Microbial L-phenylalanine ammonia-lyase. Purification, subunit structure and kinetic properties of the enzyme from Rhizoctonia solani

Phenylalanine ammonia-lyase (EC 4.3.1.5) was purified to homogeneity from the acetone-dried powders of the mycelial felts of the plant pathogenic fungus Rhizoctonia solani. 2. A useful modification in protamine sulphate treatment to get substantial purification of the enzyme in a single-step is described. 3. The purified enzyme shows bisubstrate activity towards L-phenylalanine and L-tyrosine. 4. It is sensitive to carbonyl reagents and the inhibition is not reversed by gel filtration. 5. The molecular weight of the enzyme as determined by Sephadex G-200 chromatography and sucrose-density-gradient centrifugation is around 330000. 6. The enzyme is made up of two pairs of unidentical subunits, with a molecular weight of 70000 (alpha) and 90000 (beta) respectively. 7. Studies on initial velocity versus substrate concentration have shown significant deviations from Michaelis-Menten kinetics. 8. The double-reciprocal plots are biphasic (concave downwards) and Hofstee plots show a curvilinear pattern. 9. The apparent Km value increases from 0.18 mM to as high as 5.0 mM with the increase in the concentration of the substrate and during this process the Vmax, increases by 2-2.5-fold. 10. The value of Hill coefficient is 0.5. 11. Steady-state rates of phenylalanine ammonia-lyase reaction in the presence of inhibitors like D-phenylalanine, cinnamic, p-coumaric, caffeic, dihydrocaffeic and phenylpyruvic acid have shown that only one molecule of each type of inhibitor binds to a molecule of the enzyme. These observations suggest the involvement of negative homotropic interactions in phenylalanine ammonia-lyase. 12. The enzyme could not be desensitized by treatment with HgCl2, p-chloromercuribenzoic acid or by repeated freezing and thawing.

Bromination of Intermolecular Chelate Linkage Isomers of 3-Oximino-4,9-Dimethyl (or 4,7,9-Trimethyl)-5,8-Diazadodeca-4,8-Diene-2,11-Dionato Nickel(II)

The reaction of the title complexes (FIG. 1) with N-bromosuccinimide or bromine in chloroform yields isomeric bromo complexes on substitution of the γ-CH carbon proton by bromine. The brominated products have been characterised by ir, pmr, electronic absorption spectra, conductivity and magnetic susceptibility measurements. The linkage isomerisation of the brominated products in chloroform has been shown to depend on the diamine residue.

Studies on the relative biopotencies and intestinal absorption of different apo-beta-carotenoids in rats and chickens.

1. The biopotencies relative to beta-carotene of several apocarotenoids, such as 8'-, 10'- and 12'-apo-beta-carotenal and methyl 8'-apo-beta-carotenoate, were investigated in rats, on a molar basis, by both curative-growth assay and liver-storage tests. 2. In the curative-growth assays, on a molar basis the biopotencies of 8'-, 10'- and 12'-apo-beta-carotenal and methyl 8'-apo-beta-carotenoate were 72, 78, 72 and 53% respectively, whereas on a weight basis the corresponding values were 93, 111, 111 and 63%, with respect to beta-carotene taken as 100%. In terms of yield of vitamin A, these values were much lower in the liver-storage tests. 3. When 8'-apo-beta-carotenal was fed, the unchanged aldehyde together with small amounts of the corresponding alcohol and larger proportions of the acid rapidly appeared in the tissues of both rats and chickens. The 8'-apocarotenol, 8'-apocarotenoic acid and its methyl ester were absorbed unchanged. The free acid disappeared most rapidly from the tissues, but its methyl ester persisted in the tissues longest. 4. On the basis of these observations it is suggested that most of an apocarotenal is oxidized to the corresponding acid, which, in turn, is mostly degraded to retinoic acid, with small proportions of it being attacked by the dioxygenase system giving retinal.

Direct Synthesis of Functionalized Unsymmetrical beta-Sulfonamido Disulfides by Tetrathiomolybdate Mediated Aziridine Ring-Opening Reactions

Direct synthesis of unsymmetrical beta-sulfonamido disulfides by ring-opening of aziridines by using benzyltriethyl-ammonium tetrathiomolybdate 1 as a sulfur transfer reagent in the presence of symmetrical disulfides as thiol equivalents has been reported. Reaction of benzyl and alkyl disulfides gave unsymmetrical beta-sulfonamido disulfides as the only product in very good yields. From the Study, it has been observed that aryl disulfides containing p-NO2, p-Cl, and p-CN led to the formation of the corresponding beta-aminosulfides as the exclusive products. However, un-substituted aryl disulfides and the one containing electron-donating substituents (p-Me) provide a mixture of beta-sulfonamido mono- and disulfides as the products.

Conformational Choices for the Stereochemically Constrained gamma-Amino Acid Residue Gabapentin: Theoretical Studies and Correlation With Experimental Results

Gabapentin (1-aminomethylcyclohexaneacetic acid, Gpn) is an achiral, conformationally constrained gamma amino acid residue. A survey of available crystal structures of Gpn peptides reveals that the torsion angles about the C-gamma-C-beta (theta(1)) and C-beta-C-alpha(theta(2)) bonds are overwhelmingly limited to gauche, gauche (g(+)g(+)/g(-)g(-)) conformations. The Gpn residue forms C-7 and C-9 hydrogen bonds in which the donor and acceptor atoms come from the flanking peptide units. In combination with alpha amino acid residues alpha gamma and gamma alpha segments can adopt C-12 hydrogen bonded structures. The conformational choices available to the Gpn residue have been probed using energy calculations, adopting a grid search strategy. Ramachandran phi-psi maps have been constructed for fixed values of theta(1) and theta(2), corresponding to the gauche and trans conformations. The sterically allowed and energetically favorable regions of conformational space have been defined and experimental observations compared. C-7 and C-9 hydrogen bonded conformational families have been identified using a grid search approach in which theta(1) and theta(2) values are varied over a range of +/- 10 degrees about ideal values at 1 degrees intervals. The theoretical analysis together with experimental observations for 59 Gpn residues from 35 crystal structures permits definition of the limited range of conformational possibilities at this gamma amino acid residue. .

