Allosteric regulation of serine hydroxymethyltransferase from mung bean (Phaseolus aureus)

Serine hydroxymethyltransferase, the first enzyme in the pathway for the interconversion of one carbon compounds was purified from mung bean seedlings by ammonium sulfate fractionation, DEAE-Sephadex, Blue Sepharose CL-6B affinity chromatography and gel filteration on Sephacryl S-200. The specific activity of the enzyme, 0.73 (u mol HCHO formed/min/mg protein) was 104 times larger than the highest value reported hitherto. Saturation of tetrahydrofolate was sigmoid, whereas with serine was hyperbolic, with nH values of 1.9 and 1.0 respectively. Reduced nicotinamide adenine dinucleotide, lysine and methionine decreased, whereas nicotinamide adenine dinucleotide, adenosine 5′-monophosphate and adenosine 5′-triphosphate increased the sigmoidicity. These results suggest that serine hydroxymethyltransferase from mung bean is a regulatory enzyme. H4folate; (±)-L-tetrahydrofolate

The metabolism of phenolic acids in the rat

Some of the enzyme systems in the formation of p-hydroxybenzoate from tyrosine have been studied in the rat liver in vitro. The conversion of p-hydroxycinnamate into p-hydroxybenzoate, which was found in rat liver mitochondria showed a number of differences when compared with the b-oxidation of fatty acids. Studies with p-hydroxy[U-14C]cinnamate indicated that 14CO2 was released during the formation of p-hydroxybenzoate. The formation of p-hydroxycinnamate from tyrosine of p-hydroxyphenyl-lactate could not be demonstrated in vitro. The interconversion of p-hydroxycinnamate and p-hydroxyphenylpropionate was demonstrated in rat liver mitochondria.

Purification and Regulatory Properties of Mung Bean (Vigna Radiata L.) Serine Hydroxymethyltransferase 1

Serine hydroxymethyltransferase, the first enzyme in the pathway for interconversion of C1 fragments, was purified to homogeneity for the first time from any plant source. The enzyme from 72-h mung bean (Vigna radiata L.) seedlings was isolated using Blue Sepharose CL-6B and folate-AH-Sepharose-4B affinity matrices and had the highest specific activity (1.33 micromoles of HCHO formed per minute per milligram protein) reported hitherto. The enzyme preparation was extremely stable in the presence of folate or L-serine. Pyridoxal 5'-phosphate, ethylenediaminetetraacetate and 2-mercaptoethanol prevented the inactivation of the enzyme during purification. The enzyme functioned optimally at pH 8.5 and had two temperature maxima at 35 and 55°C. The Km values for serine were 1.25 and 68 millimolar, corresponding to Vmax values of 1.8 and 5.4 micromoles of HCHO formed per minute per milligram protein, respectively. The K0.5 value for L-tetrahydrofolate (H4folate) was 0.98 millimolar. Glycine, the product of the reaction and D-cycloserine, a structural analog of D-alanine, were linear competitive inhibitors with respect to L-serine with Ki values of 2.30 and 2.02 millimolar, respectively. Dichloromethotrexate, a substrate analog of H4folate was a competitive inhibitor when H4folate was the varied substrate. Results presented in this paper suggested that pyridoxal 5'-phosphate may not be essential for catalysis.The sigmoid saturation pattern of H4folate (nH = 2.0), one of the substrates, the abolition of sigmoidicity by NADH, an allosteric positive effector (nH = 1.0) and the increase in sigmoidicity by NAD+ and adenine nucleotides, negative allosteric effectors (nH = 2.4) clearly established that this key enzyme in the folate metabolism was an allosteric protein. Further support for this conclusion were the observations that (a) serine saturation exhibited an intermediary plateau region; (b) partial inhibition by methotrexate, aminopterin, O-phosphoserine, DL-{alpha}-methylserine and DL-O-methylserine; (c) subunit nature of the enzyme; and (d) decrease in the nH value from 2.0 for H4folate to 1.5 in presence of L-serine. These results highlight the regulatory nature of mung bean serine hydroxymethyltransferase and its possible involvement in the modulation of the interconversion of folate coenzymes.

