Structure-function relationships in methionine adenosyltransferases.

Methionine adenosyltransferases (MATs) are the family of enzymes that synthesize the main biological methyl donor, S-adenosylmethionine. The high sequence conservation among catalytic subunits from bacteria and eukarya preserves key residues that control activity and oligomerization, which is reflected in the protein structure. However, structural differences among complexes with substrates and products have led to proposals of several reaction mechanisms. In parallel, folding studies begin to explain how the three intertwined domains of the catalytic subunit are produced, and to highlight the importance of certain intermediates in attaining the active final conformation. This review analyzes the available structural data and proposes a consensus interpretation that facilitates an understanding of the pathological problems derived from impairment of MAT function. In addition, new research opportunities directed toward clarification of aspects that remain obscure are also identified.

Antigenic conservation of primary structural regions of S-adenosylmethionine synthetase.

Although the physical and kinetic properties of S-adenosylmethionine (AdoMet) synthetases from different sources are quite different, it appears that these enzymes have structurally or antigenically conserved regions as demonstrated by studies with AdoMet synthetase specific antibodies. Polyclonal anti-human lymphocyte AdoMet synthetase crossreacted with enzyme from rat liver (beta isozyme), Escherichia coli and yeast. In addition, polyclonal anti-E. coli enzyme and antibodies to synthetic peptides copying several regions of the yeast enzyme reacted with the human gamma and rat beta isozymes. Antibodies to yeast SAM1 encoded protein residues 6-21, 87-113 and 87-124 inhibited the activity of human lymphocyte AdoMet synthetase, while antibodies to residues 272-287 had no effect on the enzyme activity. Our results suggest that these conserved regions may be important in enzyme activity.

Spatial proximity and sequence localization of the reactive sulfhydryls of porphobilinogen synthase.

The zinc metalloenzyme porphobilinogen synthase (PBGS) contains several functionally important, but previously unidentified, reactive sulfhydryl groups. The enzyme has been modified with the reversible sulfhydryl-specific nitroxide spin label derivative of methyl methanethiosulfonate (MMTS), (1-oxyl-2,2,5,5-tetramethyl-delta 3-pyrroline-3-methyl)methanethiosulfonate (SL-MMTS) (Berliner, L. J., Grunwald, J., Hankovszky, H. O., & Hideg, K., 1982, Anal. Biochem. 119, 450-455). EPR spectra show that SL-MMTS labels three groups per PBGS subunit (24 per octamer), as does MMTS. EPR signals reflecting nitroxides of different mobilities are observed. Two of the three modified cysteines have been identified as Cys-119 and Cys-223 by sequencing peptides produced by an Asp-N protease digest of the modified protein. Because MMTS-reactive thiols have been implicated as ligands to the required Zn(II), EPR spectroscopy has been used to determine the spatial proximity of the modified cysteine residues. A forbidden (delta m = 2) EPR transition is observed indicating a through-space dipolar interaction between at least two of the nitroxides. The relative intensity of the forbidden and allowed transitions show that at least two of the unpaired electrons are within at most 7.6 A of each other. SL-MMTS-modified PBGS loses all Zn(II) and cannot catalyze product formation. The modified enzyme retains the ability to bind one of the two substrates at each active site. Binding of this substrate has no influence on the EPR spectral properties of the spin-labeled enzyme, or on the rate of release of the nitroxides when 2-mercaptoethanol is added.(ABSTRACT TRUNCATED AT 250 WORDS)

Osteoclast radical interactions: NADPH causes pulsatile release of NO and stimulates superoxide production.

