence of resonance shifts is consistent with the postulate that 2 mol of Zn’+/mol of dimer are required to induce the tight binding of 1 mol of P, in the noncovalent complex, E .P (6).

The line width and relaxation times of resonances assigned to phosphate which is not tightly bound to the enzyme (assignment based on the observed chemical shift) can be satisfactorily explained on the basis of exchange mechanisms operative between the forms of P, present in a given sample (see below). However, at the concentrations of P, employed in these studies (2 1 mM) binding of P, at protein sites of greatly reduced affinity is suggested by radiolabelling studies (6, 17) and cannot be definitively ruled out on the basis of the NMR data.

An additional 2 g at of ZnY+ and 1 to 2 g at of Mg’+ have been shown to stabilize the structure of alkaline phosphatase (23-25). Therefore, the effect of additional Zn”+, Pi, and MgS+ on the enzyme. phosphate noncovalent complex was exam- ined. Addition of a further 2 eq of Zn”+ has no effect on the distribution of free and bound phosphate (Fig. 1D). There is a 1-ppm upfield shift of the resonance of E. P induced by the extra zinc. This would appear to reflect a modulation of enzyme structure by the binding of the extra zinc to sites on the molecule which stabilize the structure. These may be related to the partially occupied Zn”+ sites observed in the electron density map of the enzyme at 3 A resolution (26). When a second equivalent of phosphate is added to the enzyme containing four Zn”+ ions, additional intensity appears at the chemical shift position of free Pi (Fig. 1E).

Addition of Mg”+ to the system does not alter the relative stoichiometry of the bound and free phosphate (Fig. 1F). Mg’+ does induce a substantial upfield shift in the resonance of E .P which moves from 4.2 to 3.5 ppm. This shift would appear to reflect further changes in enzyme structure induced by enzyme-bound Mg”+ , an ion which stabilizes the protein against heat denaturation (23, 25) and also activates the enzyme (25).

Exchange of P, Bound at Active Site of Zn’+ Enzyme -For the series of spectra shown in Fig. 1 the rate of chemical exchange between E .P and free P, is clearly slower than the chemical shift difference (-800 s-l). In this slow exchange limit it is possible to estimate the life-times of the different phosphate species and thus the dissociation rate constant, km,. As determined from the line width of the E P resonance in Fig. lC, Iz-, is 60 + 20 s-l. This value is consistent with the value of h,.,, determined from stopped flow reaction kinetics (27) and with the value of h-, (10 to 20 s-l) reported by Hull et al. (3) using ‘“P NMR. The precision of such determinations

of rate constants is not sufficient, however, to determine to what extent dissociation of E. P contributes to the steady state rate. It is likely that the rates of phosphorylation of the enzyme, dephosphorylation of E-P, and dissociation of E ‘P are all of similar magnitude at alkaline pH.

Values of the spin-lattice relaxation time, T,, were determined for the resonances shown in Fig. 1F using the progressive saturation method. Both the resonances for E .P and free P, have equivalent T, values of 1.8 + 0.2 s. Thus in terms of relaxation rates the fast exchange condition T,,IT, oLIy < 1 applies to the rate of chemical exchange. This condition permits use of the peak areas directly as a measure of the relative concentration of the individual phosphate species (Fig. 1). The line widths and chemical shift differences of the lowfield resonances of Fig. 1B suggest that the proposed exchange of P, between enzyme species occurs at a rate somewhat greater than the chemical exchange of P, between the bound (E P) and free forms.

The results obtained with 2 two metal ionsidimer present (Fig. 1, C to F) suggest that observations of multiple forms of

E .P complexes (3) can be related to variable metal ion content of the protein. Exchange of P, between these chemically distinct enzyme species would also lead to the observed broadening of bound resonances (3). The metal ion complement of “native” alkaline phosphatase may deviate substantially from the maximum metal ion composition (four or more Zns+ ions and two Mg’+ ions) owing to the potential for removal of metal ion from the low affinity binding sites under standard methods of preparation (25). Thus “native” enzyme is often a combination of species of varying metal ion composition which in turn mediates significant structural changes which are reflected in the chemical shift of bound phosphate (Fig. 1). Thus reports of multiple species of bound phosphate observed with native enzyme which are converted to single bound forms (by the NMR chemical shift criterion) on exhaustive dialysis (3) can be rationally explained. Variable metal ion content is not a factor governing the stoichiometry of phosphate binding. Under the conditions of moderate ionic strength and low Tris concentration, one phosphate ligand is tightly bound/protein dimer. This binding requires the presence of at least two Zn’+ ions and the stoichiometry is unaffected by further metal ion (ZnS+, Mg’+ additions) (Fig. 1).

