[Improved procedure of the search for poly(ADP-ribose) polymerase-1 potential inhibitors with use of molecular docking approach].

A search for poly(ADP-ribose) polymerase-1 inhibitors by virtual screening of a chemical compound database and a subsequent experimental verification of their activities have been done. It was shown that the most efficient method to predict inhibitory properties implies a combinatorial approach joining molecular docking capabilities with structural filtration. Among more than 300000 database chemicals 9 PARP1 inhibitors were revealed; the most active ones, namely: STK031481, STK056130, and STK265022,--displayed biological effect at a micro-molar concentration (IC50 = 2.0 microM, 1.0 microM and 2.6 microM, respectively).

[A new N-acyl derivative of (S)-cysteine for quantitative determination of enantiomers of amino compounds by HPLC method with a precolumn modification with o-phthalaldehyde]. / Novoe N-atsil'noe proizvodnoe (S)-tsisteina dlia kolichestvennogo opredeleniia énantiomerov aminosoedineniii metodom VEZhKh s predkolonochnoi modifikatsiei opto-ftalevym al'degidom.

N-Phenylacetyl-(R)-phenylglycyl-(S)-cysteine (NPPC) was used for the determination of enantiomers of primary amines by rpHPLC with a precolumn modification with o-phthalaldehyde. NPPC was compared with the classic SH reagent N-acetyl-(S)-cysteine (NAC) in the analysis of stereomers of nonfunctionalized amines and amino alcohols. After the NAC modification, the resulting diastereomeric isoindoles were difficult to separate by HPLC, and satisfactory resolution was achieved only for some aliphatic amino alcohols. The use of NPPC improved the chromatographic analysis of stereomeric amino alcohols and, in addition, allowed the enantiomeric analysis of the nonfunctionalized amines. Similarity between the side radicals of the amino component and the thiol reagent favored the diastereomer separation. This method was used for determination of the absolute concentration of individual enantiomers of amines in the course of stereoselective enzymatic reactions. The optically active NPPC was prepared with a high yield by a chemoenzymatic synthesis based on a regioselective acylation of the (S)-cysteine amino group in aqueous medium by the action of penicillin acylase. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.

[Detection of nucleic amino acid enantiomers by HPLC with preliminary modification by o-phthalic aldehyde]. / Opredelenie énantiomerov nucleoaminokislot metodom vysoko'effektivnoi zhidkostnoi khromatografii s predkolonochnoi modifikatsiei o-ftalevym al'degidom.

The use of reversed-phase HPLC with UV detection at 254 nm for the separation of stereoisomers of nucleoamino acids, namely, pyrimidyl and purinyl derivatives of alanine, after premodification with o-phthalic aldehyde and N-acetyl-L-cysteine was studied. The use of 0.01 M phosphate buffer (pH 7.0) containing 5% acetonitrile as a mobile phase resulted in satisfactory separation. The range of the detectable amounts of nucleoamino acids was 0.08-1.0 nmol. This method was used for monitoring the formation of stereoisomers in the hydrolysis of N-phenylacetylcytosinylalanine.

[Regulation of the catalytic activity and supermolecular structure of penicillin acylase from Escherichia coli in an aerosol OT reversed micelle system in octane]. / Reguliatsiia kataliticheskoi aktivnosti i nadmolekuliarnoi struktury penitsillinatsilazy iz Escherichia coli v sistem obrashchennykh mitsell aérozolia OT v oktane.

The properties of penicillin acylase from E. coli solubilized by hydrated reversed micelles of Aerozol OT (AOT) in octane were studied. The catalytic activity dependence on the hydration degree, a parameter which determines the size of the micelle inner cavity, represents a curve with three optima, each corresponding to the enzyme functioning either in a dimer form (omega 0 = 23) or in the form of separate subunits--heavy, beta, and light, alpha, at omega 0 = 20 and 14, respectively. Reversible dissociation of the enzyme was confirmed by ultracentrifugation followed by electrophoresis. Preparative isolation of penicillin acylase subunits, their catalytic activity being retained, was shown to be possible.

