Structural basis for the stability of a thermophilic methionine adenosyltransferase against guanidinium chloride.

The methionine adenosyltransferase from the thermophile Methanococcus jannaschii is fully and irreversibly unfolded in the presence of guanidinium chloride. Unfolding of this dimeric protein is a three-state process in which a dimeric intermediate could be identified. The less stable secondary structural elements of the protein are the C-terminal ends of ß-strands E2 and E6, as deduced from the behavior of tyrosine to tryptophan mutants at residues 72 and 170, which are located in the subunit interface. Unraveling of these elements at the monomer interface may soften intersubunit interactions, leading to the observed 85% activity loss. Accumulation of the intermediate was associated with maintenance of residual activity, an increase in the elution volume of the protein upon gel filtration and a decrease in the sedimentation coefficient. Elimination of the remaining enzymatic activity occurred in conjunction with a 50% reduction in helicity and fluorescence alterations illustrating a transient burial of tryptophans at ß-strands E2, E3 and E9. The available 3D-model predicted that these ß-strands are involved in the central and N-terminal domains of the monomer structure. Severe perturbation of this area of the monomer-monomer interface may destroy the remaining intermolecular interactions, thus leading to dissociation and aggregation. Finally, transition to the denatured state includes completion of the changes detected in the microenvironments around tryptophans included at α-helixes H5 and H6, the loops connecting H5-E8 and E9, ß-strands E3 and E12.

Thermodynamics of boroxine formation from the aliphatic boronic acid monomers R-B(OH)2 (R = H, H3C, H2N, HO, and F): a computational investigation.

Boroxines are the six-membered cyclotrimeric dehydration products of organoboronic acids, 3R­B(OH)2 → R3B3O3 + 3H2O, and in recent years have emerged as a useful class of organoboron molecules with applications in organic synthesis both as reagents and catalysts, as structural components in boronic-acid-derived pharmaceutical agents, and as anion acceptors and electrolyte additives for battery materials [Korich, A. L.; Iovine, P. M. Dalton Trans. 2010, 39, 1423−1431]. Second-order Møller­Plesset perturbation theory, in conjunction with the Dunning­Woon correlation-consistent cc-pVDZ, aug-cc-pVDZ, cc-pVTZ, and aug-cc-pVTZ basis sets, was used to investigate the structures and relative energies of the endo­exo, anti, and syn conformers of the aliphatic boronic acids R­B(OH)2 (R = H, H3C, H2N, HO, and F), as well as the thermodynamics of their boroxine formation; single-point calculations at the MP2/aug-cc-pVQZ, MP2/aug-cc-pV5Z, and CCSD(T)/aug-cc-pVTZ levels using the MP2/aug-cc-pVTZ optimized geometries were also performed in selected cases. The endo­exo conformer was generally lowest in energy in vacuo, as well as in PCM and CPCM models of aqueous and carbon tetrachloride media. The values of ΔH(298)(0) for boroxine formation via dehydration from the endo­exo conformers of these aliphatic boronic acids ranged from −2.9 for (H2N)3B3O3 to +12.2 kcal/mol for H3B3O3 at the MP2/aug-cc-pVTZ level in vacuo; for H3B3O3, the corresponding values in PCM/UFF implicit carbon tetrachloride and aqueous media were +11.2 and +9.8 kcal/mol, respectively. On the basis of our calculations, we recommend that ΔHf(298K) for boroxine listed in the JANAF compilation needs to be revised from −290.0 to approximately −277.0 kcal/mol.

Subunit association as the stabilizing determinant for archaeal methionine adenosyltransferases.

Archaea contain a class of methionine adenosyltransferases (MATs) that exhibit substantially higher stability than their mesophilic counterparts. Their sequences are highly divergent, but preserve the essential active site motifs of the family. We have investigated the origin of this increased stability using chemical denaturation experiments on Methanococcus jannaschii MAT (Mj-MAT) and mutants containing single tryptophans in place of tyrosine residues. The results from fluorescence, circular dichroism, hydrodynamic, and enzyme activity measurements showed that the higher stability of Mj-MAT derives largely from a tighter association of its subunits in the dimer. Local fluorescence changes, interpreted using secondary structure predictions, further identify the least stable structural elements as the C-terminal ends of beta-strands E2 and E6, and the N-terminus of E3. Dimer dissociation however requires a wider perturbation of the molecule. Additional analysis was initially hindered by the lack of crystal structures for archaeal MATs, a limitation that we overcame by construction of a 3D-homology model of Mj-MAT. This model predicts preservation of the chain topology and three-domain organization typical of this family, locates the least stable structural elements at the flat contact surface between monomers, and shows that alterations in all three domains are required for dimer dissociation.

