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Effect of Substitution Inert Metal Complexes on Calcineurin
Archives of Biochemistry and Biophysics Vol. 366, No. 1, June 1, pp. 168 –176, 1999 Article ID abbi.1999.1195, available online at http://www.idealibrary.com on
Effect of Substitution Inert Metal Complexes on Calcineurin Bruce L. Martin 1 and David J. Rhode Department of Biochemistry, University of Tennessee, 858 Madison Avenue, Memphis, Tennessee 38163
Received March 30, 1998, and in revised form March 9, 1999
As a substitute for M(H 2O) 621, Co(NH 3) 631 was found to activate calcineurin with para-nitrophenyl phosphate as substrate. Kinetics for calcineurin catalyzed hydrolysis of para-nitrophenyl phosphate at pH 7.0 with Mn 21, Mg 21, Co 21, and Co(NH 3) 631 were compared. Although k cat and K m were different with the metals, values of k cat/K m were nearly identical for Mn 21 and Mg 21, but lower for Co 21 and Co(NH 3) 631. The concentration of each metal providing half-maximal activation, designated K act, was evaluated as 15.9 mM for Co(NH 3) 631, compared to K act 5 0.17 mM for Mn 21 and Co 21 and 6.3 mM for Mg 21, respectively. Comparing k cat/K cat showed that Co(NH 3) 631 was a 170-fold poorer activator of calcineurin than was Mn 21, but only 1.5fold poorer than Mg 21. Activation by Co(NH 3) 631 indicated that activation of calcineurin by exogenous metal ions can proceed via an outer coordination sphere reaction mechanism with no requirement for the direct coordination of substrate by metal. Because Co(NH 3) 631 was found to support calcineurin activity, the related compound [Co-(ethylenediamine) 3] 31 (or Co(en) 331) was tested as a possible activator. Co(en) 331 did not support calcineurin activity but did inhibit calcineurin. Co(en) 331 showed competitive inhibition kinetics with either Mn 21 or pNPP as the varied ligand and the other at a fixed, subsaturating concentration. Inorganic phosphate was used as a known competitive inhibitor to pNPP (B. L. Martin and D. J. Graves, J. Biol. Chem. 261, 14545–14550, 1986) and showed uncompetitive inhibition with Mn 21 as the varied ligand. These patterns are consistent with the mechanism of ligand binding to calcineurin being ordered with metal preceding substrate. Prior formation of a metal– substrate complex was not required for association with calcineurin. © 1999 Academic Press 1 To whom correspondence should be addressed at Department of Biochemistry, University of Tennessee, 858 Madison Ave., Suite G01, Memphis, TN 38163. Fax: 901-448-7360. E-mail: bmartin@ utmem1.utmem.edu.
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Key Words: calcineurin; protein phosphatase; metalactivated enzyme.
Protein serine and threonine protein phosphatases are involved in the phosphorylation and dephosphorylation cycles critical for the regulation of a variety of enzymes. There are four classes of cytosolic protein serine phosphatases, designated types-1, -2A, -2B, and -2C (1) with types-1, -2A, and -2B belonging to the same gene family (2). Intrinsic metals have been identified in the resolved crystal structures of types-1 and -2B (3– 6) and all classes of the type 1/2 gene family have enzyme forms that are activated by metal ions (7–10). Although distinct from the protein phosphatases, some alkaline phosphatases (11, 12) and the purple acid phosphatases (13, 14) also are metalloenzymes and likely share some characteristics involving the participation of metal ions in catalyzing the hydrolysis of phosphate ester bonds. The precise role attributable to metal is not understood. It is likely that metals serve one of the following functions resulting in enhanced phosphate ester hydrolysis: (i) activation of the nucleophilicity of coordinated water for attack on the phosphate ester, (ii) coordination and activation of the phosphate ester substrate, and (iii) stabilization of the developing leaving group. Various model studies have been done to elucidate features about the roles of metals in phosphoryl transfer reactions. From a study of the hydrolysis of paranitrophenyl phosphate (pNPP) 2 by Mg 21 or Ca 21, Herschlag and Jencks (15) suggested that stabilization of the leaving group did occur. Other studies (16 –18) have shown that chelation of metal can result in accelAbbreviations used: EGTA, ethylene glycol bis(b-amino ethyl ether) N,N9-tetraacetic acid; Mops, 3-(N-morpholino)propanesulfonic acid; pNP, para-nitrophenol; pNPP, para-nitrophenyl phosphate. 2
0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
SUBSTITUTION INERT COMPLEXES AND CALCINEURIN
erations even higher than observed with Mg 21 or Ca 21 (15). Protein phosphatase-2B, designated calcineurin, is a calmodulin-activated phosphatase that is isolated with intrinsically bound iron and zinc (6, 19) but requires exogenous metal for maximal activity (9, 19 –23). The roles of intrinsic metals are not clear. The oxidation state of the intrinsic iron has been reported to be crucial for optimal activity (24, 25), although there is disagreement whether active enzyme contains Fe 21 or Fe 31. The work of Klee and colleagues (25) suggested that Fe was present as Fe 21, but these findings were made with only a fraction of the total enzyme pool that shows instability in the presence of calmodulin and Ca 21. The Rusnak laboratory reported (24, 26, 27) that calcineurin contained Fe 31 and was active with a value of k cat comparable to other published values. Earlier studies on brain calcineurin by King (28) reported that all cysteines of the enzyme were in the reduced state with incubation with mercaptoethanol causing no effect on Mn 21 supported activity. These latter findings are suggestive that the reducing agent is not required in Mn 21-supported activation. The enzyme used in this report was not activated by incubation with ascorbate, but did show a 10% loss of activity incubated in the absence of dithiothreitol for 20 min. 3 There was no loss of activity during incubation for shorter times such as the length of activity assay. There is currently insufficient information to make final conclusions about the relationship between the oxidation state of intrinsic Fe and the activity of calcineurin. Calcineurin effectively has no catalytic activity (,1%) in the absence of exogenous metal. Mn 21, Ni 21, and Mg 21 are typically used for activating calcineurin. A curious property of the enzyme is the loss of sensitivity to Ca 21 during affinity chromatography on immobilized calmodulin as originally reported by the Klee and Cohen laboratories (29, 30). In these reports, equivalent activation was achieved with Mn 21 or Ca 21 until the affinity chromatography step. Recovered enzyme was then only activated by Mn 21. Subsequent studies by many different laboratories (9, 21–23, 31– 35) have consistently demonstrated the activation of calcineurin by Mn 21 in the absence of Ca 21. Cheung and associates demonstrated that Mn 21 activation was targeted at the catalytic subunit (35, 36). From mechanistic studies (37– 40), divalent metal has been proposed to have a direct role in the catalytic mechanism of the enzyme, not simply causing the protein to adopt an active conformation. Substituting Mg 21 for Mn 21 caused a shift in the pH optimum for the hydrolysis of pNPP. The shift was approximately 1 pH unit, comparable to the difference in pK a for water bound to Mn 21 3
