©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Investigation of Monovalent Cation Activation of S-Adenosylmethionine Synthetase Using Mutagenesis and Uranyl Inhibition (*)

(Received for publication, November 2, 1994; and in revised form, April 24, 1995)

Michael S. McQueney (§) George D. Markham (¶)

From the Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

S-Adenosylmethionine (AdoMet) synthetase catalyzes the formation of AdoMet from ATP and L-methionine with subsequent hydrolysis of the bound tripolyphosphate intermediate. Maximal activity requires the presence of two divalent and one monovalent cation per active site. Recently, the x-ray structure of the Escherichia coli AdoMet synthetase was solved, and the positions of the two Mg binding sites were identified. Based on additional spherical electron density, the K binding site was postulated to be a nearby site where the uranyl heavy atom derivative also bound in the crystal. The side chain of glutamate 42 is within ligation distance of the metals. Mutagenesis of glutamate 42 to glutamine (E42QMetK) abolished monovalent cation activation and produced an enzyme that has kinetic properties virtually identical to those of K-free wild type AdoMet synthetase in both the overall AdoMet synthetase reaction and in the hydrolysis of tripolyphosphate. Thus, there is a 100-fold decrease in the V(max) for AdoMet synthesis and large increases in the K values for both substrates. In contrast there is only a 2-fold decrease in V(max) for tripolyphosphate hydrolysis. The uranyl ion, UO(2), is a competitive inhibitor with respect to K (K = 350 nM) and is the first ion to bind at this site and inhibit the enzyme. The UO(2) inhibition is reversible and tight-binding, and results from UO(2) and not UO(2)bulletATP. Analogous to K activation, UO(2) predominantly inhibits AdoMet formation rather than tripolyphosphate hydrolysis. The kinetic results indicate that UO(2) inhibition is likely to result from interference with productive ATP binding. UO(2) remains a tight-binding inhibitor of the E42Q mutant, which suggests that K and UO(2) have different ligation preferences when bound in the monovalent cation binding pocket. The results support the model that glutamate 42 provides ligands to the K and has a major role in monovalent cation binding.


INTRODUCTION

S-Adenosylmethionine synthetase (ATP:L-methionine S-adenosyltransferase or AdoMet (^1)synthetase) is one of many enzymes that are activated by monovalent metal cations(1) . However, the modes and structural basis for monovalent cation activation are not well understood for most of these enzymes. We have studied monovalent cation activation of AdoMet synthetase in an attempt to define the structural mechanisms involved in activation. S-Adenosylmethionine synthetase catalyzes the two sequential reactions shown in Fig. SI(2) . The first reaction, AdoMet formation and the second reaction, tripolyphosphate (PPP(i)) hydrolysis, occur at the same active site. Both steps require divalent metal ions such as Mg. Two divalent metal ions bind per subunit (active site) of the tetrameric enzyme from Escherichia coli and both are required for activity(3) . One monovalent cation, K or one of similar size, binds per active site and stimulates the AdoMet formation step up to 100-fold at low substrate concentrations, decreases the K of ATP (4-fold) and L-methionine (12-fold), but has relatively little effect (2-5-fold) on the rate of the PPP(i) hydrolysis reaction. The size of the monovalent cation dictates how well it binds to, and activates, AdoMet synthetase. Spectroscopic studies have shown all three metals bind close together in the active site(4) . Tl NMR studies of the enzyme Tl complex suggest that the monovalent metal activator does not coordinate directly to either substrates or products(5) . Kinetic isotope effects indicate that the monovalent cation does not alter the transition state structure, but does enhance the commitment to catalysis of bound ATP(6) . Based upon these results, monovalent cation activation has been proposed to result from subtle alterations in protein conformation(5) . The nature of these changes remains elusive, but details about the structure of the K binding site and how it could affect the protein or active site conformation are now starting to emerge, primarily as a result of the recently determined x-ray crystal structure of AdoMet synthetase.


Figure SI: Scheme IThe two sequential reactions catalyzed by AdoMet synthetase.



The crystal structure of E. coli metK-encoded AdoMet synthetase isozyme has been solved at 3-Å resolution by Takusagawa and co-workers. (^2)The location of the two divalent metal ion binding sites have been identified from differences in electron density maps of enzyme crystals grown in the presence of either Mg or Co. A third distinct metal ion binding site, 5 and 7 Å away from the two Mg sites, was noted as a site of other spherical high electron density. This was also the site where UO(2) bound and was postulated to be the K binding site. This site contains the side chain of glutamate 42 as a potential ligand to the metal ion. The only other residue close to the K (or uranyl) ion was serine 263. These residues are conserved in all 11 reported AdoMet synthetase sequences. Guided by the x-ray data, two sets of experiments were conducted to test the importance of glutamate 42 as a ligand to the activating K ion: 1) site-directed mutagenesis of this residue was performed to assess whether there was disruption of K activation, and 2) evaluation of whether UO(2) activates like K or inhibits competitively with respect to K.