Stoichiometry of Cd’+ Binding and Formation of Phos- phoryl Enzyme-Native Zn”+ alkaline phosphatase does not form significant equilibrium concentrations of the covalent phosphoryl enzyme (E-P) at pH greater than 6.5. As the pH is lowered, the stoichiometry of phosphoryl enzyme formation increases, reaching a value of 0.5 mol of E-P/m01 of dimer at pH 5.5. A pH of 5 is, however, at the low end of the pH stability function for the active dimer which begins to lose the metal ion below this pH (28, 29). Thus equilibrium studies of the phosphoryl enzyme formed with the native Zn’+ enzyme are difficult. It has, however, previously been shown by :L’P NMR that E-P formed by the native enzyme at low pH is characterized by a ‘“P resonance with a chemical shift over 8 ppm downfield from that for phosphoric acid (l-3). This resonance is unusually far downfield for a phosphate monoester and suggests a significant distortion in the conformational state of this phosphoserine from that of unconstrained phosphate monoesters (1, 4, 5). The difficulty of studying the E-P complex of the enzyme can largely be overcome by using Ccl’+ alkaline phosphatase (1). Substitution of Cd’ for Zn’+ at the active site slows both the phosphorylation rate and dephospho-


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l13Cd NMR and 31P NMR Studies of Alkaline Phosphatase

rylation rate of the enzyme by -2 orders of magnitude (6, 30). Dephosphorylation of the Cd” enzyme is slow relative to phosphorylation so that E-P is rapidly formed. At neutral pH E-P is the only significant species present at equilibrium.

The stability of E-P at pH 6.5 with Cd’+ present at the active sites allows a clear demonstration of the stoichiometry of formation ofE-P (Fig. 2). Similar to the spectrum shown in Fig. lA, the 3’P resonance of 2 eq of inorganic phosphate in the presence of 1 eq of apoenzyme occurs at the chemical shift of free inorganic phosphate at this pH and shows no evidence of specific interaction with the protein (Fig. 2A). On the addition of 1 eq of Cd’+ to this mixture a new resonance appears at 8.07 ppm, assigned to the phosphoserine group in the Cd’+ enzyme (Fig. 2B) (1). There is a reciprocal loss of resonance from the peak for inorganic phosphate. Addition of a second equivalent of Cd’+ to the enzyme increases the ampiitude of the resonance which is now equivalent to the

A I /

Apo AP +

2 Sq Pi. pH 6.5

Apo AP + 2 eq Pi

FIG. 2. 3LP NMR spectra of phosphate binding to Cd>+ alkaline phosphatase. Variation of metal ion stoichiometry and phosphate concentration. Conditions: 0.01 M Tris, 0.01 M NaOAc, 0.1 M NaCl, pH 6.5. A, 1.72 x 10m3 M apoalkaline phosphatase (Ape Al’), 3.29 x 10m3 M K,HPO,; B, 1.69 x lo-” M apoalkaline phosphatase, 3.23 x 10m3 M K,HPO,, 1.63 x 1V3 M Cd’+; C, 1.67 x 10m3 M apoalkaline phosphatase, 3.21 x lo-:’ M K,HPO,, 3.21 x 10m3 M Cd”. Cd” was added as CdL+Cl,.

peak remaining at the resonance position for inorganic phosphate. Thus the cadmium enzyme forms a maximum of 1 eq of E -P/enzyme dimer and two Cd’+ ions/dimer are required to induce formation of 1 eq of E-P. Additional P, shows no evidence (see below) of interaction at the enzyme active center. Thus 31P NMR of the cadmium phosphoryl enzyme strongly suggests that, even though two identical Cd” binding sites, separated by 32 A, are present at the active centers, on phosphorylation of the serine at one active site the other cannot interact with phosphate. This stongly supports the hypothesis that negative homotropic interactions between the subunits of the initially symmetrical dimer occur on interaction with ligand phosphate.

