Hydrolysis of new phthalimide-derived esters catalyzed by immobilized lipase

The last step of the production of four phthalimide-derived acids, designed to act as antiasthma drugs, was performed by enzymatic hydrolysis of the respective methyl or ethyl esters. The esters 4-ethyl-[2-(1,3-dioxo-1,3-dihydro-2-isoindoylyl)]-phenoxyacetic methyl ester (PHT-MET), 4-ethyl-[2-(1,3-dioxo-1,3-dihydro-2-isoindoylyl)]-phenoxyacetic ethyl ester, 4-(1,3-dioxo-1,3-dihydro-2-isoindoylyl)-phenoxyacetic ethyl ester, and 2-(1,3-dioxo-1, 3-dihydro-2-isoindoylyl)-phenoxyacetic ethyl ester were hydrolyzed by immobilized lipase. The enzymatic reaction could be used only to produce the desired 4-substituted compounds. The best result that was found to hydrolysis of PHT-MET, and, therefore, that ester was selected for optimization experiments in a three-phase system. Reactions were performed with solid biocatalyst (Lipozyme® RM IM), organic solvent phase (ethyl acetate), and aqueous phase (saturated Na₂CO₃ solution). To optimize the reaction conditions, an experimental design optimization procedure was used. The variables studied were the amount of enzyme, the temperature, and the volume of the aqueous solution. Time course experiments were then performed for different initial enzyme concentrations (0.5, 0.9, and 1.4 U_H/mL of solvent). The optimized reaction conditions found were 20 mg of Lipozyme (0.9 U_H/mL_solvent) and 5.0 mL of Na₂CO_3(sat) at 40°C for 6 h.     

Density-functional study of mechanisms for the cofactor-free decarboxylation performed by uroporphyrinogen III decarboxylase

Uroporphyrinogen III decarboxylase catalyzes the fifth step in heme biosynthesis: the elimination of carboxyl groups from the four acetate side chains of uroporphyrinogen III to yield coproporphyrinogen III. The enzyme acts by successively protonating each of the four pyrrole rings present in the substrate, thereby allowing decarboxylation of their side chains, but the identity of the proton donors has not been established yet. Tyr164 has been suggested as a proton donor, and Asp86 has been proposed to act either as a proton donor or as an intermediate-stabilizing residue. We have performed density-functional calculations to study this reaction mechanism, and found that the rate-limiting step is substrate protonation, rather than decarboxylation. Surprisingly, whereas Tyr164 is unable to protonate the substrate, this protonation can be effected by a nearby arginine residue (Arg37), with a free energy barrier of 21.4 kcal.mol(-1), in remarkable agreement with the experimental value of 19.5 kcal.mol(-1). The central positioning of this residue in close proximity to all four pyrrole rings in the substrate may play a key role in the sequential activation of each of these moieties.     

RNA integrity as a quality indicator during the first steps of RNP purifications : A comparison of yeast lysis methods

Background  

The completion of several genome-sequencing projects has increased our need to assign functions to newly identified genes. The presence of a specific protein domain has been used as the determinant for suggesting a function for these new genes. In the case of proteins that are predicted to interact with mRNA, most RNAs bound by these proteins are still unknown. In yeast, several protocols for the identification of protein-protein interactions in high-throughput analyses have been developed during the last years leading to an increased understanding of cellular proteomics. If any of these protocols or similar approaches shall be used for the identification of mRNA-protein complexes, the integrity of mRNA is a critical factor.     

Reaction of 5,6,7,8-Tetrahydropterin with Iron(III) Acetylacetonate. Detection of Radical Cations by Electrospray Ionization Mass Spectrometry

The experimental conditions developed for the detection of rather stable radical cations in solution by electrospray-ionization mass spectrometry (ESI-MS) of a Fe^II complex of 2-amino-5,6,7,8-tetrahydro-5-methylpteridin-4 (3H)-one ( 1c ) are used to observe the formation of the more unstable radical cations formed from 2-amino-5,6,7,8-tetrahydropteridin-4(3H)-one ( 1a ) and tris(pentane-2,4-dionato)iron(III) ([Fe^III(acac)₃]; 4 ) and to monitor their oxidation to the corresponding p-quinonoid dihydropterin complexes. These results contribute to the understanding of the important role played by 6β-5,6,7,8-tetrahydro-L -biopterin ( 1b ; a homologue of 1a ) together with iron as constituent of some cofactors. The complexes obtained from 1a and iron may be considered, e.g. as a model of the cofactor of the phenylalanine hydroxylase. Moreover, we describe an improved synthesis of 1c .     