We report here results that confirm an important role of glutamate 42 in monovalent cation activation. These studies, in combination with the crystallographic data, suggest that similar structural modes of potassium activation may exist between AdoMet synthetase and two other K-activated enzymes for which crystal structures have recently been published, dialkylglycine decarboxylase (7) and pyruvate kinase(8) .


EXPERIMENTAL PROCEDURES

Materials

AdoMet synthetase (wild type) was purified by published methods(2) . Uranium oxyacetate (uranyl acetate) dihydrate was purchased from NOAH and was of the highest available purity. L-[methyl-^14C]methionine (40-60 mCi/mmol, 16.9 mM) was purchased from DuPont NEN. ACES, Tris, EDTA, ATP, Na(5)P(3)O, 8-hydroxyquinoline, lysozyme, isopropyl-beta-D-thiogalactopyranoside, beta-mercaptoethanol, streptomycin sulfate, and S-adenosyl-L-methionine (chloride form), were purchased from Sigma. HEPES and MES were purchased from U. S. Biochemical Corp. All other reagents were the highest purity available. P-81 ion exchange paper used in enzyme assays was obtained from Whatman.

Mutagenesis

Site-directed mutagenesis was done using the Muta-Gene in vitro mutagenesis kit (Bio-Rad), employing T4 DNA ligase, T4 DNA polymerase, and polynucleotide kinase from New England Biolabs. Mutagenesis was performed on the plasmid pT7-6(metK), which consists of the metK gene (9) inserted between the PstI and EcoRI sites of pT7-6. (^3)The oligonucleotide (antisense strand) used in mutagenesis was as follows: 5`-GCG CAA CGA ACG *GTT TGG ATG CAT TTT TC-3`, where the asterisk is to the left of the mutated base. Plasmids were purified by either Rapid Pure Miniprep (RPM-60, Bio101 Inc.) or QIAGEN Plasmid Prep. (QIAGEN, Inc.). Following mutagenesis the plasmid was transformed into E. coli strain DM22 (9) for propagation. The DNA sequence of the mutated gene was verified by DNA sequencing using the Sequenase kit (U. S. Biochemical Corp.). The mutagenized plasmid pT7-6(metKE42Q) was ultimately transformed by electroporation into strain RSR15(DE3), an E. coli B strain for which extracts have no detectable AdoMet synthetase activity due to a chromosomal mutation.^3 A mutant with a lysine at position 42 was prepared in an analogous fashion, yielding pT7-6(metKE42K).

Purification of E42QMetK

E. coli strain RSR15(DE3) containing the mutagenized plasmid was grown in LB medium containing ampicillin (100 µg/ml) to an absorbance at 550 nm of 1.0, at which time 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) was added. RSR15(DE3)pT7-6(metKE42Q) cells were harvested after 4 h and frozen at -80 °C. Cells were suspended in 0.1 M TrisbulletHCl, 1 mM EDTA, 0.1% beta-mercaptoethanol, pH 8, and lysed by addition of lysozyme (1.0 mg/g cells) followed by sonication. The protein purification followed previously described methods(2) . The cell free homogenate was treated with streptomycin sulfate, followed by ammonium sulfate fractionation. E42QMetK-AdoMet synthetase was purified by chromatography on phenyl-Sepharose (Sigma), hydroxylapatite (Bio-Rad), and aminohexyl-Sepharose-4B (Pharmacia). Protein purity and native structure were determined by denaturing and non-denaturing gel electrophoresis. The protein concentration was determined by absorbance at 280 nm (an A of 1.3 represents a 1.0 mg/ml solution of the enzyme). In analogous trials, the E42KMetK mutant did not appreciably accumulate after IPTG induction, apparently as a result of proteolysis. Thus E42KMetK-AdoMet synthetase was not further characterized.

AdoMet Synthesis Assay

The overall AdoMet synthetase activity was determined by methods described previously (2) with some modifications. The reaction mixture (50 µl) contained 50 mM HEPESbulletN(CH(3))(4)OH, pH 8.0, 20 mM MgCl(2), 0.1 mM ATP, L-[methyl-^14C]methionine (30-60 mCi/mmol, 0.1 mM), and where indicated 50 mM KCl. The reactions were terminated by the addition of 150 µl of 25 mM EDTA, and 150 µl of the reaction was spotted on a two-centimeter disk of P-81 filter paper (Whatman). After air drying, a group of disks were placed on a Buchner funnel and washed with 4 liters of distilled water; the retained radioactivity was quantified by liquid scintillation counting in EcoScint scintillation mixture using a Beckman LS2800 liquid scintillation counter. The effects of uranyl ion on AdoMet formation activity were determined under identical conditions, with the exception that the buffer was changed from HEPESbulletN(CH(3))(4)OH, pH 8.0, to 100 mM ACESbulletN(CH(3))(4)OH, pH 6.2, and the reactions were terminated by the addition of 150 µl of 25 mM EDTA, 100 mM TrisbulletHCl, pH 8.0. Substrate saturation data were computer-fitted using the programs of Cleland(10) . Other data were analyzed by least squares fitting in a variety of computer programs.