The rate of chemical exchange between E. P on the zinc enzyme and free phosphate is estimated to be -60 s-‘. In contrast, exchange of E-P with free P, requires hydrolysis of E-P. For the Cd’+ enzyme at pH 6.5 the rate constant for the hydrolysis of E-P is <O.Ol s-l as shown by lRO exchange studies (6). The resonance for free phosphate in the presence of the phosphorylated Cd” enzyme (Fig. 2C) is broadened (45 Hz) relative to the corresponding resonance in the presence of the apoenzyme at pH 6.5 (Fig. 24). Because of the large difference in rate constants describing the formation of E .P andE-P, the line width of free P, reflects lifetime broadening, controlled by the slowest association step in the overall process. Free Pi in solution is in equilibrium with E-P via the E ‘P complex; the latter present in negligible steady state concentration for the Cd,‘+ enzyme at pH 6.5 (Fig. 2). Association of P, with the enzyme to form E .P, based on the results obtained with the Zn’+ enzyme, must be 10” to 10’ ss’. Assuming the equilibirum distribution of E. P/E-P to be 0.01, the phosphorylation rate, he, for the conversion ofE .P to E-P is -0.1 s1 and would result in a line width on the order of 50 Hz as is observed.

Stoichionetry of Phosphate Binding to Co” Alkaline Phos- phatase at pH 8 .O -A similar titration of a mixture of apoenzyme and inorganic phosphate can be performed with Co’-. In contrast to the Cd’+ enzyme which turns over very slowly, the activity of the Co’+ enzyme is approximately 30% that of the native enzyme and %lP labeling studies show that, like the Zn’+ enzyme, the major intermediate formed at alkaline pH is E .P (6, 27). Initial studies reported previously have shown that the phosphate in E .P is bound close enough to the Co” for the NMR line to be extensively broadened by the paramagnetic component of the relaxation (11. Thus the dis- appearance of the resonance for P, can be used to follow the binding of phosphate close to the active site metal ion.

The spectrum of a mixture of apoenzyme and 1 eq of P, at pH 8.0 is shown in Fig. 3A along with an external marker of methyl phosphonate. At pH 8.0 free phosphate in the presence of the apoenzyme has a significantly narrower line width than at pH 6.5 (compare Figs. 3A and 4A), perhaps reflecting less interaction of the phosphate dianion with the protein, thus resulting in less efficient relaxation as compared to the monoanion. The T, of the phosphate resonance is sufficiently long so that, at the pulse repetition rate employed, complete recovery does not occur. On addition of 1 eq of Co”+ at pH 8.0, there is a concommitant reduction in both T, and T, of free P, (Fig. 3B), sufficient to enhance the resonance amplitude compared to that of Fig. 3A. However, the resonance observed following the Co’+ addition can be shown to represent only half the phosphate present. The resonance of Fig. 3B is directly comparable to that observed on addition of 1 eq of P,


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‘13Cd NMR and 31P NMR Studies of Alkaline Phosphatase 7057

to the reconstituted Co’+ enzyme after the full complement of phosphate is bound (Fig. 301, and is half the amplitude of the latter.

On the addition of a second equivalent of COG+ to the enzyme, the phosphate resonance is now broadened to several hundred Hz (Fig. 3C). This resonance must represent the E .P complex of the Co’+ enzyme. Thus 2 mol of Co”+/mol of enzyme dimer are required for 1 mol of phosphate to be bound to the dimer close enough to the metal ion for the resonance to be broadened by paramagnetic relaxation. This requires that the oxygen of the phosphate be within the first or second coordination sphere of the metal ion (1).

When a second equivalent of P, is added to the enzyme containing two COG+ ions and one bound phosphate anion, the resonance of the second equivalent appears at the chemical shift of free inorganic phosphate (Fig. 30). The resonance is

ApoAP + tP( +lCo(II)

APO AP + 1 Pi+2 CO(U)

APO AP + 2Pi + 2Co(II)

: \ --WV---

FIG. 3. 3’P NMR spectra of phosphate binding to Co’+ alkaline phosphatase at pH 8.0. Conditions: 0.01 M Tris, 0.01 M NaOAc, 0.1 M NaCl, pH 8.0. All spectra were obtained with a constant concentration of methyl phosphonate as an external standard (6 = 29.4 ppm). A, 1.65 x 10m3 M apoalkaline phosphatase (Ape API, 1.60 x lo-” M K,PO,; B, 1.59 x 10m3 M apoalkaline phosphatase, 1.55 x 10m3 M K,HPO,, 1.55 X 10m3 M co'+; C, 1.54 x 10m3 M apoalkaline phosphatase, 1.50 x 10m3 M K,HPO,, 3.02 x 10m3 M Co’+; D, 1.49 x 10m3 M apoalkaline phosphatase, 2.90 x 1Om3 M K,HPO,, 2.91 x 10.” M Co’+. Co’+ added as Coz+Cl,.