[Kinetic characteristics and enantioselective action of penicillinase in the hydrolysis reaction of N-phenylacetyl derivatives of 1-aminoethylphosphonic acid and its esters]. / Kineticheskie zakonomernosti i énantioselektivnost' deistviia penitsillinazy v reaktsii gidroliza N-fenilatsetil'nykh proizvodnykh 1-aminoétilfosfonovoi kisloty i ee éfirov.

Penicillin acylase from E. coli (EC 3.5.1.11) was found to hydrolyze N-phenylacetylated 1-aminoethylphosphonic acid and its esters. The enzyme preferentially converts the R-form of the substrates: the ratios of the bimolecular rate constants of penicillin acylasecatalyzed hydrolysis of R- and S-forms of 1-(N-phenylacetamino)-ethylphosphonic acid and its dimethyl- and diisopropyl-esters are 58000, 2300, 1800; these derivatives were shown to have the greatest values of the catalytic constants for enzymatic hydrolysis of all known substrates for penicillin acylase: 237, 148 and 134 s-1; the corresponding Km values are 3.7 10(-5), 6.8 10(-4) and 6.2 10(-4) M at pH 7.0. The kinetics of enzymatic hydrolysis of 1-(N-phenylacetamino)-ethylphosphonic acid was investigated up to high degrees of conversion. The inhibition of penicillin acylase by high concentrations of the R-form of the substrate (with substrate inhibition constant of 0.07 M) and competitive inhibition by the reaction product, phenylacetic acid (Ki = 3.5 10(-5) M), was observed.

[Peptide synthesis catalyzed by proteases. Elevated nucleophilic activity of naphthylamides of amino acids in acyl transfer reactions catalyzed by alpha-chymotrypsin]. / Peptidnyi sintez, kataliziruemyi proteazami. Povyshennaia nukleofil'nost' naftilamidov aminokislot v reaktsiiakh atsil'nogo perenosa, kataliziruemykh alpha-khimotripsinom.

It was found that the reactivity of alpha-amino acid naphthylamides in acyl transfer reactions catalyzed by alpha-chymotrypsin exceeds by more than two orders of magnitude the effective reactivity of other C-protected derivatives of these compounds. A detailed kinetic analysis of the acyl transfer of the tert-butyl oxycarbonyl-L-methionine residue from its p-nitrophenyl ester to L-arginine naphthylamide was carried out. A minimal kinetic scheme of acyl transfer reactions is proposed, including together with the major process, i.e., acyl residue transfer to the nucleophil, the hydrolysis of the acyl enzyme-nucleophil complex and nucleophil binding by the free enzyme. The numeric values of some kinetic constants were determined. A theoretical analysis of the effect of hydrolysis of the acyl enzyme-nucleophil complex on the degree of nucleophil conversion into the peptide at initial acyl group donor and nucleophil concentrations was carried out.

[Peptide synthesis catalyzed by proteases. Analysis of a kinetic model for enzymes with acyl-enzyme mechanism of action]. / Peptidnyi sintez, kataliziruemyi proteazami. Analiz kineticheskoi modeli dlia fermentov, deistvuiushchikh po atsilfermentnomu mekhanizmu.

The kinetics of peptide synthesis via transfer of the acyl moiety from activated derivatives of amino acids or peptides (S) to nucleophiles (N) catalyzed by proteases forming an acyl-enzyme intermediate, was analysed. A kinetic model assumes enzymatic hydrolysis of the formed peptide (P), so the kinetic curve for P has a maximum (denoted as pmax). Particular attention was given to the analysis of the effects of the initial concentrations and kinetic constants on pmax. Computer analysis demonstrated that at a given ratio of initial S and N concentrations pmax is affected only by the ratio of the second order rate constants for enzymatic hydrolysis of S and P (alpha) and the ratio of rate constants for an attack of the acyl-enzyme intermediate by nucleophile and water (beta). These conclusions apply regardless of the existence of enzyme forms other than a free enzyme and an acyl-enzyme intermediate. Thus, the kinetically controlled maximum yield of peptide (pmax) can be calculated a priori from the values of alpha and beta which can be readily evaluated from the reference data. Simple explicit expressions were obtained, allowing fairly accurate prediction of pmax for a broad spectrum of S and N initial concentrations.