Tripolyphosphate Hydrolysis Assay

The tripolyphosphate hydrolysis activity was determined as described previously(2) . Reactions (50 µl) consisted of 50 mM HEPESbulletN(CH(3))(4)OH, pH 8.0, 20 mM MgCl(2), 0.1 mM Na(5)P(3)O, and, where indicated, 0.1 mM AdoMet and 10 mM KCl. Phosphate formed as product was quantified by a molybdate-malachite green assay(11) . Tripolyphosphatase activity in the uranyl inhibition studies was determined by the same method with the exception that the buffer was changed from HEPESbulletN(CH(3))(4)OH to 100 mM ACESbulletN(CH(3))(4)OH, pH 6.2, and the reactions were terminated by the addition of 150 µl of 25 mM EDTA, 100 mM TrisbulletHCl, pH 8.0.

Stability Constants

Stability constants for UO(2)bulletATP, UO(2)bullet PPP(i), MgbulletATP, and MgbulletPPP(i) at pH 6.2 were measured by electron paramagnetic resonance (EPR) techniques (12, 13) using a Varian E-109 spectrometer. The stability constants for MnbulletATP and MnbulletPPP(i) at pH 6.2 were determined by the addition of ATP or PPP(i) to a solution of 0.1 mM MnCl(2) in 100 mM ACESbulletN(CH(3))(4)OH, pH 6.2, which resulted in a decrease in the EPR signal of free Mn(13) . The dissociation constant was determined from the concentration of ATP or PPP(i) that decreased the signal to half its total intensity, giving K values of 92 and 57 µM, respectively. The MnbulletATP and MnbulletPPP(i) complexes (0.1 mM MnCl(2), and 0.1 mM PPP(i) or 0.4 mM ATP) were then titrated with a solution of UO(2)(Ac)(2), which led to the release of Mn and an increase in the EPR signal. Values for K for uranyl complexes were determined from the concentration of uranyl acetate, which led to recovery of half of the total EPR signal; the actual K value was calculated from the relationship, K = K/(1 + A/K), where A is the concentration of the competing metal (Mn), and K is the dissociation constant for the (MnbulletATP or MnbulletPPP(i)) complex (12) . Analogous experiments were performed to obtain the K for MgbulletATP (0.30 mM) and MgbulletPPP(i) (47 µM) under these conditions.

The stability constants of UO(2)bulletATP and UO(2)bulletPPP(i) were also determined colorimetrically using 8-hydroxyquinoline as a metallochromic dye source(12) . A solution of 0.4 mM 8-hydroxyquinoline in ACESbulletN(CH(3))(4)OH pH 6.2, was titrated with a solution of UO(2)(Ac)(2) (0-60 µM final concentrations), which resulted in an increase in absorbance at 380 nm. This result was then compared to identical titrations of mixtures of 8-hydroxyquinoline and either 0.1 mM ATP or 0.1 mM PPP(i). The K values were calculated using: K = [UO(2)][ligand]/[UO(2)bulletligand], where ligand is ATP or PPP(i)(12) . Kvalues of 0.2 mM for UO(2)bulletATP and 0.07 mM UO(2)bulletPPP(i) were obtained.

Fluorescence Spectra of UO(2) Complexes with ATP or PPP(i)

Solutions contained 100 mM ACESbulletN(CH(3))(4), pH 6.2 and 1 mM UO(2)(Ac)(2). The spectra for UO(2)bulletATP and UO(2)bulletPPP(i) were obtained by the addition of either 2 mM ATP or 2 mM PPP(i). The solutions were excited by 422 nm light with a 5-nm excitation slit width; the fluorescence emission spectrum was detected with a 5-nm slit width. A Perkin-Elmer LS-50 luminescence spectrometer was used. The final spectra are the average of three scans.


RESULTS

Mutagenesis of Glutamate 42

The crystal structure of AdoMet synthetase implicated glutamate 42 as a ligand to the K activator. Glutamate 42 is the only charged ligand in the vicinity of the K binding pocket. Thus the conservative mutation of glutamate 42 to glutamine (E42QMetK) might disrupt monovalent cation binding and activation by removing the negative charge. The E42QMetK mutant was therefore prepared and characterized. During column chromatography, E42QMetK behaved indistinguishably from wild type AdoMet synthetase. Pure E42QMetK is indistinguishable from wild type AdoMet synthetase by electrophoresis under denaturing and non-denaturing conditions. However, the specific activity of the mutant is only 1% that of the K-activated wild type enzyme.

The mutant with lysine at position 42, E42KMetK, was also constructed in order to determine whether the change in side-chain charge might result in an enzyme that was fully active in the absence of a monovalent cation. However, the E42KMetK mutant protein did not accumulate in vivo, suggesting misfolding and proteolytic degradation. Thus the E42KMetK mutant was not amenable to characterization.