The CBS subdomain of inosine 5'-monophosphate dehydrogenase regulates purine nucleotide turnover.

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyses the rate-limiting step in guanine nucleotide biosynthesis. IMPDH has an evolutionary conserved CBS subdomain of unknown function. The subdomain can be deleted without impairing the in vitro IMPDH catalytic activity and is the site for mutations associated with human retinitis pigmentosa. A guanine-prototrophic Escherichia coli strain, MP101, was constructed with the subdomain sequence deleted from the chromosomal gene for IMPDH. The ATP content was substantially elevated in MP101 whereas the GTP content was slighty reduced. The activities of IMPDH, adenylosuccinate synthetase and GMP reductase were two to threefold lower in MP101 crude extracts compared with the BW25113 wild-type strain. Guanine induced a threefold reduction in the MP101 ATP pool and a fourfold increase in the GTP pool within 10 min of addition to growing cells; this response does not result from the reduced IMPDH activity or starvation for guanylates. In vivo kinetic analysis using 14-C tracers and 33-P pulse-chasing revealed mutation-associated changes in purine nucleotide fluxes and turnover rates. We conclude that the CBS subdomain of IMPDH may coordinate the activities of the enzymes of purine nucleotide metabolism and is essential for maintaining the normal ATP and GTP pool sizes in E. coli.

An investigation of the catalytic mechanism of S-adenosylmethionine synthetase by QM/MM calculations.

Catalysis by S-adenosylmethionine synthetase has been investigated by quantum mechanical/molecular mechanical calculations, exploiting structures of the active crystalline enzyme. The transition state energy of +19.1 kcal/mol computed for a nucleophilic attack of the methionyl sulfur on carbon-5' of the nucleotide was indistinguishable from the experimental (solution) value when the QM residues were an uncharged histidine that hydrogen bonds to the leaving oxygen-5' and an aspartate that chelates a Mg2+ ion, and was similar (+18.8 kcal/mol) when the QM region also included the active site arginine and lysines. The computed energy difference between reactant and product was also consistent with their equimolar abundance in co-crystals. The calculated geometrical changes support catalysis of a S(N)2 reaction through hydrogen bonding of the liberated oxygen-5' to the histidine, charge neutralization by the two Mg2+ ions, and stabilization of the product sulfonium cation through a close, non-bonded, contact between the sulfur and the ribose oxygen-4'.

Shape shifting leads to small-molecule allosteric drug discovery.

Enzymes that regulate their activity by modulating an equilibrium of alternate, nonadditive, functionally distinct oligomeric assemblies (morpheeins) constitute a recently described mode of allostery. The oligomeric equilibrium for porphobilinogen synthase (PBGS) consists of high-activity octamers, low-activity hexamers, and two dimer conformations. A phylogenetically diverse allosteric site specific to hexamers is proposed as an inhibitor binding site. Inhibitor binding is predicted to draw the oligomeric equilibrium toward the low-activity hexamer. In silico docking enriched a selection from a small-molecule library for compounds predicted to bind to this allosteric site. In vitro testing of selected compounds identified one compound whose inhibition mechanism is species-specific conversion of PBGS octamers to hexamers. We propose that this strategy for inhibitor discovery can be applied to other proteins that use the morpheein model for allosteric regulation.

Computational investigation of the oxidative deboronation of boroglycine, H2N-CH2-B(OH)2, Using H2O and H2O2.