Rotational isomerism about the Ca-CO bond in proline derivatives. 1H and 13C NMR studies of benzyloxycarbonyl-Pro-N-methylamide and pivaloyl-Pro-N-methylamide

The 270 MHz 1H n.m.r. spectrum of benzyloxycarbonyl-Pro-N-methylamide in CDCl3 is exchange broadened at 293° K. Spectral lines due to two species are frozen out at 253° K and a dynamically averaged spectrum is obtained at 323° K. A selective broadening of the Cβ and Cγ resonances in the 13C n.m.r. spectrum is observed at 253° K, with a splitting of the Cβ and Cγ resonances into a pair of lines of unequal intensity. A similar broadening of Cβ and Cγ peaks is also detected in pivaloyl-Pro-N-methylamide where cis-trans interconversion about the imide bond is precluded by the bulky t-butyl group. The rate process is thus attributed to rotation about the Cα-CO bond (ψ) and a barrier (ΔG#) of 14kcal mol-1 is estimated. 13C n.m.r. data for pivaloyl-Pro-N-methylamide in a number of solvents is presented and the differences in the Cβ and Cγ chemical shifts are interpreted in terms of rotational isomerism about the Cα-CO bond.

Regulation of monkey liver serine hydroxymethyltransferase by nicotinamide nucleotide

The positive homotropic binding of tetrahydrofolate to monkey liver serine hydroxymethyltransferase was abolished on preincubating the enzyme with NADH and NADPH. NAD+ was a negative heterotropic effector, whereas NADP+ was without effect. The allosteric effects of nicotinamide nucleotides on the serine hydroxymethyltransferase, reported for the first time, lead to a better understanding of the regulation of the metabolic interconversion of folate coenzymes.

Diproline Templates as Folding Nuclei in Designed Peptides. Conformational Analysis of Synthetic Peptide Helices Containing Amino Terminal Pro-Pro Segments

The effect of N-terminal diproline segments in nucleating helical folding in designed peptides has been studied in two model sequences Piv-Pro-Pro-Aib-Leu-Aib-Phe-OMe (1) and Boc-Aib-Pro-Pro-Aib-Val-Ala-Phe-OMe (2). The structure of 1 in crystals, determined by X-ray diffraction, reveals a helical (RR) conformation for the segment residues 2 to 5, stabilized by one 4 -> 1 hydrogen bond and two 5 -> 1 interactions. The N-terminus residue, Pro(1) adopts a polyproline II (P-II) conformation. NMR studies in three different solvent systems support a conformation similar to that observed in crystals. In the apolar solvent CDCl3, NOE data favor the population of both completely helical and partially unfolded structures. In the former, the Pro-Pro segment adopts an alpha(R)-alpha(R) conformation, whereas in the latter, a P-II-alpha(R) structure is established. The conformational equilibrium shifts in favor of the P-II-alpha(R) structure in solvents like methanol and DMSO. A significant population of the Pro(1)- Pro(2) cis conformer is also observed. The NMR results are consistent with the population of at least three conformational states about Pro- Pro segment: trans alpha(R)-alpha(R), trans P-II-alpha(R) and cis P-II-alpha(R). Of these, the two trans conformers are in rapid dynamic exchange on the NMR time scale, whereas the interconversion between cis and trans form is slow. Similar results are obtained with peptide 2. Analysis of 462 diproline segments in protein crystal structures reveals 25 examples of the alpha(R)-alpha(R) conformation followed by a helix. Modeling and energy minimization studies suggest that both P-II-alpha(R) and alpha(R)-alpha(R) conformations have very similar energies in the model hexapeptide 1

The precision of regulation in Dictyostelium discoideum: implications for cell-type proportioning in the absence of spatial pattern