Kinetic Analysis of E42QMetK

Initially K activation of E42QMetK was tested. Monovalent cation activation of wild type AdoMet synthetase is most pronounced at low substrate concentrations (K values, 0.1 mM for ATP, 0.1 mM for L-methionine). The effect of KCl concentration on the activity of wild type versus E42QMetK is shown in Fig. 1. Unlike wild type AdoMet synthetase, the activity of E42QMetK is not stimulated by KCl. The comparison of steady state kinetic parameters for wild type and E42QMetK is shown in Table 1; the results indicate that E42QMetK behaves nearly identically to K-free wild type AdoMet synthetase in terms of K and V(max) values as well as relative activities. Furthermore, the K for Mg is not detrimentally affected by the mutation, consistent with the crystallographic data, which indicate that glutamate 42 is not part of the Mg binding site. The results suggest that glutamate 42 is essential for monovalent cation activation of AdoMet synthetase, presumably by ligating K. The E42QMetK mutant was also not activated by 0.1 M LiCl, NH(4)Cl, NaCl, or CsCl, in contrast to the wild type enzyme. Unfortunately there is no direct method for measuring K binding to AdoMet synthetase, so it is not clear whether the mutation affects binding as well as activation.


Figure 1: KCl activation of the rate of AdoMet formation by wild type MetK and E42QMetK. Reactions were carried out at 22 °C and contained TrisbulletHCl (100 mM), pH 8.0. The ATP, L-methionine, and MgCl(2) concentrations were 0.1, 0.1, and 20 mM, respectively.





Uranyl Inhibition of AdoMet Synthetase

Since UO(2) binds to glutamate 42 in crystals, we investigated the effect of UO(2) on wild type AdoMet synthetase activity. If glutamate 42 is a ligand to K, then UO(2) would be expected to either activate analogously to K or competitively inhibit. No ions had been found to be competitive inhibitors with respect to K, i.e. all monovalent cations tested either activate or apparently do not bind(2) .

Uranyl Is a Reversible Tight-binding Inhibitor of AdoMet Synthetase

Experiments with uranyl acetate were carried out at pH 6.2 since at higher pH polynuclear UO(2) species and ``uranyl and amorphous hydroxide'' precipitates form(14, 15) . Uranyl acetate was soluble up to pH 6.5 in the normal AdoMet synthetase assay buffer, therefore to diminish problems associated with the chemical instability of UO(2), experiments were performed at pH 6.2. The kinetic constants for AdoMet synthetase at pH 6.2 are comparable to those at pH 8.0 (see below). The uranyl cation was found to be a potent inhibitor of AdoMet synthetase. Uranyl acetate at 25 µM fully inhibits K-free AdoMet synthetase in the presence of K concentrations of substrates. The inhibition was eliminated by the addition of 10 mM KCl but was unaffected by the addition of up to 100 mM MgCl(2), consistent with uranyl binding at the monovalent cation binding site. The relatively small amount of UO(2) required to inhibit the enzyme suggested tight-binding inhibition, which needed to be further characterized before steady state kinetic methods could be used to determine if UO(2) is a competitive inhibitor with respect to K(16) . Two conditions must hold true for complete steady state kinetic analysis of tight-binding inhibitors; the inhibition must be reversible, and the onset of inhibition must be fast with respect to the assay used(16) . Preferably, the inhibitor concentration used should be at least 10-fold higher than the enzyme concentration to avoid complications associated with depletion of free inhibitor.

We first examined whether the UO(2) inhibition was reversible. Irreversible inhibition was a particular concern since uranyl is photo-reactive (17, 36, 37, 38, 39) and could produce radical species in the active site resulting in irreversible modification(18, 40) . For AdoMet synthetase the uranyl inhibition was fully reversed by dilution into a solution containing saturating amounts of potassium. This was shown by an experiment in which 10 µM enzyme was incubated for 30 min with 50 µM UO(2)(Ac)(2) and substrates (0.1 mM each) in a potassium-free buffer and then the activity determined; the enzyme had 20% of the activity of an UO(2)-free control. Parallel uranyl-containing reaction mixtures were incubated for 30 min and then diluted 10-fold into buffer containing 50 mM KCl, and further incubated for 30 min prior to determination of the enzymatic activity. The enzyme from the potassium-containing reaction mixture had 92% of the activity of a uranyl free uninhibited control, thus demonstrating that uranyl inhibition was reversible.

Many tight-binding inhibitors are also slow-binding inhibitors (19) and require specialized kinetic analysis. Slow onset of inhibition is only relevant to the kinetic analysis if it is slow enough that the reaction time courses do not reflect the true steady state rates. The possibility of slow binding was evaluated by following the product formation from partially inhibited enzyme for 10 min, followed by addition of an additional 20 µM UO(2) (Fig. 2). There was no observable lag in the onset of additional inhibition (Fig. 2)(19) . Since the uranyl inhibition was both reversible and had a rapid onset, conventional steady state kinetic methods were used to determine the inhibition patterns with various substrates.


Figure 2: Time course of the onset of uranyl inhibition. Solutions contained 100 mM ACESbulletN(CH(3))(4)OH pH 6.2, 20 mM MgCl(2), 0.1 mM ATP, 0.1 mML-methionine, 1 µM AdoMet synthetase active sites, and 10 µM UO(2)(Ac)(2), for the first 15 min. After 15 min, additional UO(2)(Ac)(2) was added to a final concentration of 30 µM. The reaction was carried out at 22 °C. The lines are the best fits to the data from 0 to 15 min and 15 to 25 min.