D. J. Rhode and B. L. Martin, unpublished observations.
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(pK a 5 10.6) and Mg 21 (pK a 5 11.4). Li and associates likewise concluded that the pH optimum was dependent on the metal used (32). Collectively, these data suggest a role for exogenous metal in catalysis. From analysis of the resolved crystal structure of protein phosphatase-1, incorporation of exogenous metal into enzyme has been suggested as one possible mechanism for activation. Chu et al. have shown the stable incorporation of Co 21, but with limited recovery of activity (8). Greater activity was recovered with Mn 21, but the incorporation of this metal was not stable. Available data with calcineurin is not consistent with this mechanism. Using atomic absorption spectroscopy, it was found (41) that incubation of calcineurin with Mn 21 did not result in loss of intrinsic metal. This was a critical result: Exogenous metal did not physically replace intrinsic metal. Calcineurin has activity with low-molecular-weight phosphate esters (37, 42, 43), making it a useful model to examine features of phosphatase catalysis (38 – 40, 44). In this study, pNPP was used as the substrate. Using a simple phosphate ester enables the basis of metal activation of phosphatase activity to be conveniently investigated without the complication of nonspecific binding of metals by protein substrates. Using the inert metal complex Co(NH 3) 631 as a substitute for Mg(H 2O) 621, the activation by this complex was examined. Co(NH 3) 631 is regarded to function as an outer sphere complex and has been shown to substitute for Mg(H 2O) 621 in some enzyme systems, such as ribonuclease H (45) and DNA topoisomerase I (46). Inasmuch as exogenous metal does not replace intrinsic metal, the inert metal is not likely bound by the ligands of intrinsic iron and zinc. EXPERIMENTAL PROCEDURES Materials. Buffers, EGTA, and phenyl-Sepharose were purchased from Sigma Chemical. The substrate, pNPP (Sigma 104 substrate), was also purchased from Sigma. DE-52 cellulose was obtained from Whatman. Co(NH 3) 6Cl 3 was purchased from Strem Chemicals (Newburyport, MA) as 99.999% pure (Cat. No. 93-2708). Tris(ethylenediamine) cobalt (III) chloride was purchased from Alfa Aesar. Other chemicals (metal salts, etc.) were obtained from Fisher. All solutions, including those for the preparation and assay of calcineurin, were prepared from water that had been passed over a Chelex-100 column. Proteins. Bovine brain calcineurin was isolated from bovine brain to apparent homogeneity on SDS–polyacrylamide gel electrophoresis by the method of Sharma et al. (47) with Mops buffers instead of Tris buffers. Calmodulin was purified by the procedure of Sharma and Wang (48) slightly modified to include a final chromatography step on phenyl-Sepharose with elution by EGTA (49). Protein concentrations were determined by the method of Bradford (50). Calcineurin assay. Calcineurin was assayed on a Cary model 1E spectrophotometer with pNPP as the substrate by measuring the release of pNP at 410 nm at 30°C. Standard reaction conditions were 25 mM Mops, pH 7.0; 1.0 mM MnCl 2; and 10 mM pNPP in 800 ml reaction volume. Reaction times ranged from 10 to 30 min with changes at 410 nm of 0.05 to 0.35 absorbance units. Calcineurin was
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at concentrations indicated in the figure legends with calmodulin included at a fivefold molar excess of calcineurin. The model substrate pNPP was used to minimize the number of potential binding sites for the metals studied. The amino acid residues of polypeptide substrates may have different preferences for associating with metals, particularly as metals have different preferences for oxygen-, nitrogen-, or sulfur-containing ligands. The stability constants for Mn 21 (log K 1 5 1.9), Mg 21 (log K 1 5 1.3), and Co 21 (log K 1 5 1.6) with pNPP are within the same order of magnitude. Ca 21 was omitted to simplify the analysis of metal activation. Numerous studies have demonstrated that the presence of Ca 21 is not required to achieve activation by other metals whether assayed as the purified enzyme (6, 21–23, 31–33) or assayed in tissue extracts (29, 30, 34). Initial activation by Co(NH 3) 631 was determined using 10 mM pNPP as in the standard assay conditions. Likewise, Co(en) 331 was tested as an activator and as an inhibitor using 10 mM pNPP. Kinetic measurements. The parameters k cat, K m, and K act were determined by varying pNPP or varying metal as appropriate with other conditions already described. Substrate (pNPP) concentrations used were 50.0, 16.0, 10.0, 7.0, and 5.0 mM. For the different metals, the following concentration ranges were used: 0.50, 0.16, 0.10, 0.070, and 0.050 mM for MnCl 2; 1.00, 0.33, 0.20, 0.14, and 0.11 mM for CoCl 2; and 50.0, 16.0, 10.0, 7.0, and 5.0 mM for MgCl 2 and Co(NH 3) 631. In experiments with varied metal, measured activity was corrected for activity in the absence of any added exogenous metal. To monitor kinetic inhibition of calcineurin, the concentrations of pNPP and various metals were varied at fixed, different concentrations of inhibitors. The cited ranges of substrate and MnCl 2 concentrations were used. With Co(en) 331 as the inhibitor, concentrations of 0.0, 15.0, and 35.0 mM were used with Mn 21 as the varied ligand and 0.0, 15.0, 30.0, and 50.0 mM when pNPP was varied. Inhibition by inorganic phosphate with Mn 21 as the varied ligand was studied with phosphate concentrations of 0.0, 1.0, and 2.0 mM. The kinetic parameters, k cat, K m, K act, and K I, were evaluated. Data sets were plotted in double-reciprocal form for identifying the mode of inhibition. The type of inhibition was verified by fitting the data to the Michaelis–Menten equations for each mode of inhibition (Eqs. [1] to [3]).
v5
v5
k cat p @pNPP# K m~pNPP! p ~1 1 @I#/K I)1[pNPP#
competitive inhibition
[1]
k cat p @pNPP# K m~pNPP! p ~1 1 @I#/K I! 1 @pNPP# p ~1 1 @I#/K I! noncompetitive inhibition
v5
k cat p @pNPP# K m~pNPP! 1 @pNPP# p ~1 1 @I#/K I!
[2]
uncompetitive inhibition [3]
Data fitting and numerical estimates were done using EnzFitter (Elsevier BioSoft), Enzyme Kinetics (Trinity Software), or Psi-Plot (PsiPlot, Inc.) as appropriate. Double-reciprocal plots were prepared using Deltagraph (DeltaPoint, Inc.).
RESULTS
Calcineurin is typically activated by transition metals, but Mg 21 has been found to partially substitute for transition metals in the reaction mixture. Co(NH 3) 631 has been shown to substitute for Mg(H 2O) 21 in select enzyme systems, such as ribonuclease H (45) and DNA
FIG. 1. Structures of inert complexes. Co(NH 3) 631 and Co(en) 331 are shown to compare the steric restrictions of the Co(en) 331 complex. The structure of Mg(H 2O) 621 is shown to illustrate the similar metal to ligand distance for Co(NH 3) 631.
topoisomerase I (46). Ligand substitution with media is very slow with a rate constant of 5.8 3 10 212 s 21 reported in water (51), but the protons of the ammonia ligands can be abstracted as in the mechanism for substitution reactions of compounds of the family Co(NH 3) 5X 31 (52). The metal to ligand distance in this complex is 1.97 Å (Fig. 1), similar to the distance found in Mg(H 2O) 621. Cowan (53) has presented an approach for discriminating between an outer sphere and an inner sphere interaction based on the ability of Co(NH 3) 631 to activate or inhibit an enzyme. Use of Co(NH 3 ) 631 as Substitute for Mg 21 Co(NH 3) 631 was found to activate calcineurin with the concentration dependence showing hyperbolic kinetics (shown in Fig. 2). Subsequent kinetic experiments provided estimates of k cat, K m (pNPP), and K act for the Co(NH 3) 631 activation of calcineurin (Table I). Activation by other metals was characterized under the same conditions for comparison. Not surprisingly, the kinetic parameters evaluated were similar to those for Mg 21 activation and were distinct from the parameters for
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SUBSTITUTION INERT COMPLEXES AND CALCINEURIN
with an outer coordination sphere interaction using a bridging H 2O (NH 3) between metal and substrate. If k cat/K m approximates the rate constant for substrate association, the lower value with the Co(NH 3) 631activated enzyme may reflect some consequence of having ammonia groups compared to water with Mn 21 and Mg 21. First, the van der Waal radii of oxygen and nitrogen are similar, but the third hydrogen atom (radius 5 1.20 Å) in ammonia will occupy additional space. The association rate may be dampened because of increased difficulty of substrate and metal orientation in the binding site. Second, there likely would be differences between water and ammonia in transfer of a proton to the leaving group, a role previously postulated for metal (40). Effect of [Co-(ethylenediamine) 3 ] 31 on Calcineurin Activity
FIG. 2. Activation curve for Co(NH 3) 631. Calcineurin (3 mg/ml 5 37.5 nM) was assayed with varying amounts of Co(NH 3) 631 at pH 7.0 with pNPP at 10.0 mM. Other details are described in the text. Each initial velocity is the average of four replicates at each Co(NH 3) 631 concentration with the error bars showing the standard deviation of the measurements.
Co 21 (Co(H 2O) 621 in solution) and Mn 21 (Table I) and Ni 21 (54). Co(NH 3) 631 supported a slightly higher maximal activity (k cat) than did Mg 21, but the k cat/K m parameter was lower for Co(NH 3) 631 than for Mg 21. Comparison of k cat/K act values showed that Co(NH 3) 631 was an approximately 150-fold poorer activator of calcineurin than Mn 21, but comparable to Mg 21. The K m for pNPP with Co(NH 3) 631 was higher than that with Mg 21 and essentially identical to that with Mn 21 and Co 21. Comparison of k cat/K m values suggested that ligand substitution was not involved in substrate binding. The absence of any ostensible requirement for ligand substitution at the metal ion was consistent
Co(NH 3) 631 activated calcineurin with parameters comparable to activation by Mg 21. The related compound [Co-(ethylenediamine) 3] 31 (or Co(en) 331) has been well characterized as a model compound for the study of transition metal complexes. Although having a similar metal to nitrogen ligand distance (1.96 Å, Fig. 1) as Co(NH 3) 631, this chelated complex restricts the mobility of the metal ligands. The molecule is also bulkier and provides insight into the steric constraints of metal activation sites. Co(en) 331 did not activate calcineurin activity. Instead, calcineurin was inhibited (not shown) and Co(en) 331 showed competitive inhibition kinetics against Mn 21 (Fig. 3) with a K I of approximately 16.5 mM. Previously, inorganic phosphate was shown to be a competitive inhibitor with pNPP as the varied substrate (38). Having two characterized competitive inhibitors provided an opportunity to evaluate the order of ligand binding by additional kinetic studies. P i was found to be an uncompetitive inhibitor with Mn 21 as the varied substrate and pNPP maintained at a constant, subsaturating concentration of 10 mM (Fig. 4). The value of K I was evaluated as approximately 0.8
TABLE I
Activation of Calcineurin by Exogenous Metals
Metal
k cat (s 21)
Km (mM)
K act (mM)
k cat/K m (M 21 s 21)
k cat/K act (M 21 s 21)
Co(NH 3) 631 Co 21 Mn 21 Ni 21 Mg 21
0.59 6 0.02 0.81 6 0.07 1.08 6 0.06 0.69 6 0.06 0.32 6 0.02
13.5 6 0.5 17.4 6 3.2 14.0 6 1.9 10.1 6 2.1 4.8 6 1.0
15.9 6 1.7 0.17 6 0.02 0.17 6 0.02 0.41 6 0.07 6.3 6 0.74
44 47 77 68 67
37 4765 6353 1683 51
Note. The kinetic parameters were estimated from initial rates collected at 30°C for the hydrolysis of pNPP. Each substrate–velocity pair was measured at least in triplicate. The data for Mn 21, Ni 21, and Mg 21 are from Ref. (54).