broadened to the same extent as the resonance in the presence of one Co’+ ion (Fig. 3B1, but is twice the amplitude, indicating that these resonances represent 1.0 and 0.5 eq, respectively, of free phosphate in slow exchange with the phosphate of

E .P. If the exchange rate is ~100 SS’ (similar to that for the Zn’+ enzyme), then the observed broadening can be entirely accounted for by the exchange broadening. Thus no significant amount of the second equivalent of P, can be bound to a site within the first or second coordination sphere of the Co’+ ion. Hence, once 1 mol of E .P is formed at one Co’+ site on the enzyme, the second site cannot form the same complex. These data reflect the same phenomenon of negative cooperativity for E .P formation by the Co’+ enzyme as described above for the formation of E-P by the Cd’+ enzyme (Fig. 2).

C

D I ApoAP l ZP, l 2co~P)

I, I I I, I I 41 / I I I1 ! I so 40 50 20 IO 0 -10 -20

81 pm )

FIG. 4. R1P NMR spectra of phosphate binding to CoL+ alkaline phosphatase at pH 6.5. Conditions: 0.01 M Tris, 0.01 M NaOAc, 0.1 M NaCl, pH 6.5. All spectra were obtained with a constant concentration of methyl phosphonate as an external standard (6 = 29.4 ppm). A, 1.80 x lo-:’ M apoalkaline phosphatase (Ape API, 1.80 x

10m3 M K,HPO,; B, 1.76 x 10m3 M apoalkaline phosphatase, 1.77 x lo-:’ M K,HPO,, 1.73 x 10m3 M Co?+; C, 1.75 x 10m9 M alkaline phosphatase, 1.73 x 10mJ M K,HPO,, 3.40 x lo-” M Co’+; D, 1.70 x lo-” M alkaline phosphatase, 3.37 x lo-” M K,HPO,, 3.31 x 10mR M Co’+; E, 1.62 x 10m3 M apoalkaline phosphatase, 3.22 x 10m3 M K,HPO,, 3.16 x lo-” M Co’+, 0.06 x 10m3 M apoalkaline phosphatase added. Co’+ added as Co’+Cl,.


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““Cd NMR and :“P NMR Studies of Alkaline Phosphatase

Phosphate Binding to Co’+ Enzyme, pH 6.5 -The ‘“P spec-

tra observed when the corresponding titration is conducted at pH 6.5 are altered. Addition of 1 eq of Co’+ results in line broadening (AU ,,? - 120 Hz) greater than that observed under comparable conditions at pH 8.0 (compare Fig. 4B and 3B). This suggests that the free phosphate may now be in rapid exchange with E P. This conclusion is further supported by a doubling of the line width (-225 Hz) of the observed resonances when a second equivalent of Co’+ is added (Fig. 40. This requires that the dissociation rate of P, from the bound state be ~10:’ s ‘, increasing from the value of -10’ s ’ estimated for the system at pH 8.0. On the addition of a second equivalent of P,, the resonance (still at the chemical shift position of free P,):’ increases in intensity and the line width narrows to -120 Hz, that observed in Fig. 4B. The area normalized for the number of transients is approximately twice that of the resonance in Fig. 4B. This relationship is expected if the fast exchange condition pertains; the resonance in Fig. 4B representing a total of 1 eq of phosphate, 0.52 eq in excess of reconstituted enzyme; the resonance in Fig. 40 representing a total of 2 eq of phosphate, 1.02 eq in excess of reconsituted enzyme. These calculations assume a dissociation constant for E.P of the Co” enzyme of 5 x 10 ” M, a magnitude supported by I:“PJHPO,‘~ binding studies at pH 6.5 (1 x 10 I M enzyme) which show -1 eq of E.P (6). Addition of excess apoenzyme to the final mixture (Fig. 4E) does not alter the line width, suggesting that significant free Co”’ does not contribute to the line broadening.