Characterization of Uranyl Inhibition by Steady State Kinetics

Double reciprocal plots were used to reveal the type of inhibition with respect to K, ATP, and methionine. Initially, kinetic constants for AdoMet synthetase at pH 6.2 (100 mM ACESbulletN(CH(3))(4)OH, 20 mM MgCl(2), and 50 mM KCl) were determined to examine whether the enzyme activity was altered at this lower pH. Compared to the standard assay conditions at pH 8.0, the K values for ATP (0.11 ± 0.01) and L-methionine (0.10 ± 0.01 mM) are essentially unchanged, and the V(max) is reduced only 2-fold.

Plots of 1/Vversus 1/KCl at various concentrations of UO(2) intersect on the 1/V axis and thus show that uranyl is a competitive inhibitor with respect to potassium (Fig. 3a). A true inhibition constant could not be extracted from a replot of slope versus inhibitor concentration because the replot is not linear (Fig. 3a, inset), which was expected since the concentration of inhibitory species is comparable to the enzyme concentration (see below). An alternative method presented below was used to determine the inhibition constant. Nevertheless, these results demonstrate that UO(2) is a competitive inhibitor with respect to potassium, therefore confirming that both UO(2) and K bind at the same site. The double-reciprocal plot of initial velocity versusL-methionine concentration at four different levels of UO(2) showed a noncompetitive pattern with intersection on the abscissa (data not shown).


Figure 3: Double-reciprocal plots of uranyl inhibition versus KCl (a) and ATP (b). Solutions contained 100 mM ACESbulletN(CH(3))(4)OH, pH 6.2, 20 mM MgCl(2), and, for a, 0.1 mM ATP, 0.1 mML-methionine, 1 µM AdoMet synthetase active sites, and 0, 4, 6, 8, and 10 µM UO(2)(Ac)(2). The reactions for b contained 100 mM ACESbulletN(CH(3))(4)OH, pH 6.2, 20 mM MgCl(2), 0.1 mML-methionine, 1 µM AdoMet synthetase active sites and 0, 10, 15, and 20 µM UO(2)(Ac)(2). All reactions were carried out at 22 °C.



The plot of 1/Vversus 1/ATP at various UO(2) concentrations should reveal whether free UO(2) or UO(2)bulletATP is the predominant inhibitory species. Lanthanide metal ions, which inhibit of some nucleotide-utilizing enzymes (e.g. yeast phosphoglycerate kinase (20) and creatine kinase(16) ) are not inhibitory as the free ion but are inhibitory as a Lnbulletnucleotide complex. It is also known that UO(2) binds to ATP(24) . If UO(2)bulletATP is the inhibitory species one would expect to see a set of downward curved lines intersecting at the 1/V axis, as was observed in studies of EubulletATP inhibition of yeast phosphoglycerate kinase (20) and creatine kinase(16) . The downward curvature results from increased concentrations of the inhibitory EubulletATP species as the total concentrations of both ATP and Eu increase. For AdoMet synthetase the plot of 1/Vversus 1/ATP at fixed concentrations of UO(2) shows the opposite trend (Fig. 3b), with a set of upward curved lines intersecting at the 1/V axis, demonstrating competitive inhibition. Reasoning analogous to the case of EubulletATP suggests that UO(2)bulletATP is not the inhibitor, since at higher concentrations of both ATP and UO(2) less inhibition is observed than expected from the low concentration data. Rather, it is likely that ATP competes with the enzyme for UO(2).

Characterization of Tight-binding Inhibition

Inhibition constants for tight-binding inhibitors are typically obtained by methods outlined by Williams and Morrison(16) . The methods involve measuring initial velocity as a function of enzyme active site concentration at fixed concentrations of inhibitor and substrates. If the inhibitor is a tight-binding inhibitor such plots have upward curved lines, whereas for conventional inhibitors the plots are linear. For AdoMet synthetase the initial velocity was measured as a function of uranyl concentration, and a non-linear relationship was found (Fig. 4). When the data are fit to of Williams and Morrison, values for K and the purity of the enzyme can be obtained(16) .


Figure 4: Velocity of AdoMet formation as a function of AdoMet synthetase concentration in the presence of uranyl acetate. Solutions contained 100 mM ACESbulletN(CH(3))(4)OH, pH 6.2, 20 mM MgCl(2), 0.1 mM ATP, 0.1 mML-methionine, in the presence or absence of 20 µM UO(2)(Ac)(2) (by mass, 3.5 µM inhibitory species as determined using ). The line with the bullet is the best fit to the data in the presence of uranyl acetate using , K = 510 nM. The reflect data obtained in the absence of uranyl acetate. Reactions were carried out at 22 °C.



In (and , see below), R represents the reciprocal of the steady state velocity in the absence of inhibitor, S is the apparent inhibition constant, I is the inhibitor concentration, E is the concentration of total protein, and alpha is the fraction of total protein that can react with inhibitor.