Osteoclasts have been shown to destroy calcified tissue by complex developmental steps involving cell recruitment, cell attachment and deployment of multiple enzymes. They also appear to regulate resorption by several mechanisms. In particular, earlier investigations have indicated that oxygen radical metabolites may be produce by osteoclasts. These labile reactants could accelerate destruction of calcified tissue. In addition, recent studies have suggested that nitric oxide may have an inhibitory role in bone resorption. Previous studies of these radical substituents have predicted that interactions of nitric oxide and oxygen radicals could explain the conflicting roles of these radicals in the control of bone resorption. In view of the requirement of both of the enzymes, NADPH-oxidase and NO synthase (NOS), for NADPH(beta-nicotinamide adenine dinucleotide phosphate), one level of interaction could be related to competition for this necessary cofactor. To test this hypothesis, we have investigated the ability of the osteoclast to generate nitric oxide and oxygen radicals after stimulation by NADPH. Consistent with earlier diaphorase histochemistry, we have shown that resorbing osteoclasts produce NO. Addition of NADPH (10 microM) resulted in a transient burst of NO production (measured by porphyrin coated microsensor) with an amplitude of 152 +/- 43 nM and a duration of 4 seconds. Repetitive stimulation resulted in a decremental response with a partial recovery after 30 minutes. Addition of L-NAME (N omega-nitro-L-arginine methyl ester, 100 microM) to the cells resulted in at least 50% inhibition of the amplitude of NO peak and produced an extended peak duration. To compare the effect of the added NADPH on superoxide production by osteoclast NADPH-oxidase, osteoclast oxygen radicals were detected by EPR(electron paramagnetic resonance) spectrometer with the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The production of a spin adduct with a quadruplet signal was inhibited by SOD (superoxide dismutase). We were not able to demonstrate an increase in superoxide production after addition of L-NAME, another possible interaction of NOS and NADPH-oxidase. These results demonstrate that although osteoclasts produce both NO and superoxide, NOS competition for NADPH is not a major site of interaction with NADPH-oxidase under these conditions. Additionally, these initial findings set the stage for the further investigation of interactions of osteoclast radicals in modulating bone resorption.

Expression of the cloned genes encoding the putrescine biosynthetic enzymes and methionine adenosyltransferase of Escherichia coli (speA, speB, speC and metK).

The speA, speB and speC genes, which code for arginine decarboxylase (ADCase), agmatine ureohydrolase (AUHase) and ornithine decarboxylase (ODCase), respectively, and the metK gene, which encodes methionine adenosyltransferase (MATase), have been cloned. The genes were isolated from hybrid ColE1 plasmids of the Clarke-Carbon collection and were ligated into plasmid pBR322. Escherichia coli strains transformed with the recombinant plasmids exhibit a 7- to 17-fold overproduction of the various enzymes, as estimated from increases in the specific activities of the enzymes assayed in crude extracts. Minicells bearing the pBR322 hybrid plasmids and labeled with radioactive lysine synthesize radiolabeled proteins with Mrs corresponding to those reported for purified ODCase, ADCase and MATase. Restriction enzyme analysis of the plasmids, combined with measurements of specific activities of the enzymes in crude extracts of cells bearing recombinant plasmids, clarified the relative position of speA and speB. The gene order in the 62- to 64-min region is serA speB speA metK speC glc.

Flexible loop in the structure of S-adenosylmethionine synthetase crystallized in the tetragonal modification.

S-Adenosylmethionine synthetase (MAT, ATP:L-methionine S-adenosyltransferase, E.C.2.5.1.6.) plays a central metabolic role in all organisms. MAT catalyzes the two-step reaction which synthesizes S-adenosylmethionine (AdoMet), pyrophosphate (PPi) and orthophosphate (Pi) from ATP and L-methionine. AdoMet is the primary methyl group donor in biological systems. MAT from Escherichia coli was crystallized in the tetragonal modification with space group P4(3)2(1)2 using the same conditions as previously yielded crystals of the hexagonal system [Takusagawa, et al., (1996), J. Biol. Chem. 171, 136-147], except for the crystallization temperature. The structure has been determined by molecular replacement at 3.2 A resolution. The overall structure of the tetrameric MAT in the tetragonal modification is essentially the same as the structure found in the hexagonal modification. However there are two remarkable differences between the structures of two modifications. One is the contents in the active sites (holoform vs. apo-form), and the other is the conformation of the flexible loop over the active site (open vs. closed). These differences in the crystal structures are caused solely by the difference in crystallization temperatures (26 degrees C vs. 4 degrees C). We have interpreted the structural data obtained from the X-ray analyses in conjunction with the results of the mechanistic and sequencing studies in terms of possible dynamic motion of the flexible loop. When a substrate/product binds in the active site (hexagonal modification), the loop becomes disordered, apparently due to flexibility at the entrance of the active site as if it acts as a "mobile loop" during the catalytic reaction. On the other hand, when the temperature is decreased, the dynamic motion of the flexible loop may be reduced, and the loop residues enter the active site and close its entrance (tetragonal modification). Thus, the active site of the tetragonal modification is empty despite the crystals being grown in mother liquor containing a large concentration of phosphate (100 mM). There is no significant displacement of amino acid residues in the active site between the holo and apo forms, suggesting that the flexible loop plays an important role in determination of the contents in the active site. Since the functionally important amino acid residues in the active site are all conserved throughout various species, the structures of the active sites and the mechanism of the catalysis are probably essentially identical in the enzymes from a wide range of organisms. However, the substrate KM and Vmax values of MATs from various species are distributed over a wide range. The amino acid residues in the flexible loop regions are poorly conserved throughout various species. Therefore, the wide differences in catalysis rates of MATs from various speeches may be due to the differences in the composition of the flexible loop.