“:‘Cd NMR of Metal Ions in Cd” Alkaline Phosphatase and Phosphorylated Derivative- All data on alkaline phosphatase, including mapping of the tryptic peptides (32), sequence studies (33), and x-ray diffraction (26, 34), suggest that the subunits of the unliganded dimer are identical. Thus the interpretation of the ‘“P NMR spectra given above requires that while the active sites are initally identical, reaction with P, at one site destroys the 2-fold symmetry and the second site can no longer interact with ligand (phosphate). The alteration of the nonliganded site is presumably brought about by conformational changes induced by the ligand and propagated across the monomer-monomer interface. Such changes might take place primarily at the monomer-monomer interface if the active sites themselves are located along the domain of subunit contact.

Demonstration of the existence of negative homotropic interactions between the sites using phosphate or substrate as the reporter group is not completely satisfactory since such methods reflect alterations only at the liganded site. The requisite structural changes at the unliganded site can only be inferred from the observation of half the sites reactivity. A more satisfactory technique would be one in which the environments of both active centers were simultaneously observed during ligand interaction at a single site.

“Wd’+ FT (Fourier Transform) NMR is a technique uniquely satisfying these criteria for alkaline phosphatase. “Wd has a nuclear spin of r/z and a chemical shift extremely sensitive to the chemical environment of the Cd’+ ion. Chem- ical shift values for common CcP+ compounds span over 600

” The absence of a chemical shift arising from the Fermi contact contribution from the Co” ran is surprising but could be due to equal and opposite contributions from the contact and pseudocontact terms (31). Alternatively the direct through-bond contribution could be entirely absent, implying that P, is not directly liganded to the Co” ion.

ppm (13, 35). The sensitivity of this technique has recently been sufficiently improved to permit observation of ““Cd’+ bound to biological macromolecules (14).

The “:‘Cd” spectrum of phosphate-free alkaline phosphatase dimer shows a single cadmium resonance at 170 ppm (Fig. 5A). Thus the two Cd” ions, bound at the separate binding sites of the subunits, are in identical environments in the unliganded protein dimer. Therefore, the dimer must have Z-fold symmetry with respect to the immediate environment of the metal ions.

On the addition of 1 eq of phosphate to the protein, the cadmium resonance splits into two resonances, one at 142 ppm and one at 55 ppm. each with an amplitude accounting for half the cadmium initially present, The :“P NMR spectrum of this sample shows a single resonance at 8.07 ppm corresponding to the Cd”+ phosphoryl enzyme (Fig. 2). Addition of 1 eq more of P, does not alter the ‘Wd” resonances and the ‘“P intensity of the additional phosphate appears at 2 ppm, corresponding to free phosphate (Fig. 2C).

Thus, phosphorylation of one active site not only influences the chemical environment of both metal ions, but renders the chemical environment of one different from the other as indicated by the 87-ppm difference in the chemical shift for the two “Ykl”+ resonances of the phosphorylated enzyme. Neither environment is the same as that observed in the unliganded enzyme. Thus phosphorylation destroys the 2-fold symmetry of the metal ion sites, a necessary condition of negative cooperativity for this enzyme.

Binding of Cd”+ to Apophosphoryl Enzyme-The ‘“P and ““Cd NMR data above indicate that if one of the active sites on the phosphatase dimer is phosphorylated, the immediate environments of the two metal binding sites are no longer identical (Fig. 5). However, it is not possible to determine from these data if the differences in the sites require the presence of metal ions or are a property of the potential metal binding sites of the phosphorylated apoenzyme. Preparation of the apophosphoryl enzyme (30) permits experimental resolution of this question by monitoring the ‘“P NMR spectrum of E-P as the binding sites are occupied on titration with metal ion. Regeneration of the Cd+ phosphoryl enzyme using this method is not consistent with a population of metal ion binding sites in a statistical manner (Fig. 6). Although a single resonance, representing a homogeneous species corresponding to the Cd” phosphoryl enzyme is ultimately observed (Fig. 6E), the intermediate stages of the titration (Fig. 6, B to D) are not consistent with the progressive formation of the final product. Addition of 5 1 eq of Cd’+ does not alter the chemical shift of the phosphoseryl resonance (Fig. 6B), suggesting that the metal either binds preferentially at the nonphosphorylated site or is not able to induce the structural changes that result in the 2-ppm downfield chemical shift characteristic of the Cd”+ phosphoryl enzyme. Since the presence of two metal ions in one phosphorylated dimer does produce the downfield shift, prior phosphorylation must favor the binding of one metal ion/dimer when the full complement of metal ion is not available. Incremental addition of the second Ccl” ion/dimer results in gradual changes in the spectrum with the appearance of a :“P resonance at the chemical shift position of the Cd’+ phosphoryl enzyme (Fig. 6, C to E). At stoichiometries between one and two Cd” ions/ dimer additional resonances are observed suggesting that binding of the second metal ion to the dimer induces successive conformational changes in the molecule. These conformational