We have made careful counts of the exact number of spore, stalk and basal disc cells in small fruiting bodies of Dictyostelium discoideum (undifferentiated amoebae are found only rarely and on average their fraction is 4.96 x 10(-4)). (i) Within aggregates of a given size, the relative apportioning of amoebae to the main cell types occurs with a remarkable degree of precision. In most cases the coefficient of variation (c.v.) in the mean fraction of cells that form spores is within 4.86%. The contribution of stalk and basal disc cells is highly variable when considered separately (c.v.'s upto 25% and 100%, respectively), but markedly less so when considered together. Calculations based on theoretical models indicate that purely cell-autonomous specification of cell, fate cannot account for die observed accuracy of proportioning. Cell-autonomous determination to a prestalk or prespore condition followed by cell type interconversion, and stabilised by feedbacks, suffices to explain the measured accuracy. (ii) The fraction of amoebae that differentiates into spores increases monotonically with the total number of cells. This fraction rises from an average of 73.6% for total cell numbers below 30 and reaches 86.0% for cell numbers between 170 and 200 (it remains steady thereafter at around 86%). Correspondingly, the fraction of amoebae differentiating into stalk or basal disc decreases viith total size. These trends are in accordance with evolutionary expectations and imply that a mechanism for sensing the overall size of the aggregate also plays an essential role in the determination of cell-type proportions.

Diphenylantimony(V) oxo/chloro carboxylates and phosphinates: crystal structures of {SbPh2Cl[O2P(C6H11)2]}2O and [SbPh2(O2CPh)2]2O

Reaction of SbPh(2)Cl(3) (1 mol) with the silver salt of dicyclohexylphosphinic acid (2 mol) afforded {SbPh(2)Cl[O2P(C6H11)(2)]}O-2 1, a similar reaction with AgO2P(C8H15)(2) gave a product formulated as {SbPh(2)Cl[O2P(C8H15)(2)]}O-2 2. Similar reactions with silver carboxylates (1:3 stoichiometry) led to the crystalline derivatives [SbPh(2)(O(2)CR)(2)]O-2 (R = Ph 3, CHPh(2) 4, 2,4,6-Me(3)C(6)H(2) 5, 2-MeC(6)H(4) 6 or 4-MeC(6)H(4) 7), whereas the 1:2 reaction afforded crystalline SbPh(2)Cl(O(2)CR)(2) (R = Ph 8, 2-MeC(6)H(4) 9 or 4-MeC(6)H(4) 10). Interconversion of the previously known compounds [SbPh(2)(O(2)CMe)(2)]O and Sb(4)Ph(8)O(6) . 3MeCO(2)H was achieved and established by H-1 NMR spectroscopy. Compounds 1 and 3 were further characterized by X-ray diffraction; the antimony in 1 is six-co-ordinated with bridging phosphinates whereas in 3 it is seven-co-ordinated with chelating benzoates. Short Sb-O (oxo) distances (1.923 Angstrom) and near linearity at the bridging ox

The molecular dynamics study of lithium ion conduction in phosphate glasses and the role of non-bridging oxygen

Molecular dynamics (MD) simulation of lithium phosphate (Li2O-P2O5) glasses with varying Li2O content has been carried out. Two different P-O distances corresponding to phosphorus coordination with bridging oxygen (BO) and non-bridging oxygen (NBO) were identified in the simulated glasses. NBO-BO interconversion or bond switching was noted, which results in a dynamic equilibration of the tetrahedral phosphate units (P-n, n = 1,3 indicates the number of bridging oxygen atoms in the coordination of phosphorus). The NBO-BO bond switching is mildly activated with an effective activation barrier of 0.03-0.05 eV. Lithium ion jumps do not appear to be strongly coupled to bond switching. But the number of Li+ ions coordinated to an optimum number of NBOs and the number of Li+ ions jumping out of their sites appear to be correlated. Detailed analysis was made of the dynamics of P-n species and new insights have been obtained regarding ion migration in network-modified phosphate glasses.

