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.

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.

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.

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.

Structural and functional roles of cysteine 90 and cysteine 240 in S-adenosylmethionine synthetase.

Site-specific mutagenesis was performed on the structural gene for Escherichia coli S-adenosylmethionine (AdoMet) synthetase to introduce mutations at cysteines 90 and 240, residues previously implicated by chemical modification studies to be catalytically and/or structurally important. The AdoMet synthetase mutants (i.e. MetK/C90A, MetK/C90S, and MetK/C240A) retained up to approximately 10% of wild type activity, demonstrating that neither sulfhydryl is required for catalytic activity. Mutations at Cys-90 produced a mixture of noninterconverting dimeric and tetrameric proteins, suggesting a structural significance for Cys-90. Dimeric Cys-90 mutants retained approximately 1% of wild type activity, indicating a structural influence on enzyme activity. Both dimeric and tetrameric MetK/C90A had up to a approximately 70-fold increase in Km for ATP, while both dimeric and tetrameric MetK/C90S had Km values for ATP similar to the wild type enzyme, suggesting a linkage between Cys-90 and the ATP binding site. MetK/C240A was isolated solely as a tetramer and differed from wild type enzyme only in its 10-fold reduction in specific activity, suggesting that the mutation affects the rate-limiting step of the reaction, which for the wild type enzyme is the joining of ATP and L-methionine to yield AdoMet and tripolyphosphate. Remarkably all of the mutants are much more thermally stable than the wild type enzyme.

Slow binding inhibition of S-adenosylmethionine synthetase by imidophosphate analogues of an intermediate and product.

S-Adenosylmethionine (AdoMet) synthetase catalyzes the only known route of biosynthesis of the primary in vivo alkylating agent. Inhibitors of this enzyme could provide useful modifiers of biological methylation and polyamine biosynthetic processes. The AdoMet synthetase catalyzed reaction converts ATP and L-methionine to AdoMet, PP(i), and P(i), with formation of tripolyphosphate as a tightly bound intermediate. This work describes a nonhydrolyzable analogue of the tripolyphosphate (PPP(i)) reaction intermediate, diimidotriphosphate (O(3)P-NH-PO(2)-NH-PO(3)(5)(-)), as a potent inhibitor. In the presence of AdoMet, PNPNP is a slow-binding inhibitor with an overall inhibition constant (K(i)) of 2 nM and a dissociation rate of 0.6 h(-)(1). In contrast, in the absence of AdoMet PNPNP is a classical competitive inhibitor with a K(i) of 0.5 microM, a slightly higher affinity than PPP(i) itself (K(i) = 3 microM). The imido analogue of the product pyrophosphate, imidodiphosphate (O(3)P-NH-PO(3)(4)(-)) also displays slow onset inhibition only in the presence of AdoMet, with a K(i) of 0.8 microM, compared to K(i) of 250 microM for PP(i). Circular dichroism spectra of the unliganded enzyme and various complexes are indistinguishable indicating that the protein secondary structure is not greatly altered upon complex formation, suggesting local rearrangements at the active site during the slow binding process. A model based on ionization of the bridging -NH- moiety is presented which could account for the potent inhibition by PNP and PNPNP.

The conformations of a substrate and a product bound to the active site of S-adenosylmethionine synthetase.

S-Adenosylmethionine (AdoMet) is the most widely used alkyl group donor in biological systems. The formation of AdoMet from ATP and L-methionine is catalyzed by S-adenosylmethionine synthetase (AdoMet synthetase). Elucidation of the conformations of enzyme-bound substrates, product, and inhibitors is important for the understanding of the catalytic mechanism of the enzyme and the design of new inhibitors. To obtain structural data for enzyme-bound substrates and product, we have used two-dimensional transferred nuclear Overhauser effect spectroscopy to determine the conformation of enzyme-bound AdoMet and 5'-adenylyl imidodiphosphate (AMPPNP). AMPPNP, an analogue of ATP, is resistant to the ATP hydrolysis activity of AdoMet synthetase because of the presence of a nonhydrolyzable NH-link between the beta- and gamma-phosphates but is a substrate for AdoMet formation during which tripolyphosphate is produced. AdoMet and AMPPNP both bind in an anti conformation about the glycosidic bond. The ribose rings are in C3'-exo and C4'-exo conformations in AdoMet and AMPPNP, respectively. The differences in ribose ring conformations presumably reflect the different steric requirements of the C5' substituents in AMPPNP and AdoMet. The NMR-determined conformations of AdoMet and AMPPNP were docked into the E. coli AdoMet synthetase active site taken from the enzyme.ADP. Pi crystal structure. Since there are no nonexchangeable protons either in the carboxy-terminal end of the methionine segment of AdoMet or in the tripolyphosphate segment of AMPPNP, these portions of the molecules were modeled into the enzyme active site. The interactions of AdoMet and AMPPNP with the enzyme predict the location of the methionine binding site and suggest how the positive charge formed on the sulfur during AdoMet synthesis is stabilized.