We report results from a computational investigation of the oxidative deboronation of boroglycine, H2N-CH2-B(OH)2, using H2O and H2O2 as the reactive oxygen species (ROS) to yield aminomethanol, H2N-CH2-OH; these results complement our study on the protodeboronation of boroglycine to produce methylamine, H2N-CH3 (Larkin et al. J. Phys. Chem. A 2007, 111, 6489-6500). Second-order Møller-Plesset (MP2) perturbation theory with Dunning-Woon correlation-consistent (cc) basis sets were used for the calculations with comparisons made to results from density functional theory (DFT) at the PBE1PBE/6-311++G(d,p)(cc-pVDZ) levels. The effects of a bulk aqueous environment were also incorporated into the calculations employing PCM and CPCM methodology. Using H2O as the ROS, the reaction H2O + H2N-CH2-B(OH)2 --> H2N-CH2-OH + H-B(OH)2 was calculated to be endothermic; the value of DeltaH(298)(0) was +12.0 kcal/mol at the MP2(FC)/cc-pVTZ computational level in vacuo and +13.7 kcal/mol in PCM aqueous media; the corresponding value for the activation barrier, DeltaH(double dagger), was +94.3 kcal/mol relative to the separated reactants in vacuo and +89.9 kcal/mol in PCM aqueous media. In contrast, the reaction H2O2 + H2N-CH2-B(OH)2 --> H2N-CH2-OH + B(OH)3 was calculated to be highly exothermic with an DeltaH(298)(0) value of -100.9 kcal/mol at the MP2(FC)/cc-pVTZ computational level in vacuo and -99.6 kcal/mol in CPCM aqueous media; the highest-energy transition state for the multistep process associated with this reaction involved the rearrangement of H2N-CH2-B(OH)(OOH) to H2N-CH2-O-B(OH)2 with a DeltaH(double dagger) value of +23.2 kcal/mol in vacuo relative to the separated reactants. These computational results for boroglycine are in accord with the experimental observations for the deboronation of the FDA approved anticancer drug bortezomib (Velcade, PS-341), where it was found to be the principle deactivation pathway (Labutti et al. Chem. Res. Toxicol. 2006, 19, 539-546).

A computational characterization of boron-oxygen multiple bonding in HN=CH-CH=CH-NH-BO.

Structures, relative energies, and bonding characteristics for various conformers of 3-imino-N-(oxoboryl)prop-1-en-1-amine, HN=CH-CH=CH-NH-BO, and the corresponding borocycle (-HN=CH-CH=CH-NH-B-)O are discussed using results from second-order Møller-Plesset (MP2) perturbation theory with the Dunning-Woon correlation-consistent cc-pVDZ, aug-cc-pVDZ, and cc-pVTZ basis sets. These MP2 results are compared to those from computationally efficient density functional theory (DFT) calculations using the LDA, PBE, TPSS, BLYP, B3LYP, BVP86, OLYP, O3LYP, and PBE1PBE functionals in conjunction with the economical Pople-type 6-311++G(d,p) basis set to evaluate the suitability of these DFT/6-311++G(d,p) levels for use with larger boron-containing systems. The effects of an aqueous environment were incorporated into the calculations using COSMO methodology. The calculated boron-oxygen bond lengths, orbital compositions, and bond orders in all the (acyclic) HN=CH-CH=CH-NH-BO conformers were consistent with the presence of a boron-oxygen triple bond, similar to that found in H-BO and H2N-BO. The (-HN=CH-CH=CH-NH-B-)O borocycle is predicted to be planar (C2v symmetry), and it is approximately 30 kcal/mol lower in energy than any of the (acyclic) HN=CH-CH=CH-NH-BO conformers; the boron-oxygen bond in this borocycle has significant double bond character, a bonding scheme for which there has been only one experimental structure reported in the literature (Vidovic, D. ; et al. J. Am. Chem. Soc. 2005, 127, 4566- 4569).

Interactions of Metal Ions with Water: Ab Initio Molecular Orbital Studies of Structure, Bonding Enthalpies, Vibrational Frequencies and Charge Distributions. 1. Monohydrates.