When the data were fit with , the best fit resulted in a value of 10 for alpha, suggesting there was 10 times more active enzyme in the reaction than had been introduced. However, an alternative interpretation is a decrease in inhibitor concentration, which is reasonable for UO(2) due to the multiple forms present in solution(16, 18, 40) . can be rewritten so that alpha reflects the fraction of total inhibitor that can react with a known quantity of enzyme, yielding .

The nonlinear least squares fit from an experiment in which enzyme was varied at constant uranyl concentration of the data to is shown in Fig. 4. The fit gave a value for the apparent inhibition constant of 510 nM (± 60 nM) with an R^2 value of 0.99. The true inhibition constant of 350 nM can be calculated using , the observation that UO(2) binds to ATP, and the measured stability constant for UO(2)bulletATP of 0.22 mM (discussed below). The calculated concentration of inhibitory uranyl, alphaI, was 3.5 µM (18 ± 4% of the total). The difference in total (20 µM) versus inhibitory (3.5 µM) UO(2) may be attributed to the multitude of uranyl complexes present, e.g. the slowly interconverting polymeric hydrated uranyl species, [(UO(2))(2)(OH)] and [(UO(2))(3)(OH)(5)], and chelation of uranyl to ATP. As noted earlier, UO(2) is stable only at low pH, and polynuclear UO(2) species as well as ``UO(2) hydroxide'' form and precipitate at the higher pH(14, 15, 40) . O NMR and Raman spectroscopic studies have demonstrated the existence of the polymeric hydrated uranyl species [(UO(2))(2)(OH)] and [(UO(2))(3)(OH)(5)] and that they can represent the majority of uranyl in aqueous solution at pH values greater than 3.5(21, 22) . Based upon the observation that a single uranyl ion binds to glutamate 42 in the active site of the crystalline enzyme, we deduce that the free UO(2), and not one of the polymeric species, is the actual inhibitor. Due to the large number of interconverting complexes, it was not feasible to decipher the complete distribution of the uranyl species.

Characterization of UO(2)2+-ATP and UO(2)2+-PPP(i)Interactions

To determine the affinity of ATP for uranyl under our reaction conditions, we carried out fluorescence studies. Uranyl is known to be chelated by polyphosphates(24) . The fluorescence emission spectrum of the uranyl cation is extremely sensitive to the type of ligation(17, 23, 25, 36, 37, 38, 39) . Titration of 1 mM UO(2) with PPP(i) revealed formation of a 1:1 complex having a three-peak emission spectrum (Fig. 5). In contrast, titration of UO(2) with ATP produced a complex of UO(2)bulletATP that has a single emission peak at 490 nm that is clearly different than the emission spectrum of the UO(2)bulletPPP(i) complex. The difference in the fluorescence emission patterns of UO(2)bulletATP and UO(2)bulletPPP(i) suggest that the structures of the complexes are different. Feldman et al. reported that the UO(2)bulletATP complex involves chelation of UO(2) by the N(3) atom of the adenine ring, the ribose 04` oxygen, and the negatively charged oxygen of the alpha-phosphoryl group(24) . Based upon their proposal that the linear triatomic structure of UO(2) prevents tridentate ligation by the polyphosphate chain (24) and the fact that the UO(2)bulletPP(i) fluorescence emission spectrum is very similar to that of UO(2)bulletPPP(i) (data not shown), it appears that the 1:1 UO(2)bulletPPP(i) complex involves bidentate ligation. These results indicate that UO(2) complexes with ATP and PPP(i) do form and have stability constants which are less than 1 mM.


Figure 5: Fluorescence emission spectra of uranyl cation, free and in the presence of ATP or tripolyphosphate. Solutions contained 1 mM UO(2)(Ac)(2), 100 mM ACESbulletN(CH(3))(4), pH 6.2, and where present, 2.0 mM ATP or 2.0 mM PPP(i).



The stability constants of UO(2)bulletATP and UO(2)bulletPPP(i) were determined by EPR and colorimetric techniques (12, 13) (see ``Experimental Procedures''). The EPR technique, using Mn as a probe, involved titration of Mn-chelated ATP (K = 0.092 mM) and Mn-chelated PPP(i) (K = 0.057 mM) complexes with UO(2). Uranyl binding to ATP or PPP(i) caused the release of Mn from the ATP or PPP(i) complexes and an increase in the EPR signal due to free Mn. The apparent K were 0.22 mM for UO(2)bulletATP and 0.047 mM for UO(2)bulletPPP(i). The stability constants of UO(2)bulletATP and UO(2)bulletPPP(i) were also determined by UV spectroscopic titration of a UO(2)bullet8-hydroxyquinoline (12) complex with the polyphosphates; these experiments gave stability constants of 0.20 mM for UO(2)bulletATP and 0.07 mM for UO(2)bulletPPP(i), which agree well with the values obtained by EPR. The stability constant of UO(2)bulletATP confirms that ATP does chelate UO(2) under the conditions of our kinetic studies and thus ATP decreases the concentration of free uranyl species in solution.

