Biomedical subjects

E M Savitskaia

Publications and source records attributed to E M Savitskaia.

At least 19 recordsLinked to original sources

PubMed1984

[Penicillinase from B. licheniformis. Determination of the ionization constants of the enzymatic hydrolysis products of beta-lactam antibiotics].

The level of transformation of beta-lactam antibiotics hydrolysed by penicillinase from B. licheniformis on determination with the method of pH-metric titration with sodium hydrate solutions depended on the electrochemical nature of the products formed. The data on the study of the pH dependence of the penicillinase-catalysed hydrolysis of beta-lactam antibiotics were used for estimation of the ionization constants of the products of the enzymatic hydrolysis of the beta-lactam ring in the molecules of azlocillin, carfecillin, benzylpenicillin, cephalothin and 7-PADCA. Methods for quantitative determination of the compounds were developed. The methods are based on penicillinase-catalysed enzymatic hydrolysis and pH-metric titration of the products with sodium hydrate solutions at pH 7.0 with regard to their dissociation levels.

Anti-Bacterial Agents↗

PubMed1984

[Aminoacylase from Streptoverticillium microorganisms: stereo- and substrate specificity].

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.

Amidohydrolases↗

PubMed1982

[Extractive polarographic method of controlling the stereospecific hydrolysis of N-acylated derivatives of amino acid racemates].

The equilibrium distribution of acetyl-D-phenylglycine prepared with stereospecific hydrolysis of acetyl-D,L-phenylglycine (Ac-D,L-PhG) was studied in a two-phase system of benzyl alcohol-1N HCl solution. It was found that the distribution coefficient of Ac-D-PhG did not practically depend on its concentration in the aqueous phase. This means that Ac-D-PhG does not form associates in the organic phase. The studies provided development of an extraction polarimetric method for quantitative determination of Ac-D-PhG. The method implies extraction of Ac-D-PhG with benzyl alcohol from a strong acid medium (1N HCl solution), polarimetric determination of the equilibrium concentration of Ac-D-PhG in the organic phase and subsequent calculation of the initial concentration of Ac-D-PhG in the aqueous phase with regard to its distribution coefficient.

Acylation↗

PubMed1981

[E. coli penicillin amidase. Methods for estimating the close ionization constants of ionogenic groups of the enzyme complex with substrates containing free amino groups].

The possible use of various procedures for estimation of the ionization constants of the Michaelis complex by the pH dependence of the maximum enzymatic reaction rate is discussed. It is shown that the procedures described in the literature for estimation of the close ionization constants of the enzyme-substrate complexes have limitations and in some cases cannot be used. The paper presents the methods for estimation of the constants and means for quantitative description of the bell-shaped pH dependence of the kinetic and equilibrium parameters of the biocatalytic reaction. The equations recommended in the paper were used in analysis of the pH dependences of the maximum rate of the reactions during the enzymatic synthesis of cefalexin catalysed with immobilized penicillinamidase (IPA) (CE 3.5. 1.11). The ionization constants of the enzyme-substrate complexes of IPA were compared during hydrolysis and synthesis of the compounds acylated with phenylacetic and aminophenylacetic acids. The effect of the nature of the leaving substrate group and added nucleophilic gent on the electrochemical state of the Michaelis complex is discussed.

Amidohydrolases↗

PubMed1980

[Penicillin amidase from E. coli. The kinetic and equilibrium parameters of the enzymatic hydrolysis of 7-phenylacetamidodesacetoxycephalosporanic acid catalyzed by an immobile enzyme].