The formation and properties of a wide range of metal ion monohydrates, M(n)()(+)-OH(2), where n = 1 and 2, have been studied by ab initio molecular orbital calculations at the MP2(FULL)/6-311++G//MP2(FULL)/6-311++G and CCSD(T)(FULL)/6-311++G//MP2(FULL)/6-311++G computational levels. The ions M are from groups 1A, 2A, 3A, and 4A in the second, third, and fourth periods of the periodic table and the first transition series. Structural parameters, vibrational frequencies, bonding enthalpies, orbital occupancies and energies, and atomic charge distributions are reported. Trends in these properties are correlated with the progressive occupancy of the s, p, and d orbitals. Except for K(+)-OH(2) and Ca(2+)-OH(2), the O-H bond lengths and HOH angles are greater in the hydrates than in unbound water. The M-O bond lengths decrease proceeding from group 1A --> 4A but become larger in proceeding from the second --> fourth period. The bonding enthalpies, are found to be inversely linearly dependent on the M-O bond length M(n)()(+) according to equations of the form = A + B(1/M-O) for n = 1 and n = 2. Within each monohydrate the distribution of atomic charge reveals a small but definite transfer of charge from water to the metal ion. Compared to unbound water there is, in a metal-ion-bound water complex, an increase in the electronic (negative) charge on the oxygen atom, accompanied by a (significantly) larger decrease in the electronic charge on the hydrogen atoms. The bonding of the water molecule, although electrostatic in origin, is thus more complex than a simple interaction between a point charge on the metal ion, and the water dipole.

A regulatory role of the Bateman domain of IMP dehydrogenase in adenylate nucleotide biosynthesis.

The Bateman domain (CBS subdomain) of IMP dehydrogenase (IMPDH), a rate-limiting enzyme of the de novo GMP biosynthesis, is evolutionarily conserved but has no established function. Deletion of the Bateman domain has no effect on the in vitro IMPDH activity. We report that in vivo deletion of the Bateman domain of IMPDH in Escherichia coli (guaB(DeltaCBS)) sensitizes the bacterium to growth arrest by adenosine and inosine. These nucleosides exert their growth inhibitory effect via a dramatic increase in the intracellular adenylate nucleotide pool, which results in the enhanced allosteric inhibition of PRPP synthetase and consequently a PRPP deficit. The ensuing starvation for pyrimidine nucleotides culminates in growth arrest. Thus, deletion of the Bateman domain of IMPDH derepresses the synthesis of AMP from IMP. The growth inhibitory effect of inosine can be rescued by second-site suppressor mutations in the genes responsible for the conversion of inosine to AMP (gsk, purA, and purB) as well as by the prsA1 allele, which encodes a PRPP synthetase that is insensitive to allosteric inhibition by adenylate nucleotides. Importantly, the guaB(DeltaCBS) phenotype can be complemented in trans by a mutant guaB allele, which encodes a catalytically disabled IMPDH(C305A) protein containing an intact Bateman domain. We conclude that the Bateman domain of IMPDH is a negative trans-regulator of adenylate nucleotide synthesis, and that this role is independent of the catalytic function of IMPDH in the de novo GMP biosynthesis.

Discovery of novel types of inhibitors of S-adenosylmethionine synthesis by virtual screening.

S-adenosylmethionine (AdoMet) lies at an intersection of nucleotide and amino acid metabolism and performs a multitude of metabolic functions. AdoMet formation is catalyzed by S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase (MAT)), which is a target for development of anticancer and antimicrobial agents. High affinity MAT inhibitors have been found through computational docking of more than 200000 compounds for predicted binding to the crystallographically defined nucleotide binding region of the enzyme's active site. Two of the top scoring candidate compounds had IC(50) values less than 10 nM, more than 10000-fold lower than the substrates' K(M) values. The compounds are structurally unrelated to the natural ligands of the enzyme. The enzyme is protected from inhibition by ATP, but not by methionine, consistent with binding at the adenosyl region of the active site. These results validate in silico screening as a robust approach to the discovery of inhibitors of this chemotherapeutically relevant enzyme.

Dimers of boroglycine and methylamine boronic acid: a computational comparison of the relative importance of dative versus hydrogen bonding.