Characterization of the monovalent cation activator binding site of S-adenosylmethionine synthetase by 205Tl NMR of enzyme-bound Tl+.

The structure of the binding site for the monovalent cation activator of S-adenosylmethionine (AdoMet) synthetase from Escherichia coli has been characterized by 205Tl NMR of enzyme-bound Tl+. The chemical shift of the enzyme-Tl+ complex is 176 ppm downfield from aquo Tl+, a shift which is typical only of Tl+ complexes with solely oxygen ligands. The 205Tl resonance shifts upfield to 85 ppm in the enzyme-Mg(II)-Tl+ complex, to 38 ppm in the enzyme-Tl+-AdoMet complex and to 34 ppm in the enzyme-Tl+-AdoMet-Mg(II) complex. The 205Tl chemical shift of enzyme-bound Tl+ was not altered by binding of either methionine, or the Mg(II)-ATP analog Mg(II)-adenyl-5'-yl imidodiphosphate, or Mg(II)-pyrophosphate to the enzyme-Tl+-Mg(II) complex. The NMR data suggest that the substrates or products of the enzyme do not coordinate to the monovalent cation activator and imply that monovalent cation activation results from alterations in protein conformation.

Spatial proximity of two divalent metal ions at the active site of S-adenosylmethionine synthetase.

S-Adenosylmethionine synthetase from Escherichia coli is shown to require 2 divalent metal ions/enzyme subunit for maximal enzymatic activity. In the absence of substrate, the tetrameric enzyme binds 1 Mn(II) ion/subunit, whereas in the presence of a nucleotide substrate, adenylylimidodiphosphate, or the product pyrophosphate, there are two Mn(II)-binding sites/subunit. Electron paramagnetic resonance spectra of Mn(II) bound to the enzyme reveal a spin exchange interaction between 2 Mn(II) ions in complexes of enzyme and Mn(II) which also contain adenosylmethionine, K+, and either pyrophosphate or imidotriphosphate. Since a spin exchange interaction requires orbital overlap between the 2 ions, the metal ions must be bound close to one another, and they may share a common ligand.

Structure of the divalent metal ion activator binding site of S-adenosylmethionine synthetase studied by vanadyl(IV) electron paramagnetic resonance.

The structure of the divalent metal ion binding site of S-adenosylmethionine synthetase from Escherichia coli has been studied by using the vanadyl(IV) ion (VO2+) as probe. VO2+ binds at a single site per subunit in the presence or absence of substrates. Single turnover experiments measuring S-adenosylmethionine (AdoMet) formation from methionine and the ATP analogue 5'-adenylyl imidodiphosphate show that complexes containing VO2+ and either Mg2+ or Ca2+ as a second metal ion are catalytically active, while a complex containing VO2+ alone is inactive. Electron paramagnetic resonance spectra of the enzyme-VO2+ complex, as well as complexes also containing AdoMet or methionine, indicate the coordination of two water molecules and at least two protein ligands to the VO2+. In complexes with polyphosphate substrates or products (e.g., enzyme-VO2+-ATP-methionine, enzyme-VO2+-PPi-Mg2+), EPR spectral changes reveal ligand substitutions on the VO2+, and 8.5-G isotropic superhyperfine coupling to two 31P nuclei can be resolved. 17O superhyperfine coupling from [17O]pyrophosphate indicates coordination of two oxygen atoms of PPi to the VO2+ ion. Thus the polyphosphate compounds are bidentate ligands to the VO2+, demonstrating that the VO2+ binds at the active site and suggesting a catalytic role for the protein-bound metal ion.