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FIG. 3. Inhibition pattern for Co(en) 331 with Mn 21 as the variable ligand. Calcineurin (3 mg/ml, or 37.5 nM) was assayed with varying amounts of Mn 21 with different fixed concentrations of Co(en) 331 at pH 7.0 as described in the text. Co(en) 331 was included at 0.0 mM (filled circles), 15.0 mM (filled triangles), and 35.0 mM (filled squares). As the likely second substrate, pNPP was fixed at the subsaturating concentration of 10.0 mM. Each initial rate was the average of three replicate experiments with duplicate determinations within the experiment. The error bars are standard deviations of the estimated rates.
this reaction, the 1/v vs 1/pNPP plot should yield a series of lines intersecting on the 1/v-axis for a rapidequilibrium ordered mechanism. The plots of varied Mn 21 (1/v vs 1/Mn 21 at fixed, different pNPP concentrations) and varied pNPP (1/v) vs 1/pNPP at fixed, different Mn 21 concentrations) were consistent with a sequential, but not the rapid-equilibrium ordered mechanism. Shown in Fig. 6 is the plot for pNPP as the varied substrate. As drawn in this figure, the lines intersect at a common point on the 1/[pNPP]-axis, suggesting that the K m for pNPP did not change with the level of Mn 21 used in the assays. This is consistent with ordered binding with formation of the enzyme–metal complex required prior to any binding of pNPP. The primary kinetic plots best support a steadystate, rather than a rapid-equilibrium, ordered kinetic mechanism. In disagreement with a steady-state ordered mechanism is the observed competitive inhibition pattern for Co(en) 331 with pNPP as the varied ligand (Fig. 5). This profile was found with Mn 21 levels below saturation (at 0.2 mM, approximately the K act for Mn 21) with the observed kinetic pattern more consistent with noncompetitive inhibition (not shown) when characterized with Mn 21 at a concentration of 5 3 K act. Binding of the Co(en) 331 may provide additional obstacles to subsequent binding of pNPP beyond a conformational effect on the enzyme found with any metal ion. For example, if the metal site and pNPP sites are close, the spatially restricted ligands of Co(en) 331 may
mM. Likewise, inhibition by Co(en) 331 was evaluated with pNPP as the varied substrate and Mn 21 held constant and found to be competitive (Fig. 5). The modes of inhibition deduced from the shown doublereciprocal plots (Figs. 3 to 5) were verified by directly fitting the data to Eqs. [1] through [3]. In each situation, the data set was best fit by the mode of inhibition shown in the figures. These inhibition patterns seem consistent with a rapid-equilibrium ordered binding mechanism 4 with metal binding to calcineurin before substrate. Primary Kinetic Plots A characteristic of the rapid-equilibrium ordered kinetic mechanism is the intersection point in the double-reciprocal plot for the ligand binding second. For 4
Initial experiments were done with saturating Mn 21 (1.0 mM) and yielded inhibition plots more consistent with noncompetitive inhibition. These data supported ordered binding with metal first, although not for a rapid equilibrium model. Studies with subsaturating metal were done after the review of an earlier version of the manuscript. We acknowledge and thank the referees for comments resulting in an improved manuscript.
FIG. 4. Inhibition pattern for inorganic phosphate with Mn 21 as the variable ligand. Calcineurin (3 mg/ml, or 37.5 nM) was assayed with varying amounts of Mn 21 with different fixed concentrations of inorganic phosphate at pH 7.0. Phosphate was included at 0.0 mM (filled circles), 1.0 mM (filled triangles), and 2.0 mM (filled squares) concentrations. Each initial rate was the average of duplicate experiments with duplicate determinations within an experiment. The error bars are standard deviations of the estimated rates.
SUBSTITUTION INERT COMPLEXES AND CALCINEURIN
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binds directly to free enzyme preceding substrate binding as illustrated in Fig. 7. Also depicted in this figure is the random release of the products, P i and pNP, identified in an earlier study (38) of the product inhibition pattern. A corollary result of this mechanism is that no prior formation of a metal–substrate complex is required for calcineurin activity. Two types of complexes are consistent with these data: Metal may function as a bridge between enzyme and substrate (bridge structure) or all components directly interact with one another (symmetrical structure). Wong (55) has presented kinetic formulations for metal ion activators of enzymes and has developed a set of criteria (p. 67) for each of the possible mechanisms. The ordered binding order deduced from the data effectively precluded many of the possible mechanisms described by Wong, but do not distinguish between the bridged and symmetrical structures. Based on the observed binding of inorganic phosphate in the resolved crystal structure of calcineurin, it is likely that the symmetrical model is formed.
FIG. 5. Inhibition pattern for Co(en) 331 with pNPP as the variable ligand. Calcineurin (2 mg/ml, or 25 nM) was assayed with varying amounts of pNPP with different fixed concentrations of Co(en) 331 at pH 7.0 as described in the text. Co(en) 331 was included at 0.0 mM (filled circles), 15.0 mM (filled triangles), 35.0 mM (filled squares), and 50.0 mM (filled diamonds). Each initial rate was the average of three replicate experiments with duplicate determinations within the experiment. The error bars are standard deviations of the estimated rates and are shown in only one direction for clarity of the figure.
preclude effective interaction of pNPP. Such steric barriers are less likely with Mn 21 (really Mn(H 2O) 621). The effect is not because Co(en) 331 acted as a partial competitive inhibitor inasmuch as the slope replot was linear. The bulk of the evidence favors a steady-state ordered mechanism with Mn 21 being the preceding ligand. DISCUSSION
These data demonstrated that several metal complexes were able to activate calcineurin and provide insight into the interaction of exogenous metal with the enzyme. For each metal complex, all curves showed saturation kinetics (not shown). Additionally, no evidence was observed in these experiments to suggest inhibition by excess metal. Binding Scheme for Ligands From the kinetic inhibition patterns, the binding order of these ligands can be established. Free metal
FIG. 6. Kinetics of pNPP hydrolysis at different concentrations of Mn 21. Calcineurin (2 mg/ml, or 25 nM) was assayed with varying amounts of pNPP with different fixed concentrations of Mn 21 at pH 7.0 as described in the text. Mn 21 was included at 0.10 mM (filled triangles), 0.165 mM (open triangles), 0.25 mM (filled circles), and 0.50 mM (open circles). Shown is a representative experiment with duplicates for each initial rate. The error bars are standard errors.
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MARTIN AND RHODE
FIG. 7. Kinetic scheme for metal and substrate binding to calcineurin. Although calcineurin likely retains Mn 21 for a catalysis of another cycle, the figure shows the dissociation of metal to illustrate the recovery of free enzyme. Random release of the products was concluded in an earlier study (Ref. 38).