The kinetics of 7-phenylacetamidodesacetoxycephalosporanic acid (7-PADCA) catalyzed by immobilized penicillinamidase was studied. The kinetic and equilibrium parameters of the reaction were determined by analysis of the kinetic curves of the reaction product accumulation. Inhibition of the enzymatic reaction by the substrate and hydrolysis products was studied. It was found that the Michaelis complex completely lost its activity after attachment of the substrate second molecule to it. The values of the Michaelis constants, catalytic constant and constants of inhibition by the substrate and reaction products were determined: Km = (9.3 +/- 1.1) . 10(-5) M, kcat = (65 +/- 5) c-1, Ks = (1.4 +/- 0.1) . 10(-2) M, K1 (FAA) = (2.5 +/- 0.3) . 10(-4) M, K1 (7-ADCA) = (1.4 +/- 0.1) . 10(-1) M. The diffusion effect in the kinetic reaction catalyzed by immobilized penicillinamidase is discussed. The values of the Thiele modulus and the actual value of Km were calculated.

Amidohydrolases↗

PubMed1979

[Solubility and the complex-forming properties of the zwitterlytes, 6-aminopenicillanic and 7-aminodesacetoxycephalosporanic acids].

Relation between solubility of the zwitterlites 6-aminopenicillanic acid (6-APA) and 7-aminodesacetoxycephalosporamic acid (7-ADCA) and the ionic strength and pH of the solution was studied. A specific effect of benzylpenicillin (BP) and 7-phenylacetamidodesacetoxycephalosporanic acid (7-PADCA) on solubility of the above zwitterlites was found and complex formation was suggested. An explanation of the complex formation mechanism and a method for estimation of the instability constants of the complexes are presented. The method is based on determination of the changes in the zwitterlite solubility in the presence of the complex forming compound. The values of the characteristic solubility of the ampholyte zwitter ions and the instability constants of the complexes "zwitterlite-complex forming compounds" were determined. A mathematical description of the relation between the zwitterlite solubility within a wide range of pH and concentrations of the complex forming compounds is presented. Coincidence of the calculated and experimental data was observed, which provided a supposition that the substances involved in complex formation were charged.

Cephalosporins↗

PubMed1978

[Study of E. coli penicillin amidase. The pH-dependence of the enzymatic inactivation kinetics].

The pH-dependence of the inactivation rate constant of penicillin amidase at a temperature of 40 degrees C was studied. It was shown that in all cases the enzyme inactivation corresponded to the kinetics of the reaction of the 1st order. The pH-dependence profile was found to be bell-shaped, the effect of transfer from the highest to the lowest values of the inactivation rate constants increasing more than 100 times. On the basis of the data obtained and published earlier it was concluded that the enzyme inactivation proceeded in accordance with the scheme in which out of 3 equilibrium ionic forms of penicillin amidase, i.e. "acid", "neutral" and "alkaline" the neutral form of the active enzyme was most stable. Kinetic analysis of the scheme was carried out and it was shown that the dependence found was in accordance with the theoretical curve in which the pK values of the ionogenic groups controlling the interconvertions between the penicillin amidase forms were equal to 2.4 and 10.1 at a temperature of 40 degrees C. The value of the inactivation rate constant of the "acid" or "alkaline" form was equal to 5.95 min-1, while the "neutral" form of the enzyme was characterized by the inactivation rate constant equal to 5.1.10(-4) min-1. A mechanism for the enzyme inactivation was proposed. According to this mechanism, destruction of the salt bridge in the native structure of penicillin amidase resulted in production of extremely labile forms of the enzyme as compared to the native form.

Amidohydrolases↗

PubMed1978

[Study of E. coli penicillin amidase. The pH dependence of the equilibrium constant of ampicillin enzymatic hydrolysis].

The equilibrium parameters of the hydrolysis of ampicillin catalysed by penicillin amidase were determined within the pH range of 4.5 to 5.5. The values of the ionization constants of the carboxy group of D-(-)-ALPHA-AMINOPHENYLACETIC ACID (PK1=1.80) and amino group of 6-aminopenicillanic acid (pK2=4.60) were estimated and pH-dependence of the effective free energy of ampicillin hydrolysis was calculated. It was shown that the thermodynamic optimum of ampicillin synthesis was at 3.20 (the value of the effective free energy under the experimental conditions was 3.27 kcal/mole). The value of the "true", pH-independent free energy of hydrolysis (deltasigma) of the amide bond in the ampicillin molecule was determined to be equal to 9.72 kcal/mole. The thermodynamic parameters of ampicillin and benzylpenicillin hydrolysis were compared. The amino group in the alpha-position of phenylacetic acid was shown to have a significant effect on the values of "true" free energy of hydrolysis of the penicillin amide bond and free ionization energy in the system.