UO(2)2+ Inhibits AdoMet Formation and Not PPP(i)Hydrolysis

The monovalent cation activator exerts its stimulatory effect predominantly on the AdoMet formation step and not the subsequent PPP(i) hydrolysis step. If UO(2) is a monovalent cation antagonist, it might inhibit only the AdoMet formation step and not PPP(i) hydrolysis. Consistent with the role of UO(2) as a K antagonist, concentrations of uranyl that completely inhibit the overall reaction had little effect on the rate of PPP(i) hydrolysis (Table 2). The lack of inhibition of the tripolyphosphatase reaction cannot be attributed to a UO(2) sequestering effect of PPP(i) based upon the stability constants of UO(2)bulletPPP(i) and MgbulletPPP(i) complexes. UO(2)bulletPPP(i) and MgbulletPPP(i) have indistinguishable dissociation constants of 47 µM (as determined by EPR techniques). Therefore, under the assay conditions, 20 mM MgCl(2), and 0.050 mM uranyl acetate, very little of the uranyl will be sequestered in the form of UO(2)bulletPPP(i). From these results we conclude that uranyl inhibition, analogous to K activation, exerts its effect predominantly on the first half-reaction.



UO(2)Inhibits Binding of the Adenosyl Moiety

Guided by the evidence that UO(2) binds tightly and reversibly at the potassium site, uranyl inhibition was used to probe the role of potassium in activation of AdoMet formation. The effect of UO(2) on nucleotide binding to AdoMet synthetase was assessed by determining whether the hydrolysis of the ATP analog A(S)TP is inhibited by UO(2). A(S)TP is an ATP analog in which the linkage between C5` and the alpha phosphoryl group contains sulfur rather than oxygen(2, 26) . A(S)TP binds to AdoMet synthetase and is hydrolyzed to yield A(S)DP and P(i) at a rate comparable to the maximal rate of tripolyphosphate hydrolysis, 1000-fold faster than ATP hydrolysis(2) . However, A(S)TP does not react with L-methionine to form AdoMet. Apparently when A(S)TP is bound to AdoMet synthetase, it is positioned in such a way that the -phosphoryl group is poised for hydrolysis, perhaps due to the longer C-S-P bonds. If UO(2) only inhibits the AdoMet-forming step, then A(S)TP may still bind and be hydrolyzed by UO(2)-inhibited AdoMet synthetase, whereas if UO(2) prevents productive ATP binding, then A(S)TP would not be hydrolyzed by UO(2)-inhibited AdoMet synthetase. The results summarized in Table 2indicate that A(S)TP hydrolysis is significantly inhibited by UO(2), which suggests UO(2) inhibits productive binding of A(S)TP and ATP. Since increasing concentrations of A(S)TP will overcome the inhibition by removing uranyl from the enzyme, as does ATP, it is not possible to determine whether uranyl prevents A(S)TP binding by steady state kinetic methods.

UO(2)Inhibition of E42QMetK

UO(2) was found to be also a tight-binding inhibitor of the mutant E42QMetK. The K was determined by plotting velocity versus enzyme concentration at fixed concentrations of substrates and inhibitor, as described previously for wild type AdoMet synthetase. The fit of the data to yielded a value for K = 0.38 ± 0.06 µM, which is comparable to the value for wild type; similar to the case with the wild type, enzyme only 15% of the total uranyl was inhibitory. However, unlike wild type enzyme the UO(2) inhibition of E42QMetK could not be reversed by KCl (up to 100 mM). These results may indicate that the amide ligand in E42QMetK is an adequate surrogate for the carboxylate ligand for UO(2) but not for K. An amide ligand (glutamine 11) to UO(2) was observed in the crystal structure of cytochrome b(5)(27, 41) . A carboxylate group appears to be a common constituent of K binding sites, since it has been observed in all three known structures of K binding sites in enzymes^2(7, 8) .


DISCUSSION

The construction of the E42QMetK mutant was motivated by the observation that the UO(2) binding site in the x-ray structure of AdoMet synthetase was likely to be the K binding site. Since glutamate 42 was the only negatively charged residue at this site, the objective was to selectively disrupt the binding site by altering the negatively charged glutamate to the neutral, and nearly isosteric, glutamine residue. Analysis of E42QMetK revealed a mutant enzyme which maintained the structural integrity of the wild type AdoMet synthetase, yet displayed kinetic constants and activity that were nearly identical to that of wild type enzyme deprived of a monovalent cation activator. These results indicated that glutamate 42 is important for monovalent cation activation.

Uranyl was found to be a potent inhibitor of AdoMet synthetase and a competitive inhibitor with respect to K. Additionally, unlike lanthanide ions, which often form inhibitory complexes with ATP, free UO(2) rather than UO(2)bulletATP is the predominant inhibitory species. UO(2) could thus be designated as a K antagonist. In a fashion similar to the activation by K, UO(2) inhibition predominantly affected the AdoMet formation step and not the PPP(i) hydrolysis reaction. UO(2) inhibits hydrolysis of the ATP analog A(S)TP, which reinforces the notion that the K site is involved in productive binding of ATP. All of the results are consistent with glutamate 42 being a ligand to K and essential for monovalent cation activation.