Activation by Co(NH 3 ) 631 31 3 6
Calcineurin was seemingly activated by Co(NH ) , consistent with metal functioning through an outer sphere mechanism. One caveat to this conclusion is concern about the integrity of the Co(NH 3) 631 compound, particularly any Co 21 (actually Co(H 2O) 621) contamination. The kinetic parameters for Co(NH 3) 631 were approximately 100-fold higher than found for Co 21 such that Co 21 present at 1% contamination level may be responsible for the observed activation of calcineurin with Co(NH 3) 631. There is no positive evidence for the presence of such contamination. Visible spectrophotometry (not shown) revealed no characteristic peaks for Co 21 in solutions of Co(NH 3) 631, although the spectrum may have been hidden by the native spectrum of Co(NH 3) 631. Concern about contaminating Co 21 also required that Co(NH 3) 631 not have any effect on calcineurin directly or on activation by Co 21. This seemed unlikely judging from the effects of Co(en) 331 which inhibited Mn 21 activation in the same concentration range used for Co(NH 3) 631. If Co(NH 3) 631 did inhibit, the level of contaminating Co 21 would need to be greater than 1% to measure a similar k cat value and then would have been unlikely to yield a similar value of K act. The observed effects were likely because of Co(NH 3) 631, not Co 21. The chemistry of Co(NH 3) 631 favors this conclusion. The standard isolation of Co(NH 3) 631 requires selective precipitation to separate the material from Co 21 and the 13 state of Co is stabilized by the substitution inert ammonia ligands. The exchange of ammonia ligand with solvent water occurs very slowly with a rate constant of 5.8 3 10 212 s 21 (51), providing a significant barrier to the accumulation of hydrated Co 31 as a perquisite for the reduction to Co 21. It was concluded that Co(NH 3) 631 did activate calcineurin.
Organization of Exogenous Metal in Calcineurin Catalysis Even though the kinetic parameters showed that Co(NH 3) 631 was a worse activator than Mn 21, the fact that it did cause activation provided insight into the role of exogenous metal in catalysis by calcineurin. As described by Cowan (53), activation by this metal complex is limited to a function as an outer sphere complex. Thus, calcineurin activity can be activated by Mn 21 and other metals via an outer coordination sphere interaction. This indicates that it is not obligatory for the aqua-metal to (a) directly coordinate to the phosphate ester of the substrate in the catalytic reaction or (b) activate a water molecule involved in a direct nucleophilic attack on the phosphate ester. Indeed, it seems likely that the metal ion does not function through either of these paths. Instead, the effect of metal is likely through a water molecule “bridge” between the metal and the phosphate ester linkage. A role of this type is consistent with data from studies of the heavy-atom isotope effects on the calcineurincatalyzed hydrolysis of pNPP (44). The chemical step of phosphoryl transfer was found to be partially ratelimiting for pNPP hydrolysis, unlike the situation for protein-tyrosine phosphatases and dual-specific phosphatases that exhibit fully rate-limiting chemistry with pNPP. Proton transfer to the leaving group was found to lag behind the cleavage reaction, resulting in some partial negative charge in the transition state. The activating metal ion Mn 21 caused a change in the commitment factor with the chemical step becoming more rate-limiting at higher concentrations of Mn 21. The degree to which the chemical step was rate-limiting together with effect of Mn 12 indicated that some nonchemical step (steps) after substrate binding, but before catalysis, was (were) affected. A proton transfer to the leaving group would fit these criteria.
SUBSTITUTION INERT COMPLEXES AND CALCINEURIN
A model using two metal ions has been proposed for calcineurin with one metal site serving to stabilize or enhance the leaving group through the donation of a proton from water coordinated to the metal ion (40). Subsequent studies have raised questions about the involvement of both intrinsic metals in catalysis (41). An outer sphere interaction would be sufficient to satisfy this function. For Mn 21 and Mg 21, water coordinated to these metals has pK as of 10.6 and 11.4, respectively. As noted earlier, the relative pH optimum of calcineurin activity was different whether Mn 21 (approximately pH 7.0) and Mg 21 (approximately pH 8.2) was used as the activator. There was a one pH unit difference mirroring the difference in pK a values of H 2O with these metals. This difference is consistent with the expected difference in the ability to donate a proton of these species, particularly in the stabilization of the leaving group. Stabilization of the leaving group by metal was found by Herschlag and Jencks (15) in the hydrolysis of pNPP by Mg 21 or Ca 21. It is notable that Mn 21 bound to calcineurin was found (56) to retain exchangeable water within its inner coordination sphere establishing the availability of a water ligand for proton donation, albeit through an outer sphere interaction. In a study of coordination of water to Mg 21, water could be found both as an inner and an outer coordination ligand (57), suggesting that Mg 21 could also function through this interaction. Possible Consequences of an Outer Sphere Mechanism in Phosphatase Catalysis Participation of a metal ion through an outer sphere coordination interaction may be the cause of the reduced k cat observed with calcineurin compared to other phosphatases. Protein tyrosine phosphatases (58) typically have values of k cat in the range 5– 60 s 21 ('10fold greater than that found for calcineurin), the purple acid phosphatase (59) in the range 200 – 400 s 21 ('100fold greater), alkaline phosphatase (60) in the range 10 –30 s 21 ('10-fold greater), and another protein serine protein phosphatases (PP2A, Ref. 61) in the range 40 –200 s 21 ('50-fold greater) for pNPP hydrolysis. Phosphotriesterase, a dinuclear containing enzyme, catalyzes the hydrolysis of model substrates with k cat in the range 50 –100 s 21 (62). All except protein tyrosine phosphatases are metalloenzymes and generally do not require the addition of exogenous metal. Furthermore, model studies (16 –18) have shown that chelation of metal can result in greater rate accelerations than those observed with free Mg 21 or Ca 21 (15) in solution. Of the noted enzymes, alkaline phosphatases are the most well characterized. It is a zinc metalloenzyme with the Zn 21 directly coordinating phosphate as an inner sphere ligand. This mechanism enables zinc to
175
polarize one of the phosphorous– oxygen bonds for nucleophilic attack (63). Utilization of an outer vs inner sphere ligand in an enzyme mechanism may limit the magnitude of the enzymatic rate that can be achieved. If true, the difference between calcineurin and alkaline phosphatase may provide the range for distinguishing the mechanistic role of metal. Consider protein phosphatase-1, which is a member of the same gene family as calcineurin. Protein phosphatase-1 does not effectively catalyze the hydrolysis of pNPP, and the resolved crystal structure of phosphatase-1 showed the presence of bound metal (3, 4). A recombinant form of phosphatase-1 was shown to be activated by metals and could incorporate metal to form stable enzyme– metal complexes (8). On the basis of on the structural information available and the low activity with pNPP, PP-1 may use an outer coordination sphere interaction in the hydrolysis of phosphate ester bonds. The possible involvement of metals via an outer sphere interaction may provide a constraint on the reaction evidence by the lower rate constants for PP-1 and calcineurin with pNPP. Firmer characterization of the metal requirement of protein phosphatase-1 may demonstrate whether additional features are shared by these two enzymes. SUMMARY
The activation of calcineurin by Co(NH 3) 631 was comparable to activation by transition metals, suggesting a common mechanism involving an outer sphere ligand interaction. The order of binding was concluded to be metal first, followed by the substrate, consistent with exogenous metal facilitating the binding of substrate and with a proposed function of providing stabilization of the departing leaving group through proton donation or outer sphere hydrogen bonding. Isotope studies of the calcineurin-catalyzed reaction have demonstrated that there is partial negative charge on the leaving group and provided evidence for some form of metal– phosphate interaction (44). Any proposed mechanism for calcineurin must define a role for exogenous metal. ACKNOWLEDGMENT This research was supported by funds from the University of Tennessee Medical Group.