Simultaneous determination of iron, cobalt, nickel and copper by UV-visible spectrophotometry with multivariate calibration

A method for the simultaneous spectrophotometric determination of the divalent ions of iron, cobalt, nickel and copper based on the formation of their complexes with 1,5-bis(di-2-pyridylmethylene) thiocarbonohydrazide (DPTH) is proposed. The resolution of quaternary mixtures of these metallic ions was accomplished by several chemometric approaches. A comparative study of the results obtained for simultaneous determinations in mixture by using principal component regression (PCR) and partial least-squares regression (PLS-1 and PLS-2) for absorbance, first-derivative and second-derivative data is presented. In general, the best recovery values are obtained by the PLS-2 method for absorbance data. This procedure allows the simultaneous determination of the cited ions in alloys and biological materials Good reliability of the determination was proved.     

Counterion distributions and effective interactions of spherical polyelectrolyte brushes

We consider the electrosteric repulsion of colloidal particles whose surface carries a dense layer of long polyelectrolyte chains (spherical polyelectrolyte brushes). The theory of electrosteric repulsion of star polyelectrolytes developed recently is augmented to include particles with a finite core radius. It is shown that most of the counterions are confined within the brush layer. The strong osmotic pressure thus created within the brush layer dominates the repulsive interaction between two such particles. Because of this the pair interaction potential between spherical polyelectrolyte brushes can be given in terms of an analytic expression. The theoretical predictions are compared with available experimental data and semi-quantitative agreement between the two is found.     

A model for energy transfer in inelastic molecular collisions applicable at steady state or non-steady state and for an arbitrary distribution of collision energies

A new model for energy exchange between translational and internal degrees of freedom in atom-molecule collisions has been developed. It is suitable for both steady state conditions (e.g., a large number of collisions with thermal kinetic energies) and non-steady state conditions with an arbitrary distribution of collision energies (e.g., single high-energy collisions). In particular, it does not require that the collision energies be characterized by a quasi-thermal distribution, but nevertheless it is capable of producing a Boltzmann distribution of internal energies with the correct internal temperature under quasi-thermal conditions. The energy exchange is described by a transfer probability density that depends on the initial relative kinetic energy, the internal energy of the molecule, and the amount of energy transferred. The probability density for collisions that lead to excitation is assumed to decrease exponentially with the amount of transferred energy. The probability density for de-excitation is obtained from microscopic reversibility. The model has been implemented in the ion trap simulation program ITSIM and coupled with an Rice-Rampsberger-Kassel-Marcus (RRKM) algorithm to describe the unimolecular dissociation of populations of ions. Monte Carlo simulations of collisional energy transfer are presented. The model is validated for non-steady state conditions and for steady state conditions, and the effect of the kinetic energy dependence of the collision cross-section on internal temperature is discussed. Applications of the model to the problem of chemical mass shifts in RF ion trap mass spectrometry are shown.     

Active species of horseradish peroxidase (HRP) and cytochrome P450: two electronic chameleons

The active site of HRP Compound I (Cpd I) is modeled using hybrid density functional theory (UB3LYP). The effects of neighboring amino acids and of environmental polarity are included. The low-lying states have porphyrin radical cationic species (Por(*)(+)). However, since the Por(*)(+) species is a very good electron acceptor, other species, which can be either the ligand or side chain amino acid residues, may participate in electron donation to the Por(*)(+) moiety, thereby making Cpd I behave like a chemical chameleon. Thus, this behavior that was noted before for Cpd I of P450 is apparently much more wide ranging than initially appreciated. Since chemical chameleonic behavior property was found to be expressed not only in the properties of Cpd I itself, but also in its reactivity, the roots of this phenomenon are generalized. A comparative discussion of Cpd I species follows for the enzymes HRP, CcP, APX, CAT (catalase), and P450.     

Stereoselective hydroxylation of an achiral cyclopentanecarboxylic acid derivative using engineered P450s BM-3

Substrate engineered, achiral carboxylic acid derivative was biohydroxylated with various mutants of cytochrome P450 BM-3 to give two out of the four possible diastereoisomers in high de and ee. The BM-3 mutants exhibit up to 9200 total turnovers for hydroxylation of the engineered substrate, which without the protecting group is not transformed by this enzyme.     

Structure and dynamics of water at the interface with phospholipid bilayers

We have performed two molecular-dynamics simulations to study the structural and dynamical properties of water at the interface with phospholipid bilayers. In one of the simulations the bilayer contained neutral phospholipid molecules, dioleoylphosphatidylcholine (DOPC); in the second simulation the bilayer contained charged lipid molecules, dioleoylphosphatidylserine (DOPS). From the density profile of water we observe that water next to the DOPS bilayer is more perturbed as compared to water near the DOPC bilayer. Using an energetic criterion for the determination of hydrogen bonding we find that water molecules create strong hydrogen bonds with the headgroups of the phospholipid molecules. Due to the presence of these bonds and also due to the confinement of water, the translational and orientational dynamics of water at the interface are slowed down. The degree of slowing down of the dynamics depends upon the location of water molecules near a lipid headgroup.