Designed three-stranded beta-sheet in an alpha/beta hybrid peptide

The incorporation of beta-amino acid residues into the antiparallel beta-strand segments of a multi-stranded beta-sheet peptide is demonstrated for a 19-residue peptide, Boc-LV(beta)FV(D)PGL(beta)FVVL(D)PGLVL(beta)FVV-OMe (BBH19). Two centrally positioned (D)Pro-Gly segments facilitate formation of a stable three-stranded beta-sheet, in which beta-phenylalanine ((beta)Phe) residues occur at facing positions 3, 8 and 17. Structure determination in methanol solution is accomplished by using NMR-derived restraints obtained from NOEs, temperature dependence of amide NH chemical shifts, rates of H/D exchange of amide protons and vicinal coupling constants. The data are consistent with a conformationally well-defined three-stranded beta-sheet structure in solution. Cross-strand interactions between (beta)Phe3/(beta)Phe17 and (beta)Phe3/Val15 residues define orientations of these side-chains. The observation of close contact distances between the side-chains on the N- and C-terminal strands of the three-stranded beta-sheet provides strong support for the designed structure. Evidence is presented for multiple side-chain conformations from an analysis of NOE data. An unusual observation of the disappearance of the Gly NH resonances upon prolonged storage in methanol is rationalised on the basis of a slow aggregation step, resulting in stacking of three-stranded beta-sheet structures, which in turn influences the conformational interconversion between type I' and type II' beta-turns at the two (D)Pro-Gly segments. Experimental evidence for these processes is presented. The decapeptide fragment Boc-LV(beta)FV(D)PGL(beta)FVV-OMe (BBH10), which has been previously characterized as a type I' beta-turn nucleated hairpin, is shown to favour a type II' beta-turn conformation in solution, supporting the occurrence of conformational interconversion at the turn segments in these hairpin and sheet structures.

``On-the-fly'' kinetics of enzymatic racemization using deuterium NMR in DNA-based chiral oriented media

We report the in situ and real-time monitoring of the interconversion of L- and D-alanine-d(3) by alanine racemase from Bacillus stearothermophilus directly observed by H-2 NMR spectroscopy in anisotropic phase. The enantiomers are distinguished by the difference of their H-2 quadrupolar splittings in a chiral liquid crystal containing short DNA fragments. The proof-of-principle, the reliability, and the robustness of this new method is demonstrated by the determination of the turnover rates of the enzyme using the Michaelis Menten model.

Impact of Mutating the Key Residues of a Bifunctional 5,10-Methylenetetrahydrofolate Dehydrogenase-Cyclohydrolase from Escherichia coli on Its Activities

Methylenetetrahydrofolate dehydrogenase-cyclohydrolase (FolD) catalyzes interconversion of 5,10-methylene-tetrahydrofolate and 10-formyl-tetrahydrofolate in the one-carbon metabolic pathway. In some organisms, the essential requirement of 10-formyl-tetrahydrofolate may also be fulfilled by formyltetrahydrofolate synthetase (Fhs). Recently, we developed an Escherichia coli strain in which the folD gene was deleted in the presence of Clostridium perfringens fhs (E. coli Delta folD/p-fhs) and used it to purify FolD mutants (free from the host-encoded FolD) and determine their biological activities. Mutations in the key residues of E. coli FolD, as identified from three-dimensional structures (D121A, Q98K, K54S, Y50S, and R191E), and a genetic screen (G122D and C58Y) were generated, and the mutant proteins were purified to determine their kinetic constants. Except for the R191E and K54S mutants, others were highly compromised in terms of both dehydrogenase and cyclohydrolase activities. While the R191E mutant showed high cyclohydrolase activity, it retained only a residual dehydrogenase activity. On the other hand, the K54S mutant lacked the cyclohydrolase activity but possessed high dehydrogenase activity. The D121A and G122D (in a loop between two helices) mutants were highly compromised in terms of both dehydrogenase and cyclohydrolase activities. In vivo and in vitro characterization of wild-type and mutant (R191E, G122D, D121A, Q98K, C58Y, K54S, and Y50S) FolD together with three-dimensional modeling has allowed us to develop a better understanding of the mechanism for substrate binding and catalysis by E. coli FolD.