[Aminoacylase from Streptoverticillium microorganisms: stereo- and substrate specificity]. / Aminoatsilaza mikroorganizmov iz roda Streptoverticillium: izuchenie stereo- i substratnoi spetsifichnosti.

The stereo- and substrate specificity of a new aminoacylase from Streptoverticillium microorganisms was studied. The enzyme effectively hydrolyzes acetyl derivatives of aliphatic (methionine, leucine) and aromatic (phenylglycine, phenylalanine, tryptophan) amino acids. The L-enanthiomer of acetylphenylglycine is hydrolyzed by aminoacylase 8000 times more effectively than the D-enanthiomer. A procedure for determination of the enanthioselectivity of aminoacylases was elaborated. This procedure is designed for a detection and assessment of contaminations of the N-acetyl derivative of one enanthiomer by another enanthiomer of the amino acid, as well as of the degree of racemization of the substrate during hydrolysis of acetyl derivatives of D-amino acids.

[Determination of lysine amidase and alpha-aminocaprolactam hydrolase activities in cell-free extracts of Cryptococcus sp]. / Opredelenie lizinamidaznoi i al'fa-aminokaprolaktamgidrolaznoi aktivnostei v beskletochnykh ékstraktakh Cryptococcus sp.

he methods for detecting two activities, i. e. lysine amidase and alpha-aminocaprolactam hydrolase ones, in crude extracts of Cryptococcus sp. are described. The method for registering lysine amidase activity is based on the ability of Cu(II) complex to absorb at 230 nm. The products of lysine and alpha-aminocaprolactam interactions with o-phthalaldehyde in the presence of mercaptoethanol possess different molar absorption at 340 nm. This fact was used for detecting alpha-aminocaprolactam hydrolase activity. The main merit of the methods is the possibility to register the data on the course of the reaction without preliminary chromatographic separation of the reaction products and reactants. The methods proposed do not require expensive enzymes, such as lysine decarboxylase and lysine-alpha-ketoglutarate-epsilon-aminotransferase, which are used for the quantitative estimation of lysine.

[Enzymes incorporated in polyelectrolyte complexes. Effect of matrix conformational changes and phase transitions in solutions on catalytic properties]. / Fermenty, vkliuchennye v poliélektrolitnye kompleksy. Vliianie konformatsionnykh izmenenii matritsy i fazovykh perekhodov v rastvorakh na kataliticheskie svoistva.

Immobilization of enzymes (penicillin amidase and alpha-chymotrypsin) in water-soluble nonstoichiometric polyeloctrolyte complexes (PEC) formed by poly(4-vinyl-N-ethylpyridinium bromide) (polycation) and polymethacrylic acid (polyanion) was carried out. Particles of these PEC consist of a nucleus formed by sequences of salt bonds between the units of oppositely charged polyelectrolytes and the hydrophylic shell formed by ionized groups of polyanions which is in excess in PEC. Such a structure of PEC particles results in a cooperative phase transitions of these systems at slight variations of pH and ionic strength. The work demonstrates phase diagrams of PEC solutions. The values of pH and ionic strength at which phase transitions in solutions of different PEC occur were elucidated. The decrease of pH value from 6.1 to 5.7 leads to reversible phase transition followed by a saltatory increase of Km for immobilized penicillin amidase by 5-10 fold depending on substrate used. The phase transition induced by ionic strength increase up to 0,27 M NaCl doesn't change significantly the Km-value of enzymic reaction. The phenomenon observed can be accounted for by the different structure of PEC particles. The catalytic properties of immobilized chymotrypsin were shown to depend on the loci of enzyme attachment. If the enzyme is bound to polyanion, neither conformational changes of the matrix nor phase transition in solution influence its accessibility for the protein inhibitor, but rather change the binding constant. If the enzyme is attached to polycation, i.e. included in the polycomplex nucleus, two fractions of enzymes accessible and inaccessible for protein inhibitor appear.(ABSTRACT TRUNCATED AT 250 WORDS)