13C NMR studies of methylene and methine carbons of substrate bound to a 280,000-dalton protein, porphobilinogen synthase.

13C NMR has been used to observe the equilibrium complex of [5,5-2H,5-13C]-5-aminolevulinate [( 5,5-2H,5-13C]ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. [5,5-2H,5-13C]ALA (chemical shift 46.9 ppm in D2O) was prepared from [5-13C]ALA through enolization in deuteriated neutral potassium phosphate buffer. In the PBG synthase reaction [5,5-2H,5-13C]ALA forms [2,11,11-2H,2,11-13C]PBG (chemical shifts 116.2 ppm for C2 and 34.2 ppm for C11 in D2O). For the complex formed between [5,5-2H,5-13C]ALA and methyl methanethiosulfonate (MMTS) modified PBG synthase, which does not catalyze PBG formation but can form a Schiff base adduct, the chemical shift of 44.2 ppm (line width 92 Hz) identifies an imine structure as the predominant tautomeric form of the Schiff base. By comparison to model compounds, the stereochemistry of the imine has been deduced; however, the protonation state of the imine nitrogen remains unresolved. Reconstitution of the MMTS-modified enzyme-Schiff base complex with Zn(II) and 2-mercaptoethanol results in the holoenzyme-bound equilibrium complex; this complex contains predominantly enzyme-bound PBG, and spectra reveal two peaks from bound PBG and two from free PBG. For bound PBG, C2 is -2.8 ppm from the free signal and C11 is +2.6 ppm from the free signal; the line widths of the bound signals are 55 and 75 Hz, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the reactive sulfhydryl groups of S-adenosylmethionine synthetase.

S-Adenosylmethionine synthetase from Escherichia coli is rapidly inactivated by N-ethylmaleimide. In the presence of excess N-ethylmaleimide inactivation follows pseudo first-order kinetics, and loss of enzyme activity correlates with the incorporation of 2 eq of N-[ethyl-2-3H]maleimide/subunit. Preincubation of the enzyme with methionine and the ATP analog adenylylimidodiphosphate reduced the rate of N-ethylmaleimide incorporation more than 30-fold. Two N-[ethyl-2-3H]maleimide-labeled tryptic peptides were purified from the modified enzyme by reverse phase high performance liquid chromatography. The modified residues were identified as cysteine 90 and cysteine 240 by comparison of the amino acid compositions of these peptides with the protein sequence. These are the first residues to be implicated in the activity and/or structure of the enzyme. N-Ethylmaleimide-modified S-adenosylmethionine synthetase exists mainly as a dimer in conditions where the native enzyme is a tetramer. Accumulation of the dimer parallels the loss of the enzyme activity. When an enzyme sample was partially inactivated, separation of tetrameric and dimeric enzyme forms by gel filtration revealed that the residual enzyme activity was solely present in the tetramer and N-[ethyl-2-3H] maleimide was present predominantly in the dimer. Gel filtration studies of the tetramer-dimer equilibrium for the native enzyme indicated that the dissociation constant between the tetramer and dimers is less than 6 x 10(-11) M. Similar studies for the N-ethylmaleimide-modified protein indicated that the dissociation constant of the tetramer is approximately 4 x 10(-4) M. Upon modification the strength of dimer-dimer interactions is diminished by at least 9 kcal/mol.

13C NMR studies of porphobilinogen synthase: observation of intermediates bound to a 280,000-dalton protein.