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34. Ladner, C. J., Czech, J., Maurice, J., Lorens, S. A., and Lee, J. M. (1996) J. Neuropathol. Exp. Neurol. 55, 924 –931. 35. Winkler, M. A., Merat, D. L., Tallant, E. A., Hawkins, S., and Cheung, W. Y. (1984) Proc. Natl. Acad. Sci. USA 81, 3054 –3058. 36. Merat, D. L., Hu, Z. Y., Carter, T. E., and Cheung, W. Y. (1984) Biochem. Biophys. Res. Commun. 122, 1389 –1396. 37. Martin, B., Pallen, C. J., Wang, J. H., and Graves, D. J. (1985) J. Biol. Chem. 260, 14932–14937. 38. Martin, B. L., and Graves, D. J. (1986) J. Biol. Chem. 261, 14545–14550. 39. Martin, B. L., and Graves, D. J. (1993) Biochem. Biophys. Res. Commun. 194, 150 –156. 40. Martin, B. L., and Graves, D. J. (1994) Biochim. Biophys. Acta 1206, 136 –142. 41. Martin, B. L. (1997) Arch. Biochem. Biophys. 345, 332–338. 42. Pallen, C. J., and Wang, J. H. (1983) J. Biol. Chem. 258, 8550 – 8553. 43. Pallen, C. J., Brown, M. L., Matsui, H., Mitchell, K. J., and Wang, J. H. (1985) Biochem. Biophys. Res. Commun. 131, 1256 – 1261. 44. Hengge, A. C., and Martin, B. L. (1997) Biochemistry 36, 10185– 10191. 45. Jou, R., and Cowan, J. A. (1991) J. Am. Chem. Soc. USA 113, 6685– 6686. 46. Kim, S., and Cowan, J. A. (1992) Inorg. Chem. 31, 3495–3496. 47. Sharma, R. K., Taylor, W. A., and Wang, J. H. (1983) Methods Enzymol. 102, 210 –219. 48. Sharma, R. K., and Wang, J. H. (1979) Adv. Cyclic Nucleotide Res. 10, 187–198. 49. Gopalakrishna, R., and Anderson, W. G. (1982) Biochem. Biophys. Res. Commun. 104, 830 – 836. 50. Bradford, M. M. (1976) Anal. Biochem. 72, 248 –254. 51. Dixon, N. E., Jackson, W. G., Lancaster, M. J., Lawrence, G. A., and Sargeson, A. M. (1981) Inorg. Chem. 20, 470 – 476. 52. Wilkins, R. G. (1991) Kinetics and Mechanism of Reactions of Transition Metal Complexes, pp. 215–217, VCH Publishers, New York. 53. Cowan, J. A. (1992) Comments Inorg. Chem. 13, 293–312. 54. Martin, B. L., and Jurado, L. A. (1998) J. Protein Chem. 17, 473– 478. 55. Wong, J. T.-F. (1975) Kinetics of Enzyme Mechanisms, pp. 57– 72. Academic Press, New York. 56. Haddy, A., Sharp, R. R., and Rusnak, F. (1996) Biophys. J. 70, A393. [Abstract W-Pos86] 57. Bock, C. W., Kaufman, A., and Glusker, J. P. (1994) Inorg. Chem. 33, 419 – 427. 58. Zhang, Z.-Y. (1995) J. Biol. Chem. 270, 11199 –11204. 59. Wynne, C. J., Hamilton, S. E., Dionysius, D. A., Beck, J. L., and de Jersey, J. (1995) Arch. Biochem. Biophys. 319, 133–141. 60. Han, R., and Coleman, J. E. (1995) Biochemistry 34, 4238 – 4345. 61. Hermann, J., Cayla, X., Dumortier, K., Goris, J., Ozon, R., and Merlevede, W. (1988) Eur. J. Biochem. 173, 17–25. 62. Caldwell, S. R., Newcomb, J. R., Schlecht, K. A., and Raushel, R. M. (1991) Biochemistry 30, 7438 –7444. 63. Hollfelder, F., and Herschlag, D. (1995) Biochemistry 34, 12255– 12264.
Effect of Substitution Inert Metal Complexes on Calcineurin Bruce L. Martin 1 and David J. Rhode Department of Biochemistry, University of Tennessee, 858 Madison Avenue, Memphis, Tennessee 38163
Received March 30, 1998, and in revised form March 9, 1999
As a substitute for M(H 2O) 621, Co(NH 3) 631 was found to activate calcineurin with para-nitrophenyl phosphate as substrate. Kinetics for calcineurin catalyzed hydrolysis of para-nitrophenyl phosphate at pH 7.0 with Mn 21, Mg 21, Co 21, and Co(NH 3) 631 were compared. Although k cat and K m were different with the metals, values of k cat/K m were nearly identical for Mn 21 and Mg 21, but lower for Co 21 and Co(NH 3) 631. The concentration of each metal providing half-maximal activation, designated K act, was evaluated as 15.9 mM for Co(NH 3) 631, compared to K act 5 0.17 mM for Mn 21 and Co 21 and 6.3 mM for Mg 21, respectively. Comparing k cat/K cat showed that Co(NH 3) 631 was a 170-fold poorer activator of calcineurin than was Mn 21, but only 1.5fold poorer than Mg 21. Activation by Co(NH 3) 631 indicated that activation of calcineurin by exogenous metal ions can proceed via an outer coordination sphere reaction mechanism with no requirement for the direct coordination of substrate by metal. Because Co(NH 3) 631 was found to support calcineurin activity, the related compound [Co-(ethylenediamine) 3] 31 (or Co(en) 331) was tested as a possible activator. Co(en) 331 did not support calcineurin activity but did inhibit calcineurin. Co(en) 331 showed competitive inhibition kinetics with either Mn 21 or pNPP as the varied ligand and the other at a fixed, subsaturating concentration. Inorganic phosphate was used as a known competitive inhibitor to pNPP (B. L. Martin and D. J. Graves, J. Biol. Chem. 261, 14545–14550, 1986) and showed uncompetitive inhibition with Mn 21 as the varied ligand. These patterns are consistent with the mechanism of ligand binding to calcineurin being ordered with metal preceding substrate. Prior formation of a metal– substrate complex was not required for association with calcineurin. © 1999 Academic Press 1 To whom correspondence should be addressed at Department of Biochemistry, University of Tennessee, 858 Madison Ave., Suite G01, Memphis, TN 38163. Fax: 901-448-7360. E-mail: bmartin@ utmem1.utmem.edu.
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Key Words: calcineurin; protein phosphatase; metalactivated enzyme.
Protein serine and threonine protein phosphatases are involved in the phosphorylation and dephosphorylation cycles critical for the regulation of a variety of enzymes. There are four classes of cytosolic protein serine phosphatases, designated types-1, -2A, -2B, and -2C (1) with types-1, -2A, and -2B belonging to the same gene family (2). Intrinsic metals have been identified in the resolved crystal structures of types-1 and -2B (3– 6) and all classes of the type 1/2 gene family have enzyme forms that are activated by metal ions (7–10). Although distinct from the protein phosphatases, some alkaline phosphatases (11, 12) and the purple acid phosphatases (13, 14) also are metalloenzymes and likely share some characteristics involving the participation of metal ions in catalyzing the hydrolysis of phosphate ester bonds. The precise role attributable to metal is not understood. It is likely that metals serve one of the following functions resulting in enhanced phosphate ester hydrolysis: (i) activation of the nucleophilicity of coordinated water for attack on the phosphate ester, (ii) coordination and activation of the phosphate ester substrate, and (iii) stabilization of the developing leaving group. Various model studies have been done to elucidate features about the roles of metals in phosphoryl transfer reactions. From a study of the hydrolysis of paranitrophenyl phosphate (pNPP) 2 by Mg 21 or Ca 21, Herschlag and Jencks (15) suggested that stabilization of the leaving group did occur. Other studies (16 –18) have shown that chelation of metal can result in accelAbbreviations used: EGTA, ethylene glycol bis(b-amino ethyl ether) N,N9-tetraacetic acid; Mops, 3-(N-morpholino)propanesulfonic acid; pNP, para-nitrophenol; pNPP, para-nitrophenyl phosphate. 2
0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
SUBSTITUTION INERT COMPLEXES AND CALCINEURIN
erations even higher than observed with Mg 21 or Ca 21 (15). Protein phosphatase-2B, designated calcineurin, is a calmodulin-activated phosphatase that is isolated with intrinsically bound iron and zinc (6, 19) but requires exogenous metal for maximal activity (9, 19 –23). The roles of intrinsic metals are not clear. The oxidation state of the intrinsic iron has been reported to be crucial for optimal activity (24, 25), although there is disagreement whether active enzyme contains Fe 21 or Fe 31. The work of Klee and colleagues (25) suggested that Fe was present as Fe 21, but these findings were made with only a fraction of the total enzyme pool that shows instability in the presence of calmodulin and Ca 21. The Rusnak laboratory reported (24, 26, 27) that calcineurin contained Fe 31 and was active with a value of k cat comparable to other published values. Earlier studies on brain calcineurin by King (28) reported that all cysteines of the enzyme were in the reduced state with incubation with mercaptoethanol causing no effect on Mn 21 supported activity. These latter findings are suggestive that the reducing agent is not required in Mn 21-supported activation. The enzyme used in this report was not activated by incubation with ascorbate, but did show a 10% loss of activity incubated in the absence of dithiothreitol for 20 min. 3 There was no loss of activity during incubation for shorter times such as the length of activity assay. There is currently insufficient information to make final conclusions about the relationship between the oxidation state of intrinsic Fe and the activity of calcineurin. Calcineurin effectively has no catalytic activity (,1%) in the absence of exogenous metal. Mn 21, Ni 21, and Mg 21 are typically used for activating calcineurin. A curious property of the enzyme is the loss of sensitivity to Ca 21 during affinity chromatography on immobilized calmodulin as originally reported by the Klee and Cohen laboratories (29, 30). In these reports, equivalent activation was achieved with Mn 21 or Ca 21 until the affinity chromatography step. Recovered enzyme was then only activated by Mn 21. Subsequent studies by many different laboratories (9, 21–23, 31– 35) have consistently demonstrated the activation of calcineurin by Mn 21 in the absence of Ca 21. Cheung and associates demonstrated that Mn 21 activation was targeted at the catalytic subunit (35, 36). From mechanistic studies (37– 40), divalent metal has been proposed to have a direct role in the catalytic mechanism of the enzyme, not simply causing the protein to adopt an active conformation. Substituting Mg 21 for Mn 21 caused a shift in the pH optimum for the hydrolysis of pNPP. The shift was approximately 1 pH unit, comparable to the difference in pK a for water bound to Mn 21 3