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.

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.

Probing the mechanism of inosine monophosphate dehydrogenase with kinetic isotope effects and NMR determination of the hydride transfer stereospecificity.

The mechanism of human type II inosine monophosphate dehydrogenase has been probed by measurements of primary deuterium kinetic isotope effects, and by determination of the stereochemical course of the reaction. The deuterium isotope effects on Vmax from [2-deutero]-IMP are unity for reactions with a variety of monovalent cation activators (K+, NH4+, Na+, Rb+) of various efficacy. In each case normal effects on Vmax/K(m) in the range of 1.9 to 3.5 are observed for both IMP and NAD, and are larger for NAD. These results demonstrate that both substrates can dissociate from the E.M+.IMP.NAD complex, therefore the kinetic mechanism is not ordered as previous steady-state kinetic studies have suggested. Comparison of reaction rates in D2O and H2O show no 2H isotope effect on Vmax, and a < or = twofold decrease in Vmax/K(m); thus, a proton transfer from solvent is not rate-limiting in turnover. The NMR spectrum of the [4-deutero]NADH produced in the reaction of [2-deutero]-IMP and NAD shows that the hydrogen is transferred to the B, or pro-S, side of the nicotinamide ring. Presteady-state kinetic experiments reveal a burst of NADH formation in the first turnover, demonstrating that a late step in the mechanism is rate-limiting. The rate of the burst phase is reduced approximately twofold with [2-deutero]IMP as substrate, indicating that the hydride transfer step is kinetically significant early in the reaction.

The bifunctional active site of s-adenosylmethionine synthetase. Roles of the active site aspartates.

S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively. In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the smaller Mg(2+).

Magnetic resonance studies of three forms of creatine kinase. Comparison of the properties of native, CH-S-blocked, and H2NCOCH-blocked enzymes.

Magnetic resonance methods are applied in a comparative study of native creatine kinase from rabbit muscle with two sulfhydryl-modified forms of the enzyme--one inactive form obtained by reaction of the enzyme with iodoacetamide and one form with reduced activity obtained by reaction of the iodoacetamide-sensitive sulfhydryl group with methyl methanethiolsulfonate, which blocks the sulfhydryl with a CH3S-group. Water proton relaxation rate (PRR) titrations with the CH3S-blocked enzyme show that the modification does not alter appreciably the affinities of the enzyme for MnADP and for creatine in the presence of MnADP. Similar measurements for the H2NCOCH2-blocked enzyme indicate that this modification weakens the affinity of the enzyme for MnADP. In agreement with previous findings, there is no observable change in the PRR enhancement upon additions of creatine to solutions of the ternary complex, enzyme-MnADP, for the H2NCOCH2-blocked enzyme. PRR titrations enabled the measurement of binding of creatine to the ternary CH3S-enzyme-MnADP complex and show that specific anions such as nitrate, formate, and thiocyanate decrease the apparent dissociation constant for creatine in its complex with the CH3S-blocked enzyme and MnADP, as is observed with native creatine kinase. However, the change in the PRR enhancement for the CH3S-enzyme-MnADP upon binding of creatine in the presence or absence of anions was appreciably smaller than for the native enzyme. For the H2NCOCH2-blocked enzyme, these anions failed to bring about any influence of creatine on the PRR enhancement. Consistent with the diminished influence of these anions on the PRR enhancement of the CH3S-enzyme-MnADP-creatine complex, EPR spectra of bound Mn(II) show that the CH3S-blocking group interferes with the pronounced anion-induced spectral changes which are observed with the native enzyme. EPR spectra for the H2NCOCH2-enzyme-MnADP complex were not influenced upon additions of creatine, even in the presence of anions. These results suggest that the altered catalytic properties of the CH3S-blocked enzyme arise from structural perturbations at the active site which are also reflected in the PRR enhancement factors and EPR spectral features of the Mn(II) complexes. Moreover, the results clearly indicate that the H2NCOCH2-blocking group, which completely inactivates the enzyme, also eliminates the ability of the MnADP site to sense the presence of the second substrate, creatine, alone and in combination with anions which are structural analogs of the migrating phosphoryl group.