13C NMR has been used to observe the equilibrium complex of [4-13C]-5-aminolevulinate ([4-13C]ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. [4-13C]ALA (chemical shift = 205.9 ppm) forms [3,5-13C]PBG (chemical shifts = 121.0 and 123.0 ppm). PBG prepared from a mixture of [4-13C]ALA and [15N]ALA was used to assign the 121.0 and 123.0 ppm resonances to C5 and C3, respectively. For the enzyme-bound equilibrium complex formed from holoenzyme and [4-13C]ALA, two peaks of equal area with chemical shifts of 121.5 and 127.2 ppm are observed (line widths approximately 50 Hz), indicating that the predominant species is probably a distorted form of PBG. When excess free PBG is present, it is in slow exchange with bound PBG, indicating an exchange rate of less than 10 s-1, which is consistent with the turnover rate of the enzyme. For the complex formed from [4-13C]ALA and methyl methanethiosulfonate (MMTS) modified PBG synthase, which does not catalyze PBG formation, the predominant species is a Schiff base adduct (chemical shift = 166.5 ppm, line width approximately 50 Hz). Free ALA is in slow exchange with the Schiff base. Activation of the MMTS-modified enzyme-Schiff base complex with 113Cd and 2-mercaptoethanol results in the loss of the Schiff base signal and the appearance of bound PBG with the same chemical shifts as for the bound equilibrium complex with Zn(II) enzyme. Neither splitting nor broadening from 113Cd-13C coupling was observed.

Adenosine-5'-phosphosulfate kinase from Escherichia coli K12. Purification, characterization, and identification of a phosphorylated enzyme intermediate.

Adenosine-5'-phosphosulfate kinase (ATP:adenylylsulfate 3'-phosphotransferase), the second enzyme in the pathway of sulfate activation, has been purified (approximately 300-fold) to homogeneity from an Escherichia coli K12 strain, which overproduces the enzyme activity (approximately 100-fold). The purified enzyme has a specific activity of 153 mumol of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) formed/min/mg of protein at 25 degrees C. The enzyme is remarkably efficient with a Vmax/Km(APS) of greater than 10(8) M-1 s-1, indicating that at physiologically low substrate concentrations the reaction is essentially diffusion limited. Upon incubation with MgATP a phosphorylated enzyme is formed; the isolated phosphorylated enzyme can transfer its phosphoryl group to adenosine 5'-phosphosulfate (APS) to form PAPS or to ADP to form ATP. The phosphorylated enzyme exists as a dimer of identical 21-kilodalton subunits, while the dephosphorylated form primarily exists as a tetramer. Divalent cations are required for activity with Mg(II), Mn(II), Co(II), and Cd(II) activating. Studies of the divalent metal-dependent stereoselectivity for the alpha- and beta-phosphorothioate derivatives of ATP indicate metal coordination to at least the alpha-phosphoryl group of the nucleotide. Steady state kinetic studies of the reverse reaction indicate a sequential mechanism, with a rapid equilibrium ordered binding of MgADP before PAPS. In the forward direction APS is a potent substrate inhibitor, competitive with ATP, complicating kinetic studies. The primary kinetic mechanism in the forward direction is sequential. Product inhibition studies at high concentrations of APS suggest an ordered kinetic mechanism with MgATP binding before APS. At submicromolar concentrations of APS, product inhibition by both MgADP and PAPS is more complex and is not consistent with a solely ordered sequential mechanism. The formation of a phosphorylated enzyme capable of transferring its phosphoryl group to APS or to MgADP suggests that a ping-pong pathway in which the rate of MgADP dissociation is comparable to the rate of APS binding might contribute at very low concentrations of APS. The substrate inhibition by APS is consistent with APS binding to the enzyme, to form a dead-end E.APS complex.

The conformation of inosine 5'-monophosphate (IMP) bound to IMP dehydrogenase determined by transferred nuclear overhauser effect spectroscopy.

IMP dehydrogenase (IMPDH) catalyzes the NAD-dependent synthesis of xanthosine 5'-monophosphate which is the rate-limiting step in guanine nucleotide biosynthesis. Although IMPDH is the target of numerous chemotherapeutic agents, nothing has been known about the conformation of the enzyme-bound substrates. The conformation of IMP bound to human type II IMP dehydrogenase has been determined by two-dimensional transferred nuclear Overhauser effect NMR spectroscopy at 600 MHz. NOE buildup rates were determined by recording NOESY spectra at numerous mixing times. The cross-relaxation rates determined from the initial NOE build-up rates were used to calculate inter-proton distances of bound IMP. The conformation of the enzyme-bound IMP was obtained by molecular modeling with energy minimization using the experimentally determined inter-proton distance constraints. The glycosidic torsion angle of the bound nucleotide is anti and the sugar is in the C2-endo-conformation. This conformation places H2 of IMP, which is transferred to NAD in the reaction, in a position clear of the rest of the molecule in order to facilitate the reaction.

