Active-site amino acid residues in γ-glutamyltransferase and the nature of the γ-glutamyl-enzyme bond

Active-site residues in rat kidney γ-glutamyltransferase (EC 2.3.2.2) were investigated by means of chemical modification. 1. In the presence of maleate, the activity was inhibited by phenylmethanesulphonyl fluoride, and the inhibition was not reversed by β-mercaptoethanol, suggesting that a serine residue is close to the active site, but is shielded except in the presence of maleate. 2. Treatment of the enzyme with N-acetylimidazole modified an amino group, exposed a previously inaccessible cysteine residue and inhibited hydrolysis of the γ-glutamyl-enzyme intermediate, but not its formation. 3. After reaction of the enzyme successively with N-acetylimidazole and with non-radioactive iodoacetamide/serine/borate, two active-site residues reacted with iodo[¹⁴C]acetamide. One of these possessed a carboxy group, which formed a [¹⁴C]glycollamide ester, and the other was cysteine, shown by isolation of S-[¹⁴C]carboxymethylcysteine after acid hydrolysis. When N-acetylimidazole treatment was omitted, only the carboxy group reacted with iodo[¹⁴C]acetamide. 4. Isolation of the γ-[¹⁴C]glutamyl-enzyme intermediate was made easier by prior treatment of the enzyme with N-acetylimidazole. The γ-glutamyl-enzyme bond was stable to performic acid, and to hydroxylamine/urea at pH10, but was hydrolysed slowly at pH12, indicating attachment of the γ-[¹⁴C]glutamyl group in amide linkage to an amino group on the enzyme. Proteolysis of the γ-[¹⁴C]glutamyl-enzyme after performic acid oxidation gave rise to a small acidic radioactive peptide that was resistant to further proteolysis and was not identical with γ-glutamyl-ε-lysine. 5. A scheme for the catalytic mechanism is proposed.     

Age-dependent deamidation of glutamine residues in human γS crystallin: deamidation and unstructured regions

Human aging is associated with the deterioration of long-lived proteins. Gradual cumulative modifications to the life-long proteins of the lens may ultimately be responsible for the pronounced alterations to the optical and physical properties that characterize lenses from older people. γS crystallin, a major human lens protein, is known to undergo several age-dependent changes. Using proteomic techniques, a site of deamidation involving glutamine 92 has been characterized and its time course established. The proportion of deamidation increased from birth to teen-age years and then plateaud. Deamidation at this site increased again in the eighth decade of life. There was no significant difference in the extent of deamidation between cataract and age-matched normal lenses. Gln92 is located in the linker region between the two domains, and the introduction of a negative charge at this site may alter the interaction between the two regions of the protein. Gln170, which is located in another unstructured part of γS crystallin, showed a similar deamidation profile to that of Gln92. As the other Gln residues in β-sheet regions of γS crystallin appear to remain as amides, modification of Gln92 and Gln170 thus conforms to a pattern whereby deamidation is localized to the unstructured regions of long-lived proteins.     

Synthesis and reactivity of novel γ-phosphate modified ATP analogues

We hereby present a simple yet novel chemical synthesis of a family of γ-modified ATPs bearing functional groups on the γ-phosphate that are amenable to further derivatization by highly selective chemical manipulations (e.g., click chemistry, Staudinger ligations). A preliminary screen of these compounds as phosphate donors with a typical wild type protein kinase (cdk2) and one of its known substrates p27^kip1 is also presented.     

Synthesis of 2-amino-2-deoxy- and 3-amino-3-deoxy-α-d-mannopyranosyl α-d-mannopyranoside by sequential osmylations of a dienic disaccharide

Partial oxyamination of 4,6-di-O-acetyl-2,3-dideoxy-α-d-erythro-hex-2-enopyranosyl 4,6-di-O-acetyl-2,3-dideoxy-α-d-erythro-hex-2-enopyranoside with chloramine-T and osmium tetraoxide gave 4,6-di-O-acetyl-2-deoxy-2-(p-toluene-sulfonamido)-α-d-mannopyranosyl 4,6-di-O-acetyl-2,3-dideoxy-α-d-erythro-hex-2-enopyranoside and its 3-deoxy-3-(p-toluenesulfonamido) regioisomer, each in 18–19% isolated yield. Osmium tetraoxide-catalyzed cis-hydroxylation of the remaining alkenic residue in these products led in high yields to the corresponding triols having the α-d-manno, α-d-manno configuration. These were N-desulfonylated (and simultaneously O-deacetylated) by the action of sodium in liquid ammonia to furnish 2-amino-2-deoxy-α-d-mannopyranosyl α-d-mannopyranoside and 3-amino-3-deoxy-α-d-mannopyranosyl α-d-mannopyranoside as new, trehalose-type amino sugars.     

