Search

Two of the most popular positron emission tomography (PET) tracers, [ 11 C]PE2I and [ 18 F]FE-PE2I, used to quantify dopamine transporters (DAT), display dissimilar kinetic behavior in in vivo assays. This difference can be explained by comparing values of kinetic rate constants, which characterize interaction of these tracers with DAT sites in vitro . At the same time, this kinetic analysis showed that the overall binding mechanism is similar for these two tracers and includes a fast step of complex formation followed by a slow isomerization step of this complex. Comparison with previous PE2I data revealed that isomerization of the DAT complex with PE2I occurs three times faster than in the case of FE-PE2I, which leads to the slower onset of peak specific binding of the former tracer in the DAT-rich regions. Therefore, ligands with slower isomerization on-rate, including [ 18 F]FE-PE2I, seem to be better tracers in vivo , and their properties can be predicted in vitro ., Competing Interests: The authors declare no competing financial interest.

The protein kinase A (PKA)-catalyzed phosphorylation of peptide substrate RRASVA analogs, containing Nβ-Me-aza-β3-amino acid residues in all subsequent positions, was studied. This work follows along the lines of our previous research of the phosphorylation of aza-β3-analogs of RRASVA (the shortest active substrate of PKA) and allows characterizing the influence of Nβ-methylation of aza-β3-amino acid residues on substrate recognition by PKA on substrate binding and phosphorylation steps. It was found that the effect of Nβ-methylation was dependent upon the position of the structure alteration. Moreover, the presence of a single Nβ-methylation site in the substrate changed the recognition pattern of this series of peptidomimetics, strongly affecting the phosphorylation step. Structure modeling of aza-β3- and Nβ-Me-aza-β3-containing substrates revealed that Nβ-methylation of aza-β3-moieties changed the peptide bond geometry from trans- to cis-configuration in -CO-NMe- fragments, with an exception for the N-terminally methylated Nβ-Me-aza-β3-RRRASVA (with the N-terminal amino group not participating in the peptide bond) and RRAS-Nβ-Me-aza-β3-VA. As has been shown in literature, this conformational preference of the backbone has a significant influence on the flexibility of the peptide substrate chain. Following our results, this property seems to have significant influence on the recognition of the amino acid side groups by the enzyme binding site, and in the case of PKA this structural modification was decisive for the phosphate transfer step of the catalytic process.

Peptidomimetic analogs of the hexapeptide RRASVA, containing simultaneously two aza-β(3)-amino acid residues in different positions of this sequence, except for the phosphorylatable serine residue, were synthesized and tested as substrates for the cAMP-dependent protein kinase catalytic subunit. All these peptidomimetics were phosphorylated by the enzyme and this reaction was characterized by the K(m) and k(cat) values as well as by the second-order rate constants k(II). Affinity and reactivity of all peptidomimetics was lower than that for the parent peptide RRASVA. The effect of backbone modification was dependent upon the positions where these two aza-β(3) residues were located, although the sequence of amino acid side groups remained the same in all compounds. It was found that the influence of two backbone modifications in the substrate structure was not described additively, i.e. the effect of each structural alteration was dependent upon the position of the second modification. The results were in agreement with the concept of specificity-determining clusters in the sequence of peptide and peptidomimetic ligands, which predominantly determine the molecular recognition of these ligands by their target sites and therefore serve as major modification points for the design of activity of peptidomimetic ligands., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Peptidomimetic analogs of the peptide RRASVA, known as the "minimal substrate" of the catalytic subunit of the cAMP-dependent protein kinase (PKA), were synthesized by consecutive replacement of natural amino acids by their aza-beta(3) analogs. The peptidomimetics were tested as PKA substrates and the kinetic parameters of the phosphorylation reaction were determined. It was found that the interaction of these peptidomimetics with the enzyme active center was sensitive to the location of the backbone modification, while the maximal rate of the reaction was practically not affected by the structure of substrates. The pattern of molecular recognition of peptidomimetics was in agreement with the results of structure modeling and also with the results of computational docking study of peptide and peptidomimetic substrates with the active center of PKA. It was concluded that the specificity determining factors which govern substrate recognition by the enzyme should be grouped along the phosphorylatable substrate, and such clustering might open new perspectives for pharmacophore design of peptides and peptide-like ligands., (2010 Elsevier Inc. All rights reserved.)

Kinetic mechanism and stoichiometry of scavenging the 2,2-diphenyl-1-picrylhydrazyl radical by glutathione and its novel analog, containing alpha-glutamyl residue in place of the gamma-glutamyl moiety, were studied using different ratios of reagents. At low concentrations of the peptides, the process was described as a bimolecular reaction obeying the stoichiometric ratio 1:1. However, at excess of peptides the formation of a non-covalent complex between the reagents was discovered and characterized by dissociation constants K = 0.61 mM for glutathione and K = 0.27 mM for the glutathione alpha-glutamyl analog, respectively. The complex formation was followed by a reaction step that was characterized by the similar rate constant k = 0.02 s(-1) for both peptides. Thus, the apparently different antioxidant activity of these two peptides, observed under common assay conditions, was determined by differences in the formation of this non-covalent complex.