Autoinhibitory mechanism and activity-related structural changes in a mycobacterial adenylyl cyclase

An adenylyl cyclase from Mycobacterium avium, Mal 120, is a functional orthologue of a pseudogene Rv1120c from Mycobacterium tuberculosis. We report the crystal structure of Mal 120 in a monomeric form and its truncated construct as a dimer. Mal 120 exists as a monomer in solution and crystallized as a monomer in the absence of substrate or inhibitor. An additional alpha-helix present at the N-terminus of the monomeric structure blocks the active site by interacting with the substrate binding residues and occupying the dimer interface region. However, the enzyme has been found to be active in solution, indicating the movement of the helix away from the interface to facilitate the formation of active dimers in conditions favourable for catalysis. Thus, the N-terminal helix of Ma1120 keeps the enzyme in an autoinhibited state when it is not active. Deletion of this helix enabled us to crystallize the molecule as an active homodimer in the presence of a P-site inhibitor 2',5'-dideoxy-3'-ATP, or pyrophosphate along with metal ions. The substrate specifying lysine residue plays a dual role of interacting with the substrate and stabilizing the dimer. The dimerization loop region harbouring the second substrate specifying residue, an aspartate, shows significant differences in conformation and position between the monomeric and dimeric structures. Thus, this study has not only revealed that significant structural transitions are required for the interconversion of the inactive and the active forms of the enzyme, but also provided precise nature of these transitions. (C) 2015 Elsevier Inc. All rights reserved.

Aqueous ring-opening metathesis polymerizations

Past workers in this group as well as in others have made considerable progress in the understanding and development of the ring-opening metathesis polymerization (ROMP) technique. Through these efforts, ROMP chemistry has become something of an organometallic success story. Extensive work was devoted to trying to identify the catalytically active species in classical reaction mixtures of early metal halides and alkyl aluminum compounds. Through this work, a mechanism involving the interconversion of metal carbenes and metallacyclobutanes was proposed. This preliminary work finally led to the isolation and characterization of stable metal carbene and metallacyclobutane complexes. As anticipated, these well-characterized complexes were shown to be active catalysts. In a select number of cases, these catalysts have been shown to catalyze the living polymerization of strained rings such as norbornene. The synthetic control offered by these living systems places them in a unique category of metal catalyzed reactions. To take full advantage of these new catalysts, two approaches should be explored. The first takes advantage of the unusual fact that all of the unsaturation present in the monomer is conserved in the polymer product. This makes ROMP techniques ideal for the synthesis of highly unsaturated, and fully conjugated polymers, which find uses in a variety of applications. This area is currently under intense investigation. The second aspect, which should lend itself to fruitful investigations, is expanding the utility of these catalysts through the living polymerization of monomers containing interesting functional groups. Polymer properties can be dramatically altered by the incorporation of functional groups. It is this latter aspect which will be addressed in this work.

After a general introduction to both the ring-opening metathesis reaction (Chapter 1) and the polymerization of fuctionalized monomers by transition metal catalysts (Chapter 2), the limits of the existing living ROMP catalysts with functionalized monomers are examined in Chapter 3. Because of the stringent limitations of these early metal catalysts, efforts were focused on catalysts based on ruthenium complexes. Although not living, and displaying unusually long induction periods, these catalysts show high promise for future investigations directed at the development of catalysts for the living polymerization of functionalized monomers. In an attempt to develop useful catalysts based on these ruthenium complexes, efforts to increase their initiation rates are presented in Chapter 4. This work eventually led to the discovery that these catalysts are highly active in aqueous solution, providing the opportunity to develop aqueous emulsion ROMP systems. Recycling the aqueous catalysts led to the discovery that the ruthenium complexes become more activated with use. Investigations of these recycled solutions uncovered new ruthenium-olefin complexes, which are implicated in the activation process. Although our original goal of developing living ROMP catalysts for the polymerization of fuctionalized monomers is yet to be realized, it is hoped that this work provides a foundation from which future investigations can be launched.