Inhibitors of Adenosine Deaminase: Synthesis of Coformycin and 3′-Deoxycoformycin

Abstract

Glycosylation of the heterocycle, 6,7-dihydro-imidazo [4,5-d] [1,3] diazepin-8(3H)-one, with suitably protected sugars under the influence of Lewis acid catalysts gave the β-D-ribo- and 3′-deoxy-β-D-erythropento-furanosyl nucleosides. Deprotection and reduction of the keto nucleosides with sodium borohydride gave the (8R)- and (8S)-3-β-D-glycofuranosyl-3,6,7,8-tetrahydroimidazo [4,5-d]-[1,3] diazepin-8-ols, the (8R)-isomers of which are potent inhibitors of adenosine deaminase.     

Synthesis,binding and bioactivity of γ-methylene γ-lactam ecdysone receptor ligands: Advantages of QSAR models for flexible receptors

Nuclear hormone receptors, such as the ecdysone receptor, often display a large amount of induced fit to ligands. The size and shape of the binding pocket in the EcR subunit changes markedly on ligand binding, making modelling methods such as docking extremely challenging. It is, however, possible to generate excellent 3D QSAR models for a given type of ligand, suggesting that the receptor adopts a relatively restricted number of binding site configurations or ‘attractors’. We describe the synthesis, in vitro binding and selected in vivo toxicity data for γ-methylene γ-lactams, a new class of high-affinity ligands for ecdysone receptors from Bovicola ovis (Phthiraptera) and Lucilia cuprina (Diptera). The results of a 3D QSAR study of the binding of methylene lactams to recombinant ecdysone receptor protein suggest that this class of ligands is indeed recognised by a single conformation of the EcR binding pocket.     

Distinct Effects of Fatty Acids on Translocation of γ- and ε-Subspecies of Protein Kinase C

Effects of fatty acids on translocation of the γ- and ε-subspecies of protein kinase C (PKC) in living cells were investigated using their proteins fused with green fluorescent protein (GFP). γ-PKC–GFP and ε-PKC–GFP predominated in the cytoplasm, but only a small amount of γ-PKC–GFP was found in the nucleus. Except at a high concentration of linoleic acid, all the fatty acids examined induced the translocation of γ-PKC–GFP from the cytoplasm to the plasma membrane within 30 s with a return to the cytoplasm in 3 min, but they had no effect on γ-PKC–GFP in the nucleus. Arachidonic and linoleic acids induced slow translocation of ε-PKC–GFP from the cytoplasm to the perinuclear region, whereas the other fatty acids (except for palmitic acid) induced rapid translocation to the plasma membrane. The target site of the slower translocation of ε-PKC–GFP by arachidonic acid was identified as the Golgi network. The critical concentration of fatty acid that induced translocation varied among the 11 fatty acids tested. In general, a higher concentration was required to induce the translocation of ε-PKC–GFP than that of γ-PKC–GFP, the exceptions being tridecanoic acid, linoleic acid, and arachidonic acid. Furthermore, arachidonic acid and the diacylglycerol analogue (DiC8) had synergistic effects on the translocation of γ-PKC–GFP. Simultaneous application of arachidonic acid (25 μM) and DiC8 (10 μM) elicited a slow, irreversible translocation of γ-PKC– GFP from the cytoplasm to the plasma membrane after rapid, reversible translocation, but a single application of arachidonic acid or DiC8 at the same concentration induced no translocation.These findings confirm the involvement of fatty acids in the translocation of γ- and ε-PKC, and they also indicate that each subspecies has a specific targeting mechanism that depends on the extracellular signals and that a combination of intracellular activators alters the target site of PKCs.     

Synthesis and biological evaluation of antitumor-active γ-butyrolactone substituted betulin derivatives

The plant triterpenes betulin and betulinic acid (BA) are triterpenes featuring interesting pharmacological properties. Starting from substituted betulinic aldehydes, we used them as lead structures for the synthesis of several γ-butyrolactones and butenolides. Their antitumor activity was examined for 15 cancer cell lines using a SRB-assay and their apoptotic action was documented by trypan-blue test and DNA laddering. Several compounds revealed a higher activity than betulinic acid.     

Synthesis and antitumor activity of novel 2′,3′-dideoxy-2′,3′-diethanethionucleosides bearing 1,2,3-triazole residues

A series of novel 2′,3′-dideoxy-2′,3′-diethanethioribonucleosides and those modified with a triazole ring were prepared in excellent yields and their antitumor activity was evaluated. Nucleosides with a triazole ring, 16a–16c, showed significantly improved activity towards a broad range of tumor cell lines.     