Although glutamate 42 is necessary for K activation, the fact that UO(2) inhibits E42QMetK shows that glutamate 42 is not essential for UO(2) binding. UO(2) binds to amines, amides, and ketones, as well as carboxylic acids(28) . UO(2) binds 10^4-fold tighter than K to the wild type enzyme and may be able to reorder the metal binding pocket in the E42Q mutant, whereas K cannot. Similarly, Falke and co-workers (29) have found mutations in the Ca binding site of the E.coli galactose-binding protein that cause up to a four order magnitude decrease in the affinity for Ca without detrimental effects on the affinity for certain trivalent lanthanide ions. Alternatively, a different ligation geometry may be induced upon UO(2) binding in the monovalent cation site. This postulate is supported by the fact that the central uranium ion of UO(2) is significantly smaller than K (cation radius of U(VI) = 0.87 Å versus K(I) = 1.52 Å) and uranyl binds more tightly than K. Enzymes are known to minimize vacant space by changing ligation patterns when binding a smaller metal(7, 30) . The perturbed structure may in fact give rise to the inhibition of the wild type enzyme. This proposal is analogous to the mechanism of Na deactivation of K activated dialkylglycine decarboxylase(7) . The smaller Na ion induces a different ligation pattern when it binds to dialkylglycine decarboxylase, which in turn translates into adverse alterations in the structure of the active site. The fact that K is an activator of AdoMet synthetase and UO(2) binds at the same site and completely inhibits seems to indicate that UO(2) locks the enzyme in a unproductive conformation, while K promotes a productive conformation. The absence of a monovalent cation may leave the enzyme free to interconvert between the two states.

Why uranyl, a divalent cation, binds tightly at the K binding site as opposed to the Mg site warrants some consideration. A literature survey summarized in Table 3indicates the U(VI) ion is comparable in size to Mg (0.86 Å). However, the average metal-to-oxygen (MbulletbulletO) ligand bond distance of uranyl (2.48 Å) is much larger than that of magnesium (2.04 Å), which may explain why the uranyl cation prefers to bind at the K rather than the Mg site of AdoMet synthetase. Furthermore, like K, UO(2) may have more than six ligands. UO(2) binds 10^4-fold tighter than K, probably due to the additional positive charge. M metal ions have been shown to bind at divalent metal binding sites in enzymes typically with binding constants 10^3-fold tighter than their M counterparts (31) . The tight-binding inhibition by UO(2) is a novel observation, which may be useful in future studies. The uranyl cation is spectroscopically versatile having highly characteristic UV and fluorescence spectra (23, 24) and is photo-reactive, generating free radicals that may cleave the peptide backbone(17, 18, 36, 37, 38, 39, 40) . Combined with the possibility of very tight-binding, uranyl may be useful as a probe to study other K-activated proteins.



Finally, it is useful to make some comparisons between the K binding site of AdoMet synthetase and those in the two other known structures of K-activated enzymes, pyruvate kinase and dialkylglycine decarboxylase(7, 8) . In all three enzymes, the K binding site is close to or in the active site, but K does not interact directly with any substrates or products. The binding pocket contains one carboxylate residue. The structure of the K binding pocket in dialkylglycine decarboxylase depends on the size of the bound monovalent cation. Changes in the K binding pocket structure translate to changes in the structure of the active site, thereby affecting activity (7) . The K binding sites of pyruvate kinase (8) and AdoMet synthetase^2 reveal an even more notable similarity. The carboxylate ligand to the K in each case is also hydrogen-bonded to an arginine residue, which in pyruvate kinase forms a hydrogen bond to the P of ATP. Therefore, in both pyruvate kinase and AdoMet synthetase, the K may be linked to the ATP substrate via an intervening carboxylate-arginine amino acid bridge. As Reed and co-workers pointed out, the carboxylate-arginine bridge provides a path by which cation size-dependent changes in the monovalent cation binding pocket can be readily translated to changes in the active site structure and activity. Whether this turns out to be a common motif in the structural basis for monovalent cation activation awaits further investigation.


FOOTNOTES

*
This work was supported by the National Institutes of Health Grants GM-15790 (to M. S. M.), GM-31186 (to G. D. M.), and CA-06927 and by an appropriation from the Commonwealth of Pennsylvania. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Dept. of Protein Biochemistry, SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd., King of Prussia, PA 19406-0939.

To whom correspondence should be addressed: Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111. Tel.: 215-728-2439; Fax: 215-728-3574.

^1
The abbreviations used are: AdoMet, S-adenosyl-L-methionine; ACES, 2-[(2-amino-2-oxoethyl)amino]ethanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; PPP(i), tripolyphosphate; PP(i), pyrophosphate; P(i), inorganic phosphate; IPTG, isopropyl-beta-D-thiogalactopyranoside; UO(2)(Ac)(2), uranyl acetate; A(S)TP, 5`-mercapto-5`-deoxy-ATP.

^2
S. Kamitori, G. D. Markham, and F. Takusagawa, submitted for publication.

^3
The preparation of pT7-6(metK) and RSR15(DE3) are described elsewhere (Reczkowski, R. S., and Markham, G. D., J. Biol. Chem., in press.


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