D. J. Rhode and B. L. Martin, unpublished observations.
169
(pK a 5 10.6) and Mg 21 (pK a 5 11.4). Li and associates likewise concluded that the pH optimum was dependent on the metal used (32). Collectively, these data suggest a role for exogenous metal in catalysis. From analysis of the resolved crystal structure of protein phosphatase-1, incorporation of exogenous metal into enzyme has been suggested as one possible mechanism for activation. Chu et al. have shown the stable incorporation of Co 21, but with limited recovery of activity (8). Greater activity was recovered with Mn 21, but the incorporation of this metal was not stable. Available data with calcineurin is not consistent with this mechanism. Using atomic absorption spectroscopy, it was found (41) that incubation of calcineurin with Mn 21 did not result in loss of intrinsic metal. This was a critical result: Exogenous metal did not physically replace intrinsic metal. Calcineurin has activity with low-molecular-weight phosphate esters (37, 42, 43), making it a useful model to examine features of phosphatase catalysis (38 – 40, 44). In this study, pNPP was used as the substrate. Using a simple phosphate ester enables the basis of metal activation of phosphatase activity to be conveniently investigated without the complication of nonspecific binding of metals by protein substrates. Using the inert metal complex Co(NH 3) 631 as a substitute for Mg(H 2O) 621, the activation by this complex was examined. Co(NH 3) 631 is regarded to function as an outer sphere complex and has been shown to substitute for Mg(H 2O) 621 in some enzyme systems, such as ribonuclease H (45) and DNA topoisomerase I (46). Inasmuch as exogenous metal does not replace intrinsic metal, the inert metal is not likely bound by the ligands of intrinsic iron and zinc. EXPERIMENTAL PROCEDURES Materials. Buffers, EGTA, and phenyl-Sepharose were purchased from Sigma Chemical. The substrate, pNPP (Sigma 104 substrate), was also purchased from Sigma. DE-52 cellulose was obtained from Whatman. Co(NH 3) 6Cl 3 was purchased from Strem Chemicals (Newburyport, MA) as 99.999% pure (Cat. No. 93-2708). Tris(ethylenediamine) cobalt (III) chloride was purchased from Alfa Aesar. Other chemicals (metal salts, etc.) were obtained from Fisher. All solutions, including those for the preparation and assay of calcineurin, were prepared from water that had been passed over a Chelex-100 column. Proteins. Bovine brain calcineurin was isolated from bovine brain to apparent homogeneity on SDS–polyacrylamide gel electrophoresis by the method of Sharma et al. (47) with Mops buffers instead of Tris buffers. Calmodulin was purified by the procedure of Sharma and Wang (48) slightly modified to include a final chromatography step on phenyl-Sepharose with elution by EGTA (49). Protein concentrations were determined by the method of Bradford (50). Calcineurin assay. Calcineurin was assayed on a Cary model 1E spectrophotometer with pNPP as the substrate by measuring the release of pNP at 410 nm at 30°C. Standard reaction conditions were 25 mM Mops, pH 7.0; 1.0 mM MnCl 2; and 10 mM pNPP in 800 ml reaction volume. Reaction times ranged from 10 to 30 min with changes at 410 nm of 0.05 to 0.35 absorbance units. Calcineurin was
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at concentrations indicated in the figure legends with calmodulin included at a fivefold molar excess of calcineurin. The model substrate pNPP was used to minimize the number of potential binding sites for the metals studied. The amino acid residues of polypeptide substrates may have different preferences for associating with metals, particularly as metals have different preferences for oxygen-, nitrogen-, or sulfur-containing ligands. The stability constants for Mn 21 (log K 1 5 1.9), Mg 21 (log K 1 5 1.3), and Co 21 (log K 1 5 1.6) with pNPP are within the same order of magnitude. Ca 21 was omitted to simplify the analysis of metal activation. Numerous studies have demonstrated that the presence of Ca 21 is not required to achieve activation by other metals whether assayed as the purified enzyme (6, 21–23, 31–33) or assayed in tissue extracts (29, 30, 34). Initial activation by Co(NH 3) 631 was determined using 10 mM pNPP as in the standard assay conditions. Likewise, Co(en) 331 was tested as an activator and as an inhibitor using 10 mM pNPP. Kinetic measurements. The parameters k cat, K m, and K act were determined by varying pNPP or varying metal as appropriate with other conditions already described. Substrate (pNPP) concentrations used were 50.0, 16.0, 10.0, 7.0, and 5.0 mM. For the different metals, the following concentration ranges were used: 0.50, 0.16, 0.10, 0.070, and 0.050 mM for MnCl 2; 1.00, 0.33, 0.20, 0.14, and 0.11 mM for CoCl 2; and 50.0, 16.0, 10.0, 7.0, and 5.0 mM for MgCl 2 and Co(NH 3) 631. In experiments with varied metal, measured activity was corrected for activity in the absence of any added exogenous metal. To monitor kinetic inhibition of calcineurin, the concentrations of pNPP and various metals were varied at fixed, different concentrations of inhibitors. The cited ranges of substrate and MnCl 2 concentrations were used. With Co(en) 331 as the inhibitor, concentrations of 0.0, 15.0, and 35.0 mM were used with Mn 21 as the varied ligand and 0.0, 15.0, 30.0, and 50.0 mM when pNPP was varied. Inhibition by inorganic phosphate with Mn 21 as the varied ligand was studied with phosphate concentrations of 0.0, 1.0, and 2.0 mM. The kinetic parameters, k cat, K m, K act, and K I, were evaluated. Data sets were plotted in double-reciprocal form for identifying the mode of inhibition. The type of inhibition was verified by fitting the data to the Michaelis–Menten equations for each mode of inhibition (Eqs. [1] to [3]).
v5
v5
k cat p @pNPP# K m~pNPP! p ~1 1 @I#/K I)1[pNPP#
competitive inhibition
[1]
k cat p @pNPP# K m~pNPP! p ~1 1 @I#/K I! 1 @pNPP# p ~1 1 @I#/K I! noncompetitive inhibition
v5
k cat p @pNPP# K m~pNPP! 1 @pNPP# p ~1 1 @I#/K I!
[2]
uncompetitive inhibition [3]
Data fitting and numerical estimates were done using EnzFitter (Elsevier BioSoft), Enzyme Kinetics (Trinity Software), or Psi-Plot (PsiPlot, Inc.) as appropriate. Double-reciprocal plots were prepared using Deltagraph (DeltaPoint, Inc.).
RESULTS
Calcineurin is typically activated by transition metals, but Mg 21 has been found to partially substitute for transition metals in the reaction mixture. Co(NH 3) 631 has been shown to substitute for Mg(H 2O) 21 in select enzyme systems, such as ribonuclease H (45) and DNA
FIG. 1. Structures of inert complexes. Co(NH 3) 631 and Co(en) 331 are shown to compare the steric restrictions of the Co(en) 331 complex. The structure of Mg(H 2O) 621 is shown to illustrate the similar metal to ligand distance for Co(NH 3) 631.
topoisomerase I (46). Ligand substitution with media is very slow with a rate constant of 5.8 3 10 212 s 21 reported in water (51), but the protons of the ammonia ligands can be abstracted as in the mechanism for substitution reactions of compounds of the family Co(NH 3) 5X 31 (52). The metal to ligand distance in this complex is 1.97 Å (Fig. 1), similar to the distance found in Mg(H 2O) 621. Cowan (53) has presented an approach for discriminating between an outer sphere and an inner sphere interaction based on the ability of Co(NH 3) 631 to activate or inhibit an enzyme. Use of Co(NH 3 ) 631 as Substitute for Mg 21 Co(NH 3) 631 was found to activate calcineurin with the concentration dependence showing hyperbolic kinetics (shown in Fig. 2). Subsequent kinetic experiments provided estimates of k cat, K m (pNPP), and K act for the Co(NH 3) 631 activation of calcineurin (Table I). Activation by other metals was characterized under the same conditions for comparison. Not surprisingly, the kinetic parameters evaluated were similar to those for Mg 21 activation and were distinct from the parameters for
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SUBSTITUTION INERT COMPLEXES AND CALCINEURIN
with an outer coordination sphere interaction using a bridging H 2O (NH 3) between metal and substrate. If k cat/K m approximates the rate constant for substrate association, the lower value with the Co(NH 3) 631activated enzyme may reflect some consequence of having ammonia groups compared to water with Mn 21 and Mg 21. First, the van der Waal radii of oxygen and nitrogen are similar, but the third hydrogen atom (radius 5 1.20 Å) in ammonia will occupy additional space. The association rate may be dampened because of increased difficulty of substrate and metal orientation in the binding site. Second, there likely would be differences between water and ammonia in transfer of a proton to the leaving group, a role previously postulated for metal (40). Effect of [Co-(ethylenediamine) 3 ] 31 on Calcineurin Activity
FIG. 2. Activation curve for Co(NH 3) 631. Calcineurin (3 mg/ml 5 37.5 nM) was assayed with varying amounts of Co(NH 3) 631 at pH 7.0 with pNPP at 10.0 mM. Other details are described in the text. Each initial velocity is the average of four replicates at each Co(NH 3) 631 concentration with the error bars showing the standard deviation of the measurements.