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““Cd NMR and :“P NMR Stu

isomers are sufficiently stable to be detected as discrete resonances in the ‘“P NMR spectrum, further evidence that control of metal ion stoichiometry is critical to the appearance and interpretation of the “‘P resonances of phosphate complexes of alkaline phosphatase. Once all molecules contain 2 eq of Cd’, only a single resonance at the chemical shift of the phosphorylated Cd”+ enzyme is observed (Fig. 6E). Thus phosphate (or the phosphoseryl moiety) and the metal ion appear to be reciprocally involved in the induction of asymmetric interactions of the subunits of the alkaline phosphatase dimer

Phosphate Binding to Alkaline Phosphatasr Containing Mixture of Co” and Cd” -Another indication that the presence of phosphate can induce nonequivalent interactions between the two metal ion sites is provided by following the ‘“P resonance of phosphate during the sequential addition of 1 eq of Co”+ and 1 eq of Cd”+ to a mixture of apoenzyme plus 1 eq

of inorganic phosphate (Fig. 7). The first two stages of this experiment (Fig. 7, A and B) parallel and confirm the results of the experiment depicted in Fig. 4. On addition of 1 eq of Cd”+ to the sample containing apoenzyme, phosphate and 1 eq of Co”. surprisingly little phosphoryl enzyme is formed (Fig. 7C). In the absence of Co”, addition of 1 eq of Cd’+ would be expected to result in formation of 0.5 eq of phosphoryl enzyme (Fig. 2B), generating a low field resonance in the X’P NMR spectrum of appropriate intensity. Instead, the phosphate resonance is broadened and unshifted. Thus most of the phosphate in the sample must be bound at a Co’+ site in a

dies of Alkaline Phosphatase 7059

condition of fast exchange with free phosphate (compare Figs. 4C and 7C). AS a corollary it may also be concluded that in the presence of phosphate the enzyme must be driven to form a Co’+-Cd’+ hybrid which prefers to bind phosphate at the Co” site. Other possible distributions of metalloalkaline phosphatases which could form in this sample would result in the presence of from 0.25 to 0.50 eq of Cd,” alkaline phosphatase. Generation of Cd,‘+ phosphoryl enzyme with the appearance of a ‘“P resonance of corresponding intensity at 8 ppm would be the necessary consequence which is not, however, observed. It is difficult, however, in the absence of exact knowledge of the rate constants governing P, exchange at Co”’ and Cd’+ sites on the enzyme to rigorously exclude alternative expla- nations. Preferential association of phosphate at the Co” site is plausible, since the conformational relationships at the Co’+ site promote rapid enzyme turnover while those at the Cd”+ site do not (6, 27). The changes at the CW+ site induced by the asymmetric binding of phosphate to the Co’)+ site can also be monitored by ““Cd NMR and are the object of current investigations.

CONCLUSIONS

The ““Cd NMR and :“P NMR results described above demonstrate the existence of negative homotropic interactions in the ligand (phosphate) binding of alkaline phosphatase. The chemical environment of the metal ions bound at the two active centers of the enzyme dimer are identical in the absence of phosphate (Fig. 5A). Interaction of phosphate at one site

A I “SCd(4Alholine Phosphotose,

pH65

FIG. 5. “:‘CdL+ NMR spectra of Cd,” alkaline phosphatase and CdZY+ phosphoryl alkaline phosphatase. Condi- tions: 0.01 M Tris. 0.01 M NaOAc. 0.1 M NaCl, pH 6.5. A, 3.65 x 10m3 M Cd2+ alkaline phasphatase lCd’+l/lenzymel = 2.01; B, 4.05 x lo-’ M Cd’+ alkaline

phosphatase, 4.05 x lo-:’ M K,HPO, 8 [Cd’+l/lenzymel = 2.00.