[Substrate specificity of Bacillus subtilis intracellular serine protease. Hydrolysis of insulin beta-chain, native ribonuclease A and p-nitroanilide peptide substrates]. / O substratnoi spetsifichnosti vnutrikletochnoi sepinovoi proteinazy v Bacillus subtlis. Proteoliz v-tsepi insulina i ribonukleazy A i kinetika gidroliza p-nitoranilidnykh substratov.

Intracellular serine protease, termed ISP-103, was isolated from Bacillus subtilis, strain 103. The substrate specificity of the enzyme was compared to that of secretory subtilisins. Similar to subtilisins, ISP-103 cleaves a single peptide bond Ala20-Ser21 within the native pancreatic ribonuclease A, which results in the accumulation of trypsin-sensitive ribonuclease S, consisting of a non-covalently bound S-peptide (20 amino acid residues) and S-protein (104 amino acid residues). The enzyme hydrolyzes a single peptide bond Leu15-Tyr16 of the B-chain of oxidized bovine insulin, in contrast to the subtilisins cleaving four additional bonds. ISP prefers Leu rather than Phe in the P1 binding site of the rho-nitroanilide peptide substrates and shows a more strict dependence of the activity on the presence of the hydrophobic residues in the P2 and P3 sites. The data obtained indicate that the substrate specificity of ISP, being within the borders of subtilisin specificity, is nevertheless much more restricted.

[Method of measuring activity of amino acid acylase]. / Metod opredeleniia aktivnosti atsilaz aminokislot.

The paper describes a method to follow acylase activity. The method is based on spectrophotometry of the amino acid released, using o-phthalic aldehyde and mercaptoethanol. The major advantage of the method are its high sensitivity and speed. With the aid of the method kinetic parameters of hydrolysis of N-acetyl-L-methionine and N-acetyl-D,L-methionine catalyzed by pig kidney acylase were determined. Michaelis constants at pH 7.5 were estimated to be 5 +/- 1 and 10 +/- 2, respectively; this being in consistency with the data in the literature. It was shown that N-acetyl-D-methionine, acetate ion and methionine at the concentrations tested (0.01-0.05 M) did not inhibit acylase from a pig kidney.

[Study of penicillin amidase for E. coli. Kinetics of enzymatic hydrolysis of 7-phenylacetamidodeacetoxycephalosporanic acid]. / Izuchenie penitsillinamidazy iz E. coli. Kinetika fermentativnogo gidroliza 7-fenilatsetamidodezatsetoksitsefalosporanovoi kisloty

Kinetics of hydrolysis of 7-henylacetamidodeacetoxycephalosporanic acid catalyzed by penicillin amidase as a result of which phenylacetic and 7-aminodeacetoxycephalosporanic acids are formed was studied. The kinetic parameters of the reaction were determined on the basis of both the dependence of the initial rate of enzymatic hydrolysis on the substrate concentration and the analysis of progress kinetic curves of the product accumulation. The values of Km determined by the two methods were equal to (10 +/- 1) muM and kcat (50 +/- 5) sec-1 and (50 +/- 10) sec-1 respectively. The study of the inhibition of the enzymatic hydrolysis by the reaction products showed that phenylacetic and 7-aminodeacetoxycephalosporanic acids were competitive and non-competitive inhibitors of the penicillin amidase activity respectively. The inhibition constants were (55 +/- 8) muM and (12 +/- 1) mM respectively. The physiological role of the enzyme and the effect of the structure of the substrate on the specificity of the enzyme are discussed.