Co 21 (Co(H 2O) 621 in solution) and Mn 21 (Table I) and Ni 21 (54). Co(NH 3) 631 supported a slightly higher maximal activity (k cat) than did Mg 21, but the k cat/K m parameter was lower for Co(NH 3) 631 than for Mg 21. Comparison of k cat/K act values showed that Co(NH 3) 631 was an approximately 150-fold poorer activator of calcineurin than Mn 21, but comparable to Mg 21. The K m for pNPP with Co(NH 3) 631 was higher than that with Mg 21 and essentially identical to that with Mn 21 and Co 21. Comparison of k cat/K m values suggested that ligand substitution was not involved in substrate binding. The absence of any ostensible requirement for ligand substitution at the metal ion was consistent
Co(NH 3) 631 activated calcineurin with parameters comparable to activation by Mg 21. The related compound [Co-(ethylenediamine) 3] 31 (or Co(en) 331) has been well characterized as a model compound for the study of transition metal complexes. Although having a similar metal to nitrogen ligand distance (1.96 Å, Fig. 1) as Co(NH 3) 631, this chelated complex restricts the mobility of the metal ligands. The molecule is also bulkier and provides insight into the steric constraints of metal activation sites. Co(en) 331 did not activate calcineurin activity. Instead, calcineurin was inhibited (not shown) and Co(en) 331 showed competitive inhibition kinetics against Mn 21 (Fig. 3) with a K I of approximately 16.5 mM. Previously, inorganic phosphate was shown to be a competitive inhibitor with pNPP as the varied substrate (38). Having two characterized competitive inhibitors provided an opportunity to evaluate the order of ligand binding by additional kinetic studies. P i was found to be an uncompetitive inhibitor with Mn 21 as the varied substrate and pNPP maintained at a constant, subsaturating concentration of 10 mM (Fig. 4). The value of K I was evaluated as approximately 0.8
TABLE I
Activation of Calcineurin by Exogenous Metals
Metal
k cat (s 21)
Km (mM)
K act (mM)
k cat/K m (M 21 s 21)
k cat/K act (M 21 s 21)
Co(NH 3) 631 Co 21 Mn 21 Ni 21 Mg 21
0.59 6 0.02 0.81 6 0.07 1.08 6 0.06 0.69 6 0.06 0.32 6 0.02
13.5 6 0.5 17.4 6 3.2 14.0 6 1.9 10.1 6 2.1 4.8 6 1.0
15.9 6 1.7 0.17 6 0.02 0.17 6 0.02 0.41 6 0.07 6.3 6 0.74
44 47 77 68 67
37 4765 6353 1683 51
Note. The kinetic parameters were estimated from initial rates collected at 30°C for the hydrolysis of pNPP. Each substrate–velocity pair was measured at least in triplicate. The data for Mn 21, Ni 21, and Mg 21 are from Ref. (54).
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FIG. 3. Inhibition pattern for Co(en) 331 with Mn 21 as the variable ligand. Calcineurin (3 mg/ml, or 37.5 nM) was assayed with varying amounts of Mn 21 with different fixed concentrations of Co(en) 331 at pH 7.0 as described in the text. Co(en) 331 was included at 0.0 mM (filled circles), 15.0 mM (filled triangles), and 35.0 mM (filled squares). As the likely second substrate, pNPP was fixed at the subsaturating concentration of 10.0 mM. Each initial rate was the average of three replicate experiments with duplicate determinations within the experiment. The error bars are standard deviations of the estimated rates.
this reaction, the 1/v vs 1/pNPP plot should yield a series of lines intersecting on the 1/v-axis for a rapidequilibrium ordered mechanism. The plots of varied Mn 21 (1/v vs 1/Mn 21 at fixed, different pNPP concentrations) and varied pNPP (1/v) vs 1/pNPP at fixed, different Mn 21 concentrations) were consistent with a sequential, but not the rapid-equilibrium ordered mechanism. Shown in Fig. 6 is the plot for pNPP as the varied substrate. As drawn in this figure, the lines intersect at a common point on the 1/[pNPP]-axis, suggesting that the K m for pNPP did not change with the level of Mn 21 used in the assays. This is consistent with ordered binding with formation of the enzyme–metal complex required prior to any binding of pNPP. The primary kinetic plots best support a steadystate, rather than a rapid-equilibrium, ordered kinetic mechanism. In disagreement with a steady-state ordered mechanism is the observed competitive inhibition pattern for Co(en) 331 with pNPP as the varied ligand (Fig. 5). This profile was found with Mn 21 levels below saturation (at 0.2 mM, approximately the K act for Mn 21) with the observed kinetic pattern more consistent with noncompetitive inhibition (not shown) when characterized with Mn 21 at a concentration of 5 3 K act. Binding of the Co(en) 331 may provide additional obstacles to subsequent binding of pNPP beyond a conformational effect on the enzyme found with any metal ion. For example, if the metal site and pNPP sites are close, the spatially restricted ligands of Co(en) 331 may
mM. Likewise, inhibition by Co(en) 331 was evaluated with pNPP as the varied substrate and Mn 21 held constant and found to be competitive (Fig. 5). The modes of inhibition deduced from the shown doublereciprocal plots (Figs. 3 to 5) were verified by directly fitting the data to Eqs. [1] through [3]. In each situation, the data set was best fit by the mode of inhibition shown in the figures. These inhibition patterns seem consistent with a rapid-equilibrium ordered binding mechanism 4 with metal binding to calcineurin before substrate. Primary Kinetic Plots A characteristic of the rapid-equilibrium ordered kinetic mechanism is the intersection point in the double-reciprocal plot for the ligand binding second. For 4
Initial experiments were done with saturating Mn 21 (1.0 mM) and yielded inhibition plots more consistent with noncompetitive inhibition. These data supported ordered binding with metal first, although not for a rapid equilibrium model. Studies with subsaturating metal were done after the review of an earlier version of the manuscript. We acknowledge and thank the referees for comments resulting in an improved manuscript.
FIG. 4. Inhibition pattern for inorganic phosphate with Mn 21 as the variable ligand. Calcineurin (3 mg/ml, or 37.5 nM) was assayed with varying amounts of Mn 21 with different fixed concentrations of inorganic phosphate at pH 7.0. Phosphate was included at 0.0 mM (filled circles), 1.0 mM (filled triangles), and 2.0 mM (filled squares) concentrations. Each initial rate was the average of duplicate experiments with duplicate determinations within an experiment. The error bars are standard deviations of the estimated rates.
SUBSTITUTION INERT COMPLEXES AND CALCINEURIN
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binds directly to free enzyme preceding substrate binding as illustrated in Fig. 7. Also depicted in this figure is the random release of the products, P i and pNP, identified in an earlier study (38) of the product inhibition pattern. A corollary result of this mechanism is that no prior formation of a metal–substrate complex is required for calcineurin activity. Two types of complexes are consistent with these data: Metal may function as a bridge between enzyme and substrate (bridge structure) or all components directly interact with one another (symmetrical structure). Wong (55) has presented kinetic formulations for metal ion activators of enzymes and has developed a set of criteria (p. 67) for each of the possible mechanisms. The ordered binding order deduced from the data effectively precluded many of the possible mechanisms described by Wong, but do not distinguish between the bridged and symmetrical structures. Based on the observed binding of inorganic phosphate in the resolved crystal structure of calcineurin, it is likely that the symmetrical model is formed.
FIG. 5. Inhibition pattern for Co(en) 331 with pNPP as the variable ligand. Calcineurin (2 mg/ml, or 25 nM) was assayed with varying amounts of pNPP with different fixed concentrations of Co(en) 331 at pH 7.0 as described in the text. Co(en) 331 was included at 0.0 mM (filled circles), 15.0 mM (filled triangles), 35.0 mM (filled squares), and 50.0 mM (filled diamonds). Each initial rate was the average of three replicate experiments with duplicate determinations within the experiment. The error bars are standard deviations of the estimated rates and are shown in only one direction for clarity of the figure.
preclude effective interaction of pNPP. Such steric barriers are less likely with Mn 21 (really Mn(H 2O) 621). The effect is not because Co(en) 331 acted as a partial competitive inhibitor inasmuch as the slope replot was linear. The bulk of the evidence favors a steady-state ordered mechanism with Mn 21 being the preceding ligand. DISCUSSION
These data demonstrated that several metal complexes were able to activate calcineurin and provide insight into the interaction of exogenous metal with the enzyme. For each metal complex, all curves showed saturation kinetics (not shown). Additionally, no evidence was observed in these experiments to suggest inhibition by excess metal. Binding Scheme for Ligands From the kinetic inhibition patterns, the binding order of these ligands can be established. Free metal
FIG. 6. Kinetics of pNPP hydrolysis at different concentrations of Mn 21. Calcineurin (2 mg/ml, or 25 nM) was assayed with varying amounts of pNPP with different fixed concentrations of Mn 21 at pH 7.0 as described in the text. Mn 21 was included at 0.10 mM (filled triangles), 0.165 mM (open triangles), 0.25 mM (filled circles), and 0.50 mM (open circles). Shown is a representative experiment with duplicates for each initial rate. The error bars are standard errors.