‘%d(ID, Alkolinr Phorphotosr + Pi. ptt 6.5

i

I , , .I, , , .I , ., , I ,* ,.,,,,,I i -250 200 Is0 loo SO 0

8 (ppm)


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7060 “Td NMR and ‘“P NMR Studies of Alkaline Phosphatase

f

pi- [

0 i i

0.7

0 \

1.9

E

I 2.6

L I I, I I Ii 1 I I 16 12 6 4 0

8. wm

FIG. 6. :“P NMR spectra of the phosphoryl enzyme on Cd”+ titration of apophosphoryl alkaline phosphatase (Ape APT). Con- ditions: 0.01 M Tris, 0.01 M NaOAc, 0.1 M NaCl, pH 6.5. A, 1.20 x lo-:’ M apophosphoryl alkaline phosphatase; B, 1.19 x lo-:’ M apophosphoryl alkaline phosphatase, 7.63 x 10m4 M Cd”; C, 1.18 x 10m3 M apophosphoryl alkaline phosphatase, 1.52 x lo-” M Cd’+; D, 1.17 x 10m3 M apophosphoryl alkaline phosphatase, 2.25 x lo-” M Cd’+; E, 1.16 x lo-” M apophosphoryl alkaline phosphatase, 2.98 x 10.” M Cd2+. Data aquisition was begun immediately after metal ion additions for a -10-h sampling time. CcP+ was added as CdCl,‘+.

differentially alters the environment of both metal ions and therefore the conformational structure of the active centers (Fig. 5B). Covalent (E-P) or noncovalent (E .P) complex formation with phosphate at one site induces structural changes which prevent tight specific binding of ligand at the second site (Figs. 1 to 4). Thus even at relatively high (> 1 mM) ligand concentrations the enzyme displays absolute negative cooperativity. Communication between the subunits giving rise to this phenomenon is apparently mediated by conformational changes propagated across the monomer-monomer interface.

Whether negative cooperativity is a phenomenon incidental to some rearrangement of the enzyme subunits required for phosphorylation or dephosphorylation or serves a functional purpose is unclear at present. If the in uivo function of the

Ape AP + 1 Pi

ApoAP + 1 P, + I Co(lI)

AwAP+IP, +lCo(lI)+lCd(II)

-.

11 1 I I I , I, I , 1, I , , , -30 40 30 20 IO 0 -10 -20

6 (ppml

FIG. 7. :“P NMR spectra of phosphate binding on sequential additions of Co’+ and Cd” to apoalkaline phosphatase (Ape Al’). Conditions: 0.01 M Tris, 0.01 M NaOAc, 0.1 M NaCl, pH 6.5. A, 1.80 x 10mR M apoalkaline phosphatase, 1.80 x lo-” M K,HPO,; B, 1.76 x lo-” M apoalkaline phosphatase, 1.77 x lo+ M K,HPO,, 1.73 x 10 :’ M Co’+; C, 1.70 x lo-’ M apoalkaline phosphatase, 1.71 x 10 ” M K,HPO,, 1.67 x 10 :I M Co’+, 1.74 x lo-” M Cd’+. Metal ions added as Me’+Cl,.

enzyme is a selective phosphate transfer rather than nonspe- cific monoesterase activity, conformational change and negative cooperativity might exert some spatially selective function. There is the possibility that negative cooperativity could aid in the ejection of tightly bound phosphate from the opposite site on interaction of substrate at the nonliganded site. To be effective such a mechanism would require that there be significant affGty of the non-phosphate-containing site for substrate prior to dissociation of the tightly bound phosphate. The NMR data for phosphate binding do not indicate significant affinity for the second site (Figs. 1 to 3). Initial binding of phosphate monoester, however, might be different and this mechanism therefore remains a possibility. Conformational changes associated with the phenomenon of negative cooperativity may affect solvent access to the metal ion site or the phosphoserine. Such changes can be detected by determination of alterations in the spin-lattice relaxation times and nuclear Overhauser enhancements of ’ Wd and :“P in H,O and D,O and will be the subject of further publication.

REFERENCES

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3. Hull, W. E., Halford, S. E., Gutfreund, H., and Sykes, B. D. (1976) Biochemistry 15, 1547-1561


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‘13Cd NMR and 3’P NMR Studies of Alkaline Phosphatase

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J F Chlebowski, I M Armitage and J E Colemanalkaline phosphatase as determined by 31P NMR and 113Cd NMR.

Allosteric interactions between metal ion and phosphate at the active sites of

1977, 252:7053-7061.J. Biol. Chem.

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