Boronic acids are widely used in materials science, pharmacology, and the synthesis of biologically active compounds. In this Article, geometrical structures and relative energies of dimers of boroglycine, H2N-CH2-B(OH)2, and its constitutional isomer H3C-NH-B(OH)2, were computed using second-order Møller-Plesset perturbation theory and density functional theory; Dunning-Woon correlation-consistent cc-pVDZ, aug-cc-pVDZ, cc-pVTZ, and aug-cc-pVTZ basis sets were employed for the MP2 calculations, and the Pople 6-311++G(d,p) basis set was employed for a majority of the DFT calculations. Effects of an aqueous environment were incorporated into the results using PCM and COSMO-RS methodology. The lowest-energy conformer of the H2N-CH2-B(OH)2 dimer was a six-membered ring structure (chair conformation; Ci symmetry) with two intermolecular B:N dative-bonds; it was 14.0 kcal/mol lower in energy at the MP2/aug-cc-pVDZ computational level than a conformer with the classic eight-centered ring structure (Ci symmetry) in which the boroglycine monomers are linked by a pair of H-O...H bonds. Compared to the results of MP2 calculations with correlation-consistent basis sets, DFT calculations using the PBE1PBE and TPSS functionals with the 6-311++G(d,p) basis set were significantly better at predicting relative conformational energies of the H2N-CH2-B(OH)2 and H3C-NH-B(OH)2 dimers than corresponding calculations using the BLYP, B3LYP, OLYP, and O3LYP functionals, particularly with respect to dative-bonded structures.

Metal ion activation of S-adenosylmethionine decarboxylase reflects cation charge density.

S-Adenosylmethionine decarboxylase from Escherichia coli is a pyruvoyl cofactor-containing enzyme that requires a metal cation for activity. We have found that the enzyme is activated by cations of varying charge and ionic radius, such as Li+, A13+, Tb3+, and Eu3+, as well as the divalent cations Mg2+, Mn2+, and Ca2+. All of the activating cations provide kcat values within 30-fold of one another, showing that the charge of the cation does not greatly influence the rate-limiting step for decarboxylase turnover. Cation concentrations for half-maximal activation decrease by >100-fold with each increment of increase in the cation charge, ranging from approximately 300 mM with Li+ to approximately 2 microM with trivalent lanthanide ions. The cation affinity is related to the charge/radius ratio of the ion for those ions with exchangeable first coordination sphere ligands. The exchange-inert cation Co(NH3)63+ activates in the presence of excess EDTA (and NH4+ does not activate), indicating that direct metal coordination to the protein or substrate is not required for activation. The binding of metal ions (monitored by changes in the protein tryptophan fluorescence) and enzyme activation are both cooperative with Hill coefficients as large as 4, the active site stoichiometry of this (alphabeta)4 enzyme. The Hill coefficients for Mg2+ binding and activation increase from 1 to approximately 4 as the KCl concentration increases, which is also observed with NaCl or KNO3; neither Na+ nor K+ activates the enzyme. The single tryptophan in the protein is located 16 residues from the carboxyl terminus of the pyruvoyl-containing alpha chain, in a 70-residue segment that is not present in metal ion independent AdoMet decarboxylases from other organisms. The results are consistent with allosteric metal ion activation of the enzyme, congruent with the role of the putrescine activator of the mammalian AdoMet decarboxylase.

A computational investigation of the geometrical structure and protodeboronation of boroglycine, H2N-CH2-B(OH)2.

In this article the geometrical structure of the simple, achiral, alpha-amino boronic acid boroglycine, H2N-CH2-B(OH)2, was investigated using density functional theory (DFT), second-order Møller-Plesset (MP2) perturbation theory, and coupled cluster methodology with single- and double-excitations (CCSD); the effects of an aqueous environment were incorporated into the results by using a few explicit water molecules and/or self-consistent reaction field (SCRF) calculations with the IEF polarizable continuum model (PCM). Neutral reaction mechanisms were investigated for the direct protodeboronation (hydrolysis) of boroglycine (H2O+H2N-CH2-B(OH)2-->B(OH)3+H2N-CH3), for which DeltaH degrees 298 was -21.9 kcal/mol at the MP2(FC)/aug-cc-pVDZ level, and for the 1,2-carbon-to-nitrogen shift of the -B(OH)2 moiety (H2N-CH2-B(OH)2-->H3C-NH-B(OH)2), for which the corresponding value of DeltaH degrees 298 was -18.2 kcal/mol. A boron-oxygen double-bonded intermediate was found to play an important role in the 1,2-rearrangement mechanism.