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FIG. 7. Kinetic scheme for metal and substrate binding to calcineurin. Although calcineurin likely retains Mn 21 for a catalysis of another cycle, the figure shows the dissociation of metal to illustrate the recovery of free enzyme. Random release of the products was concluded in an earlier study (Ref. 38).
Activation by Co(NH 3 ) 631 31 3 6
Calcineurin was seemingly activated by Co(NH ) , consistent with metal functioning through an outer sphere mechanism. One caveat to this conclusion is concern about the integrity of the Co(NH 3) 631 compound, particularly any Co 21 (actually Co(H 2O) 621) contamination. The kinetic parameters for Co(NH 3) 631 were approximately 100-fold higher than found for Co 21 such that Co 21 present at 1% contamination level may be responsible for the observed activation of calcineurin with Co(NH 3) 631. There is no positive evidence for the presence of such contamination. Visible spectrophotometry (not shown) revealed no characteristic peaks for Co 21 in solutions of Co(NH 3) 631, although the spectrum may have been hidden by the native spectrum of Co(NH 3) 631. Concern about contaminating Co 21 also required that Co(NH 3) 631 not have any effect on calcineurin directly or on activation by Co 21. This seemed unlikely judging from the effects of Co(en) 331 which inhibited Mn 21 activation in the same concentration range used for Co(NH 3) 631. If Co(NH 3) 631 did inhibit, the level of contaminating Co 21 would need to be greater than 1% to measure a similar k cat value and then would have been unlikely to yield a similar value of K act. The observed effects were likely because of Co(NH 3) 631, not Co 21. The chemistry of Co(NH 3) 631 favors this conclusion. The standard isolation of Co(NH 3) 631 requires selective precipitation to separate the material from Co 21 and the 13 state of Co is stabilized by the substitution inert ammonia ligands. The exchange of ammonia ligand with solvent water occurs very slowly with a rate constant of 5.8 3 10 212 s 21 (51), providing a significant barrier to the accumulation of hydrated Co 31 as a perquisite for the reduction to Co 21. It was concluded that Co(NH 3) 631 did activate calcineurin.
Organization of Exogenous Metal in Calcineurin Catalysis Even though the kinetic parameters showed that Co(NH 3) 631 was a worse activator than Mn 21, the fact that it did cause activation provided insight into the role of exogenous metal in catalysis by calcineurin. As described by Cowan (53), activation by this metal complex is limited to a function as an outer sphere complex. Thus, calcineurin activity can be activated by Mn 21 and other metals via an outer coordination sphere interaction. This indicates that it is not obligatory for the aqua-metal to (a) directly coordinate to the phosphate ester of the substrate in the catalytic reaction or (b) activate a water molecule involved in a direct nucleophilic attack on the phosphate ester. Indeed, it seems likely that the metal ion does not function through either of these paths. Instead, the effect of metal is likely through a water molecule “bridge” between the metal and the phosphate ester linkage. A role of this type is consistent with data from studies of the heavy-atom isotope effects on the calcineurincatalyzed hydrolysis of pNPP (44). The chemical step of phosphoryl transfer was found to be partially ratelimiting for pNPP hydrolysis, unlike the situation for protein-tyrosine phosphatases and dual-specific phosphatases that exhibit fully rate-limiting chemistry with pNPP. Proton transfer to the leaving group was found to lag behind the cleavage reaction, resulting in some partial negative charge in the transition state. The activating metal ion Mn 21 caused a change in the commitment factor with the chemical step becoming more rate-limiting at higher concentrations of Mn 21. The degree to which the chemical step was rate-limiting together with effect of Mn 12 indicated that some nonchemical step (steps) after substrate binding, but before catalysis, was (were) affected. A proton transfer to the leaving group would fit these criteria.
SUBSTITUTION INERT COMPLEXES AND CALCINEURIN
A model using two metal ions has been proposed for calcineurin with one metal site serving to stabilize or enhance the leaving group through the donation of a proton from water coordinated to the metal ion (40). Subsequent studies have raised questions about the involvement of both intrinsic metals in catalysis (41). An outer sphere interaction would be sufficient to satisfy this function. For Mn 21 and Mg 21, water coordinated to these metals has pK as of 10.6 and 11.4, respectively. As noted earlier, the relative pH optimum of calcineurin activity was different whether Mn 21 (approximately pH 7.0) and Mg 21 (approximately pH 8.2) was used as the activator. There was a one pH unit difference mirroring the difference in pK a values of H 2O with these metals. This difference is consistent with the expected difference in the ability to donate a proton of these species, particularly in the stabilization of the leaving group. Stabilization of the leaving group by metal was found by Herschlag and Jencks (15) in the hydrolysis of pNPP by Mg 21 or Ca 21. It is notable that Mn 21 bound to calcineurin was found (56) to retain exchangeable water within its inner coordination sphere establishing the availability of a water ligand for proton donation, albeit through an outer sphere interaction. In a study of coordination of water to Mg 21, water could be found both as an inner and an outer coordination ligand (57), suggesting that Mg 21 could also function through this interaction. Possible Consequences of an Outer Sphere Mechanism in Phosphatase Catalysis Participation of a metal ion through an outer sphere coordination interaction may be the cause of the reduced k cat observed with calcineurin compared to other phosphatases. Protein tyrosine phosphatases (58) typically have values of k cat in the range 5– 60 s 21 ('10fold greater than that found for calcineurin), the purple acid phosphatase (59) in the range 200 – 400 s 21 ('100fold greater), alkaline phosphatase (60) in the range 10 –30 s 21 ('10-fold greater), and another protein serine protein phosphatases (PP2A, Ref. 61) in the range 40 –200 s 21 ('50-fold greater) for pNPP hydrolysis. Phosphotriesterase, a dinuclear containing enzyme, catalyzes the hydrolysis of model substrates with k cat in the range 50 –100 s 21 (62). All except protein tyrosine phosphatases are metalloenzymes and generally do not require the addition of exogenous metal. Furthermore, model studies (16 –18) have shown that chelation of metal can result in greater rate accelerations than those observed with free Mg 21 or Ca 21 (15) in solution. Of the noted enzymes, alkaline phosphatases are the most well characterized. It is a zinc metalloenzyme with the Zn 21 directly coordinating phosphate as an inner sphere ligand. This mechanism enables zinc to
175
polarize one of the phosphorous– oxygen bonds for nucleophilic attack (63). Utilization of an outer vs inner sphere ligand in an enzyme mechanism may limit the magnitude of the enzymatic rate that can be achieved. If true, the difference between calcineurin and alkaline phosphatase may provide the range for distinguishing the mechanistic role of metal. Consider protein phosphatase-1, which is a member of the same gene family as calcineurin. Protein phosphatase-1 does not effectively catalyze the hydrolysis of pNPP, and the resolved crystal structure of phosphatase-1 showed the presence of bound metal (3, 4). A recombinant form of phosphatase-1 was shown to be activated by metals and could incorporate metal to form stable enzyme– metal complexes (8). On the basis of on the structural information available and the low activity with pNPP, PP-1 may use an outer coordination sphere interaction in the hydrolysis of phosphate ester bonds. The possible involvement of metals via an outer sphere interaction may provide a constraint on the reaction evidence by the lower rate constants for PP-1 and calcineurin with pNPP. Firmer characterization of the metal requirement of protein phosphatase-1 may demonstrate whether additional features are shared by these two enzymes. SUMMARY
The activation of calcineurin by Co(NH 3) 631 was comparable to activation by transition metals, suggesting a common mechanism involving an outer sphere ligand interaction. The order of binding was concluded to be metal first, followed by the substrate, consistent with exogenous metal facilitating the binding of substrate and with a proposed function of providing stabilization of the departing leaving group through proton donation or outer sphere hydrogen bonding. Isotope studies of the calcineurin-catalyzed reaction have demonstrated that there is partial negative charge on the leaving group and provided evidence for some form of metal– phosphate interaction (44). Any proposed mechanism for calcineurin must define a role for exogenous metal. ACKNOWLEDGMENT This research was supported by funds from the University of Tennessee Medical Group.
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