Synthesis and anti-acetylcholinesterase properties of novel β- and γ-substituted alkoxy organophosphonates

Activated organophosphate (OP) insecticides and chemical agents inhibit acetylcholinesterase (AChE) to form OP-AChE adducts. Whereas the structure of the OP correlates with the rate of inhibition, the structure of the OP-AChE adduct influences the rate at which post-inhibitory reactivation or aging phenomena occurs. In this report, we prepared a panel of β-substituted ethoxy and γ-substituted propoxy phosphonoesters of the type p-NO₂PhO-P(X)(R)[(O(CH₂)_nZ] (R = Me, Et; X = O, S; n = 2, 3; Z = halogen, OTs) and examined the inhibition of three AChEs by select structures in the panel. The β-fluoroethoxy methylphosphonate analog (R = Me, Z = F, n = 2) was the most potent anti-AChE compound comparable (k_i ～6 × 10⁶ M^?1 min^?1) to paraoxon against EEAChE. Analogs with Z = Br, I, or OTs were weak inhibitors of the AChEs, and methyl phosphonates (R = Me) were more potent than the corresponding ethyl phosphonates (R = Et). As expected, analogs with a thionate linkage (PS) were poor inhibitors of the AChEs.     

The conformation of end-groups is one determinant of carotenoid topology suitable for high fidelity molecular recognition: A study of β- and ε-end-groups

Conformation affects a carotenoid’s ability to bind selectively to proteins. We calculated adiabatic energy profiles for rotating the ring end-groups around the C6C7 bond and for flexing of the ring with respect to the polyene chain. The choice of computational methods is important. A low, 4.2 kcal/mol barrier to rotation exists for a β-ring. An 8.3 kcal/mol barrier exists for rotation of an ε-ring. Rotation of the ε-ring is sensitive to substitution at C3. In the absence of external forces neither β- nor ε-rings are rotationally constrained. The nearly parallel alignment of the β-ring to the C6C7 bond axis contrasts to the more perpendicular orientation of the ε-ring. Flexion of a β-ring to the minimized ε-ring conformation requires ∼23 kcal/mol; extension of the ε-ring to the minimized β-ring conformation requires ∼8 kcal/mol. Selectivity associated with β- versus ε-rings is dominated by the inability of the β-ring to flex to minimize protein/ring steric interactions and maximize van der Waal’s attractions with the binding site.     

Foldamers containing γ-amino acid residues or their analogues: structural features and applications

Over the past 20 years, the field of foldamers has rapidly increased. Many β-peptides have already been described and shown interesting properties. γ-Peptides have more recently emerged but seem to be very interesting as well. In this review, we will cover every peptidomimetic oligomer that contains a γ-amino acid or an analogue and presents a structural feature. It includes γ-peptides but also hybrid α–γ peptides, β–γ peptides and analogues such as oligoureas or aminoxy acids. We will present the biological properties of these oligomers.     

Pregabalin peptides: conformational comparison of γ3- and γ4-substituted γ-amino acids in αγααα pentapeptides

Amino Acids - Gamma-aminobutyric acid (GABA, gammaAbu), an unsubstituted gamma-amino acid, is an important inhibitory neurotransmitter in the mammalian brain. The role of GABA in the treatment of...     

Linear oligopeptides: c.d. and n.m.r. study of Dnp-pNA derivatives of Aib-containing tetrapeptides in a β-bend conformation

A circular dichroism investigation of two β-bend forming, Aib-containing tetrapeptides, with the -Aib-l-Ala- and -l-Ala-Aib- central sequences, occurring in peptaibol antibiotics, blocked at the N-terminal end with the 2,4-dinitrophenyl group and at the C-terminal end with the p-nitroanilino group, is described. A comparison is made with a tetrapeptide containing the -l-Ala-l-Ala- central sequence, also observed in peptaibol antibiotics. The amount of β-bend conformers, determined from the intensities of the exciton couplets arising from the intramolecular interaction of the p-nitroanilino chromophores, has been assessed as a function of the hydrogen-bonding properties of the solvent. The conformational analysis in dimethylsulphoxide has been extended to ¹H nuclear magnetic resonance. Some information on the type of β-bend formed has additionally been obtained. The preparation and characterization of the three chromophoric tetrapeptides, along with some analogues and synthetic precursors, are also reported.     

A reinvestigation of residues 64–68 and 175 in papain. Evidence that residues 64 and 175 are asparagine

The tryptophan-containing peptides were isolated from the chymotryptic digest of S-carboxymethylated papain. Residue 175, which is strongly hydrogen-bonded to the active-site histidine residue in the tertiary structure of papain, is asparagine, confirming the work of Kimmel, Rogers & Smith (1965). Its function is probably to maintain the orientation and tautomeric state of the imidazole ring of histidine-159. The amino acid sequence predicted from the electron-density map of papain for residues 64-68 was confirmed, but residue 64 is asparagine, not aspartic acid. This residue, which is about 10 A from the thiol group of the active-site cysteine-25, cannot therefore be a site of electrostatic attraction for substrates of basic amino acids.     

Synthesis and SAR study of tricyclic sulfones as γ-secretase inhibitors: C-6 and C-8 positions

SAR exploration at C-6 and C-8 positions of the tricyclic sulfone series was carried out. Several functional groups were found to be well tolerated at C-6 and C-8 positions. Selective combination of C-6 and C-8 modification resulted in new tricyclic sulfone analogs with efficacy in in vivo mouse Aβ₄₀ lowering model.