Structure of the boronic acid dimer and the relative stabilities of its conformers.

Despite the widespread use of boronic acids in materials science and as pharmaceutical agents, many aspects of their structure and reactivity are not well understood. In this research the boronic acid dimer, [HB(OH)(2)](2), was studied by second-order Møller-Plesset (MP2) perturbation theory and coupled cluster methodology with single and double excitations (CCSD). Pople split-valence 6-31+G*, 6-311G**, and 6-311++G** and Dunning-Woon correlation-consistent cc-pVDZ, aug-cc-pVDZ, cc-pVTZ, and aug-cc-pVTZ basis sets were employed for the calculations. A doubly hydrogen-bonded conformer (1) of the dimer was consistently found to be lowest in energy; the structure of 1 was planar (C(2h)) at most computational levels employed but was significantly nonplanar (C(2)) at the MP2/6-311++G** and CCSD/6-311++G** levels, the result of an intrinsic problem with Pople-type sp-diffuse basis functions on heavy atoms. The dimerization energy, enthalpy, and free energy for the formation of (1) from the exo-endo conformer of the monomer were -10.8, -9.2, and +1.2 kcal/mol, respectively, at the MP2/aug-cc-pVTZ level. Several other hydrogen-bonded conformers of the dimer were local minima on the potential energy surface (PES) and ranged from 2 to 5 kcal/mol higher in energy than 1. Nine doubly OH-bridged conformers, in which the boron atoms were tetracoordinated, were also local minima on the PES, but they were all greater than 13 kcal/mol higher in energy than 1; doubly H-bridged structures proved to be transition states. MP2 and CCSD results were compared to those from the BLYP, B3LYP, OLYP, O3LYP, PBE1PBE, and TPSS functionals with the 6-311++G** and aug-cc-pVTZ basis sets; the PBE1PBE functional performed best relative to the MP2 and CCSD results. Self-consistent reaction field (SCRF) calculations predict that boronic acid dimerization is less favorable in solution than in vacuo.

Catalytic properties of the archaeal S-adenosylmethionine decarboxylase from Methanococcus jannaschii.

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl cofactor-dependent enzyme that participates in polyamine biosynthesis. AdoMetDC from the Archaea Methanococcus jannaschii is a prototype for a recently discovered class that is not homologous to the eucaryotic enzymes or to a distinct group of microbial enzymes. M. jannaschii AdoMetDC has a Km of 95 microm and the turnover number (kcat) of 0.0075 s(-1) at pH 7.5 and 22 degrees C. The turnover number increased approximately 38-fold at a more physiological temperature of 80 degrees C. AdoMetDC was inactivated by treatment with the imine reductant NaCNBH3 only in the presence of substrate. Mass spectrometry of the inactivated protein showed modification solely of the pyruvoyl-containing subunit, with a mass increase corresponding to reduction of a Schiff base adduct with decarboxylated AdoMet. The presteady state time course of the AdoMetDC reaction revealed a burst of product formation; thus, a step after CO2 formation is rate-limiting in turnover. Comparable D2O kinetic isotope effects of were seen on the first turnover (1.9) and on kcat/Km (1.6); there was not a significant D2O isotope effect on kcat, suggesting that product release is rate-limiting in turnover. The pH dependence of the steady state rate showed participation of acid and basic groups with pK values of 5.3 and 8.2 for kcat and 6.5 and 8.3 for kcat/Km, respectively. The competitive inhibitor methylglyoxal bis(guanylhydrazone) binds at a single site per (alphabeta) heterodimer. UV spectroscopic studies show that methylglyoxal bis(guanylhydrazone) binds as the dication with a 23 microm dissociation constant. Studies with substrate analogs show a high specificity for AdoMet.

Structural studies of inhibition of S-adenosylmethionine synthetase by slow, tight-binding intermediate and product analogues.