New structural insights and molecular-modelling studies of 4-methyl-5-beta-hydroxyethylthiazole kinase from Pyrococcus horikoshii OT3 (PhThiK)

4-Methyl-5-beta-hydroxyethylthiazole kinase (ThiK) catalyses the phosphorylation of the hydroxyl group of 4-methyl-5-beta-hydroxyethylthiazole. This work reports the first crystal structure of an archaeal ThiK: that from Pyrococcus horikoshii OT3 (PhThiK) at 1.85 angstrom resolution with a phosphate ion occupying the position of the beta-phosphate of the nucleotide. The topology of this enzyme shows the typical ribokinase fold of an alpha/beta protein. The overall structure of PhThiK is similar to those of Bacillus subtilis ThiK (BsThiK) and Enterococcus faecalis V583 ThiK (EfThiK). Sequence analysis of ThiK enzymes from various sources indicated that three-quarters of the residues involved in interfacial regions are conserved. It also revealed that the amino-acid residues in the nucleotide-binding, magnesium ion-binding and substrate-binding sites are conserved. Binding of the nucleotide and substrate to the ThiK enzyme do not influence the quaternary association (trimer) as revealed by the crystal structure of PhThiK.

Mapping of assembled epitopic regions of human chorionic gonadotropin reveals proximity of CTP alpha to the determinant loop beta 93-100

Three overlapping assembled epitopes of beta hCG have been mapped using MAb probes and a single step solid phase radioimmunoassay. These epitopes have been shown to be at receptor binding region comprising of the loop region beta Cys93-Cys100. Importance of disulphide bonds in maintaining integrity of these epitopes is assessed. Two MAbs (INN 58 and INN 22) interact with the beta region as well as the alpha C-terminal peptide, while the other MAb INN 24 interacts with only the beta region. Cross-reactivity pattern with beta hCG and hLH as web as the reported crystal structure of hCG substantiates the epitope identification. The results demonstrate utility of MAbs as probes in investigations on three-dimensional structure of gonadatropins.

Drift mobility of holes in single-crystal beta-AgI

The drift mobility of photoexcited holes in single-crystal beta-AgI has been measured between 260 and 312 °K. In this range the drift mobility µd increased with temperature due to trap-limited behavior. At 300 °K µd=12 cm2/V sec, the concentration and energy of the dominant traps being given by Nt=3×109 to 5×109/cm3 and Et=0.52 to 0.50 eV, respectively. Electron drift mobilities could not be determined due to low electron lifetimes. Journal of Applied Physics is copyrighted by The American Institute of Physics.

A common epitope of beta-lactoglobulin and serum retinol-binding proteins: Elucidation of its core sequence using synthetic peptides

One of the monoclonal antibodies raised against bovine beta-lactoglobulin reacted with human serum retinol binding protein. The finding that this monoclonal antibody also reacted with the serum retinol binding proteins isolated from other animals, suggested that this epitopic conformation is conserved among these proteins. Using ELISA and various synthetic peptides of defined sequence, we show in this paper that the epitope defined by this monoclonal antibody comprises of the highly conserved core sequence of DTDY present in beta-lactoglobulin and retinol binding proteins.

Orthogonal beta-beta motifs in proteins

A super-secondary structural motif comprising two orthogonally oriented beta-strands connected by short linking segments of <5 residues has been identified from a data set of 65 independent protein crystal structures. Of the 42 examples from 14 proteins, a vast majority have only a single residue as the linking element. Analysis of the conformational angles at the junction reveals that the recently described type VIII beta-turn occurs frequently at the connecting hinge, while the type II beta-turn is also fairly common.

Crystal state conformation of three model monomer units for the beta-bend ribbon structure

The molecular and crystal structures of three compounds, representing the repeating units of the -bend ribbon (an approximate 310-helix, with an intramolecular hydrogen-bonding donor every two residues), have been determined by x-ray diffraction. They are Boc-Aib-Hib-NHBzl, Z-Aib-Hib-NHBzl, and Z-L-Hyp-Aib-NHMe (Aib, -aminoisobutyric acid; Bzl, benzyl; Boc, t-butyloxycarbonyl; Hyp, hydroxyproline Hib, -hydroxyisobutyric acid; Z, benzyloxycarbonyl). The two former compounds are folded in a -bend conformation: type III (III) for Boc-Aib-Hib-NHBzl, while type II (II) for the Z analogue. Conversely, the structure of Z-L-Hyp-Aib-NHMe, although not far from a type II -bend, is partially open.

Chiral Synthons from Monoterpenes.1 Stereoselective Syntheses of (-)5S, 6S, 9R-6-Isopropyl-9-Methyl-2-Oxaspiro[4.4]Nonan-3-One and (-) 3aR, 6R, 7aR-3a-Methyl-6-Isopropenylhexahydrobenzofuran-2-One

Highly stereoselective syntheses of two C-12 chiral synthons 3 and 9, mentioned in the title, starting from the monoterpenes R-1 imonene and R-carvone, using radical cyc 1 sation as key reaction, are described.