The kinetics of KCN-catalyzed benzoin condensation of benzaldehyde in water and ethanol-water binary mixtures was investigated both under ultrasound at 22 kHz and without sonication. Thermodynamic activation parameters were calculated from kinetic data obtained at 35, 50, and 65 degrees C. Evidence that ultrasound can retard reactions is reported and hence a direct proof that sonochemical processes occur in the bulk solution. Former results and literature data for ester hydrolyses and tert-butyl chloride solvolysis are involved in the discussion. A quantitative relationship between sonication effects and the hydrophobicity of reagents is presented for the first time. Ultrasound affects hydrophobic interactions with the solvent, which are not manifested in conventional kinetics. When it suppresses the stabilization of the encounter complexes between reagents, sonication hinders the reaction but accelerates it when it perturbs the hydrophobic stabilization of the ground state of a reagent.

A peptide library approach based on electrospray mass-spectrometric (ESI-MS) detection of phosphopeptides was designed for rapid and quantitative characterization of protein kinase specificity. The k(cat)/K(m) values for the protein kinase Cbeta (PKCbeta) were determined for a systematically varied set of individual substrate peptides in library mixtures by the ESI-MS method. The analysis revealed a complex structural specificity profile in positions around the phosphorylated serine with hydrophobic and/or basic residues being mostly preferred. On the basis of the kinetic parameters, a highly efficient peptide substrate for PKCbeta (K(m)value below 100 nM) FRRRRSFRRR and its alanine substituted pseudosubstrate-analog inhibitor (K(i) value of 76 nM) were designed. The quantitative specificity profiles obtained by the new approach contained more information about kinase specificity than the conventional substrate consensus motifs. The new method presents a promising basis for design of substrate-site directed peptide or peptidomimetic inhibitors of protein kinases. Second, highly specific substrates could be designed for novel applications such as high-throughput protein kinase activity screens on protein kinase chips.

Kinetic analysis of the inhibition of the phosphorylation of Kemptide, (LRRASLG), catalyzed by the catalytic subunit of cAMP-dependent protein kinase, by a peptide-nucleoside conjugate inhibitor AdcAhxArg6 was carried out over a wide range of ATP and peptide concentrations. A simple procedure was proposed for characterization of the interaction of this inhibitor with the free enzyme, and with the enzyme-ATP and enzyme-peptide complexes. The second-order rate constants, calculated from the steady-state reaction kinetics, were used for this analysis to avoid the complications related to the complex catalytic mechanism of the protein kinase catalyzed reaction.

Kinetics of the reactions of butylmagnesium chloride and phenylmagnesium bromide with tetraethoxysilane and methyltrichlorosilane was investigated in diethyl ether and diethyl ether-toluene mixtures. Replacement of ether by toluene significantly accelerates the reaction with alkoxysilanes, while no effect was found for the reaction with chlorosilanes. We established that the reaction with alkoxysilanes consists of replacement of a donor molecule at the magnesium center by the silane followed by subsequent rearrangement of the complex to products through a four-center transition state. Chlorosilanes react differently without solvent molecule replacement but also via a four-center transition state. Large negative activation entropies are consistent with formation of cyclic transition states. Small activation enthalpy values together with remarkable exothermicity point to early transition states of the reactions.

Novel affinity ligands, consisting of ATP-resembling part coupled with specificity determining peptide fragment, were proposed for purification of protein kinases. Following this approach affinity sorbents based on two closely similar ligands AdoC-Aoc-Arg4-Lys and AdoC-Aoc-Arg4-NH(CH2)6NH2, where AdoC stands for adenosine-5'-carboxylic acid and Aoc for amino-octanoic acid, were synthesized and tested for purification of recombinant protein kinase A catalytic subunit directly from crude cell extract. Elution of the enzyme with MgATP as well as L-arginine yielded homogeneous protein kinase A preparation in a single purification step. Also protein kinase A from pig heart homogenate was selectively isolated using MgATP as eluting agent. Protein kinase with acidic specificity determinant (CK2) as well as other proteins possessing nucleotide binding site (L-type pyruvate kinase) or sites for wide variety of different ligands (bovine serum albumin) did not bind to the column, pointing to high selectivity of the bi-functional binding mode of the affinity ligand.

Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings, and its substrate specificity studied using a set of synthetic peptides derived from the phosphorylatable sequence RVLSRLHS15VRER of maize sucrose synthase 2. The decapeptide LARLHSVRER was found to be efficiently phosphorylated as a minimal substrate. The same set of peptides were found to be phosphorylated by mammalian protein kinase Cbeta (PKC), but showed low reactivity with protein kinase A (PKA). Proceeding from the sequence LARLHSVRER, a series of cellulose-membrane-attached peptides of systematically modified structure was synthesised. These peptides had hydrophobic (Ala, Leu) and ionic (Arg, Glu) amino acids substituted in each position. The phosphorylation of these substrates by CDPK-1 was measured and the substrate specificity of the maize protein kinase characterised by the consensus sequence motif A/L-5X-4R-3X-2X-1SX+1R+2Z+3R+4, where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine compared with the other three varied amino acids. This motif had a characteristic sequence element RZR at positions +2 to +4 and closely resembled the primary structure of the sucrose synthase phosphorylation site. The sequence surrounding the phosphorylatable serine in this consensus motif was similar to the analogous sequence K/RXXS/TXK/R proposed for mammalian PKC, but different from the consensus motif RRXS/TX for PKA.

Regulation of inositol phospholipid hydrolysis by UTP and UDP in neuroblastoma x glioma hybrid cell line NG108-15 was potentiated in the presence of ATP. The effect of ATP was dose dependent and shifted the EC50 value for these uracil nucleotides up to three powers of magnitude, having no influence on the maximal value of the response. Adenine nucleotides (ADP, AMP, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), beta,gamma-methyleneadenosine 5'-triphosphate (betagammaMeATP), 3'-O-(4-benzoyl)benzoyl ATP (BzATP) and 3'-deoxyadenosine 5'-O-(1-thio)triphosphate (dATPalphaS)) as well as adenosine, had no influence on the pyrimidinoceptor response. The potentiation effect was abolished by excess of EDTA. The results were in agreement with the hypothesis of pyrimidinoceptor affinity regulation via extracellular phosphorylation of the receptor protein, initiated by ATP. This mechanism may have physiological implication for functioning of uracil nucleotides as endogenous signaling molecules.

A new method for biosensor calibration and data processing, allowing the prediction of steady state parameters from the analysis of transient response curves (Rinken et al., 1996. Analytical Letters 29, 859), has been evaluated in the case of an oxygen sensor based two-substrate enzyme electrode for glucose determination. The electrochemical glucose biosensor was prepared by covering the surface of oxygen sensor with glucose oxidase (EC 1.1.3.4) immobilized in nylon mesh. This decreased the oxygen flow to the sensor in the presence of glucose and resulted in time-dependent decrease of the biosensor signal. Except the lag period of the response in the beginning of the assay, the oxygen consumption by the immobilized enzyme was described by an exponential function: [formula: see text] The parameter C, which corresponded to the steady-state output of the biosensor, was found to be the most suitable for glucose determination. The non-linear fitting for data of over 1000 independent experiments to the equation above always revealed correlation coefficients greater than 0.97. The calculation of the steady state parameter from the transient phase data makes the analysis fast and precise, especially for sensors with thick membranes, being convenient to use in the case of enzyme electrodes. The theoretical essence of the parameter C also gives valuable information for the optimal design of biosensors.

The site and sequence specificity of protein kinases, as well as the role of the secondary structure and surface accessibility of the phosphorylation sites on substrate proteins, was statistically analyzed. The experimental data were collected from the literature and are available on the World Wide Web at http://www.cbs.dtu.dk/databases/PhosphoBase/. The set of data involved 1008 phosphorylatable sites in 406 proteins, which were phosphorylated by 58 protein kinases. It was found that there exists almost absolute Ser/Thr or Tyr specificity, with rare exceptions. The sequence specificity determinants were less strict and were located between positions -4 and +4 relative to the phosphorylation site. Secondary structure and surface accessibility predictions revealed that most of the phosphorylation sites were located on the surface of the target proteins.

The Michaelis-Menten parameters kcat, Ks(app) and the second-order rate constants kII = k2/Ks of acetylcholinesterase-catalyzed hydrolysis of 25 acetic esters with non-ionic leaving groups have been determined at 25 degree C and pH 7.5 in 0.15 M KCL. A linear relationship between the substrate noncovalent binding capacity and the leaving group hydrophobicity, and a multiparameter correlation of the acetylation reaction rate constant logarithm with the leaving group inductive effect, hydrophobicity, and steric effect, have been established. The acetyl-enzyme deacetylation rate constant has been calculated. Taken together, a fairly complete understanding of acetylcholinesterase specificity is possible. The data are consistent with a model of the acetylcholinesterase active site, in which the catalytically active groups are located at the bottom of a jaws-like slit with a limited range of hydrophobic walls that provide the sorption of the substrate leaving groups not longer than that in n-butyl acetate.