S-Adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase) catalyzes a two-step reaction in which tripolyphosphate (PPPi) is a tightly bound intermediate. Diimidotriphosphate (O(3)P-NH-PO(2)-NH-PO(3); PNPNP), a non-hydrolyzable analogue of PPPi, is the most potent known inhibitor of AdoMet synthetase with a K(i) of 2 nM. The structural basis for the slow, tight-binding inhibition by PNPNP has been investigated by spectroscopic methods. UV difference spectra reveal environmental alterations of aromatic protein residues upon PNPNP binding to form the enzyme.2Mg(2+).PNPNP complex, and more extensive changes upon formation of the enzyme.2Mg(2+).PNPNP.AdoMet complex. Stopped-flow kinetic studies of complex formation revealed that two slow isomerizations follow PNPNP binding in the presence of AdoMet, in contrast to the lower affinity, rapid-equilibrium binding in the absence of AdoMet. (31)P NMR spectra of enzyme complexes with PNPNP revealed electronic perturbations of each phosphorus atom by distinct upfield chemical shifts for each of the three phosphoryl groups in the enzyme.2Mg(2+).PNPNP complex, and further upfield shifts of at least 2 resonances in the complex with AdoMet. Comparison of the chemical shifts for the enzyme-bound PNPNP with the enzyme complexes containing either the product analogue O(3)P-NH-PO(3) or O(3)P-O-PO(2)-NH-PO(3) indicates that the shifts on binding are largest at the binding sites corresponding to those for the alpha and gamma phosphoryl groups of the nucleotide (-3.1 to -4.1 ppm), while the resonance at the beta phosphoryl group position shifts by -2.1 ppm. EPR spectra of Mn(2+) complexes demonstrate spin coupling between the two Mn(2+) in both enzyme.2Mn(2+).PNPNP and enzyme.2Mn(2+).PNPNP.AdoMet, indicating that the metal ions have comparable distances in both cases. The combined results indicate that formation of the highest affinity complex is associated with protein side chain rearrangements and increased electron density at the ligand phosphorus atoms, likely due to ionization of an -NH- group of the inhibitor. The energetic feasibility of ionization of a -NH- group when two Mg(2+) ions are bound to O(3)P-NH-PO(3) is supported by density functional theoretical calculations on model chelates. This mode of interaction is uniquely available to compounds with P-NH-P linkages and may be possible with other proteins in which multiple cations coordinate a polyphosphate chain.

Conformational dynamics of the active site loop of S-adenosylmethionine synthetase illuminated by site-directed spin labeling.

S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase, methionine adenosyltransferase, a.k.a. MAT) is one of numerous enzymes that have a flexible polypeptide loop that moves to gate access to the active site in a motion that is closely coupled to catalysis. Crystallographic studies of this tetrameric enzyme have shown that the loop is closed in the absence of bound substrates. However, the loop must open to allow substrate binding and a variety of data indicate that the loop is closed during the catalytic steps. Previous kinetic studies indicate that during turnover loop motion occurs on a time scale of 10(-2)s, ca. 10-fold faster than chemical transformations and turnover. Site-directed spin labeling has been used to introduce nitroxide groups at two positions in the loop to illuminate how the motion of the loop is affected by substrate binding. The two loop mutants constructed, G105C and D107C, retain wild type levels of MAT activity; attachment of a methanethiosulfonate spin label to convert the cysteine to the "R1" residue reduced the k(cat) only for the labeled D107R1 form (7-fold). The K(m) value for methionine increased 2- to 4-fold for the cysteine mutants and 2- to 7-fold for the labeled proteins, whereas the K(m) for ATP was changed by at most 2-fold. EPR spectra for both labeled proteins are nearly identical and show the presence of two major spin label environments with rotational diffusion rates differing by approximately 10-fold; the slower rate is ca. 4-fold faster than the estimated protein rotational rate. The spectra are not altered by addition of substrates or products. At both positions the less mobile conformation constitutes ca. 65% of the total species, indicating an equilibrium that only slightly favors one form, that in which the label is more immobilized. The equilibrium constant that relates the two forms is comparable to the equilibrium constant of 1.5 for a conformational change that was previously deduced from the viscosity dependence of the rate of AdoMet formation. The results suggest that the motion of the loop may be an intrinsic property of the protein and not be strictly ligand modulated.