Mechanistic studies of Escherichia coli adenosine-5'-phosphosulfate kinase.

Adenosine-5'-phosphosulfate kinase (APS kinase) catalyzes the formation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the major form of activated sulfate in biological systems. The enzyme from Escherichia coli has complex kinetic behavior, including substrate inhibition by APS and formation of a phosphorylated enzyme (E-P) as a reaction intermediate. The presence of a phosphorylated enzyme potentially enables the steady-state kinetic mechanism to change from sequential to ping-pong as the APS concentration decreases. Kinetic and equilibrium binding measurements have been used to evaluate the proposed mechanism. Equilibrium binding studies show that APS, PAPS, ADP, and the ATP analog AMPPNP each bind at a single site per subunit; thus, substrates can bind in either order. When ATPgammaS replaces ATP as substrate the V(max) is reduced 535-fold, the kinetic mechanism is sequential at each APS concentration, and substrate inhibition is not observed. The results indicate that substrate inhibition arises from a kinetic phenomenon in which product formation from ATP binding to the E. APS complex is much slower than paths in which product formation results from APS binding either to the E. ATP complex or to E-P. APS kinase requires divalent cations such as Mg(2+) or Mn(2+) for activity. APS kinase binds one Mn(2+) ion per subunit in the absence of substrates, consistent with the requirement for a divalent cation in the phosphorylation of APS by E-P. The affinity for Mn(2+) increases 23-fold when the enzyme is phosphorylated. Two Mn(2+) ions bind per subunit when both APS and the ATP analog AMPPNP are present, indicating a potential dual metal ion catalytic mechanism.

The bifunctional active site of S-adenosylmethionine synthetase. Roles of the basic residues.

S-adenosylmethionine (AdoMet) synthetase catalyzes a unique two-step enzymatic reaction leading to formation of the primary biological alkylating agent. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site, which lies between two subunits, contains four lysines and one histidine as basic residues. In order to test the proposed charge and hydrogen bonding roles in catalytic function, each lysine has been changed to an uncharged methionine or alanine, and the histidine has been altered to asparagine. The resultant enzyme variants are all tetramers like the wild type enzyme; however, circular dichroism spectra show reductions in helix content for the K245*M and K269M mutants. (The asterisk denotes that the residue is in the second subunit.) Four mutants have k(cat) reductions of approximately 10(3)-10(4)-fold in AdoMet synthesis; however, the k(cat) of K165*M variant is only reduced 2-fold. In each mutant, there is a smaller catalytic impairment in the partial reaction of tripolyphosphate hydrolysis. The K165*A enzyme has a 100-fold greater k(cat) for tripolyphosphate hydrolysis than the wild type enzyme, but this mutant is not activated by AdoMet in contrast to the wild type enzyme. The properties of these mutants require reassessment of the catalytic roles of these residues.

Investigation of monovalent cation activation of S-adenosylmethionine synthetase using mutagenesis and uranyl inhibition.

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 Mg2+ 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 approximately 100-fold decrease in the Vmax for AdoMet synthesis and large increases in the Km values for both substrates. In contrast there is only a 2-fold decrease in Vmax for tripolyphosphate hydrolysis. The uranyl ion, UO2(2+), is a competitive inhibitor with respect to K+ (Ki = 350 nM) and is the first ion to bind at this site and inhibit the enzyme. The UO2(2+) inhibition is reversible and tight-binding, and results from UO2(2+) and not UO2(2+)-ATP. Analogous to K+ activation, UO2(2+) predominantly inhibits AdoMet formation rather than tripolyphosphate hydrolysis. The kinetic results indicate that UO2(2+) inhibition is likely to result from interference with productive ATP binding. UO2(2+) remains a tight-binding inhibitor of the E42Q mutant, which suggests that K+ and UO2(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.