In the last chapter, the ionophoric properties of the poly(7-oxanobornene) materials is briefly discussed. Their limited use as acyclic host polymers led to investigations into the fabrication of ion-permeable membranes fashioned from these materials.

Bis(thiophenolate)pyridine pincer ligands and trivalent zirconocene complexes relevant to early transition metal polymerization catalysts

Two major topics are covered: the first chapter is focused on the development of post-metallocene complexes for propylene polymerization. The second and third chapters investigate the consequences of diisobutylaluminum hydride (HAlⁱBu₂) additives in zirconocene based polymerization systems.

The synthesis, structure, and solution behavior of early metal complexes with a new tridentate LX₂ type ligand, bis(thiophenolate)pyridine ((SNS) = (2-C₆H₄S)₂-2,6-C₅H₃N) are investigated. SNS complexes of Ti, Zr, and Ta having dialkylamido coligands were synthesized and structurally characterized. The zirconium complex, (SNS)Zr(NMe₂)₂, displays C₂ symmetry in the solid state. Solid-state structures of tantalum complexes (SNS)Ta(NMe₂)₃ and (SNS)TaCl(NEt₂)₂ also display pronounced C₂ twisting of the SNS ligand. 1D and 2D NMR experiments show that (SNS)Ta(NMe₂)₃ is fluxional with rotation about the Ta N(amide) bonds occurring on the NMR timescale. The fluxional behavior of (SNS)TaCl(NEt₂)₂ in solution was also studied by variable temperature ¹H NMR. Observation of separate signals for the diastereotopic protons of the methylene unit of the diethylamide indicates that the complex remains locked on the NMR timescale in one diastereomeric conformation at temperatures below -50 °C.

Reduction of Zr(IV) metallocenium cations with sodium amalgam (NaHg) produces EPR signals assignable to Zr(III) metallocene complexes. Thus, chloro-bridged heterobinuclear ansa-zirconocenium cation [((SBI))Zr(μ-Cl)₂AlMe₂]⁺B(C₆F₅)_4¯ (SBI = rac-dimethylsilylbis(1-indenyl)), gives rise to an EPR signal assignable to the complex (SBI)Zr^III(μ-Cl)₂AlMe₂, while (SBI)Zr^III-Me and (SBI)Zr^III(-H)2AlⁱBu₂ are formed by reduction of [(SBI)Zr(μ-Me)₂AlMe₂]⁺B(C₆F₅)_4¯ and [(SBI)Zr(μ-H)₃(AlⁱBu₂)₂]⁺B(C₆F₅)4¯, respectively. These products are also formed, along with (SBI)Zr^III-ⁱBu and [(SBI)Zr^III]⁺ AlR4¯ when (SBI)ZrMe₂ reacts with HAlⁱBu₂, eliminating isobutane en route to the Zr(III) complex. Studies concerning the interconversion reactions between these and other (SBI)Zr(III) complexes and reaction mechanisms involved in their formation are also reported.

The addition of HAlⁱBu₂ to precatalyst [(SBI)Zr(µ-H)₃(AlⁱBu₂)₂]⁺ significantly slows the polymerization of propylene and changes the kinetics of polymerization from 1st to 2nd order with respect to propylene. This is likely due to competitive inhibition by HAlⁱBu₂. When the same reaction is investigated using [(ⁿBuCp)₂Zr(μ-H)₃(AlⁱBu₂)₂]⁺, hydroalumination between propylene and HAlⁱBu₂ is observed instead of propylene polymerization.