Amidohydrolases↗

PubMed1978

[Extraction and polarimetric method of determining phenacetyl-D(-)-alpha-aminophenylacetic acid].

Equilibrium distribution of phenacetyl-D-(--)-alpha-aminophenylacetic acid obtained on fermentative hydrolysis of D,L-aminophenylacetic acid in a two-phase system (chloroform-water) was studied within a wide range of the substance concentration in the organic phase. It was shown than in the organic phase the distributing substance formed associates. The empirical equation: y = A + Bx describing the dependence of the effective distribution of the non-dissociated form of phenylacetyl-D-(--)-alpha-aminophenylacetic acid on its concentration in the organic phase was suggested. Coefficients A and B of the equation were determined and an equation for evaluating equilibrium concentrations in the equeous phase was suggested. On the basis of the studies an extraction-polarometric method for quantitative determination of phenacetyl-D-(--)-alpha-aminophenylacetic acid concentration was developed. The method consists of extraction of the non-dissociated form of phenylacetyl-D-(--)-alpha-aminophenylacetic acid with chloroform, determination of the equilibrium concentration of the distributing substance in the organic phase by the polarimetric method and subsequent estimation of the equilibrium and initial concentrations of the electrolyte in the aqueous phase.

Chloroform↗

PubMed1977

[Penicillin amidase from E. coli. Some physicochemical properties of the soluble enzyme].

Homogeneity of the enzyme was shown with the methods of gel filtration and disc electrophoresis. The molecular mass of penicillinamidase (PA) was determined. Sorption of PA by a carboxylic ion exchanger within a wide range of pH was studied. The values of pH in the ion exchanger phase under the conditions of the enzyme sorption were estimated. The ion exchange technique for determination of the isoelectric points of the proteins is described and the isoelectric point of PA is determined. It is proposed to use the method for estimation of close ionization constants of amphoteric an weak electrolites for interpretation of the bell-like pH dependence of kinetic and equilibrium parameters of the enzymatic reaction. The ionization constants of Michaelis complex of PA were evaluated. The activation energy of benzylpenicillin hydrolysis catalized by PA was determined.

Amidohydrolases↗

PubMed1977

[Penicillin amidase from E. coli. Some physicochemical properties of the enzyme incorporated into polyacrylamide gel].

The physico-chemical properties of penicillinamidase (PA) immobilized in polyacrylamide gel (IPA) were investigated. It was shown that simple incorporation of PA into polyacrylamide gel was not effective because of gradual washing out of the enzyme. The use of a complex method for the immobilization (immobilization in the presence of a linking agent) resulted in higher stability of IPA, the choice of the optimal ratio of the reagents being of paramount importance. The mechanical strength of IPA was studied in model experiments.

Acrylamides↗

PubMed1977

[E. coli penicillin amidase. Physico-chemical properties of the enzyme covalently bound to the 2-(3'-amino-4'-methoxyphenyl)-sulfonylethyl ester of cellulose].

The effect of the procedure of the enzyme binding with the carrier on the properties of the heterogenous catalyst obtained by covalent binding of penicillinamidase (PA) with cellulose 2-(3'-amino-4'-methoxyphenyl)-sulphonylethyl ether by means of the bifunctional reagent, i.e. glutaric aldehyde was studied. It was shown that the amount of the bound enzyme increased with a rise in the amount of the enzyme taken for the binding, while the binding efficiency characterizing the part of the active enzyme in the total amount of the bound PA decreased practically 2 times. The use of the enzyme preparations with different purify levels for the binding provided differentiation of the effects resulting in the activity loss on immobilization. In other words it provided separate estimation of the inactivation effect of the matrix and the immobilization procedure, as well as the interaction of the enzyme molecules with each other and other protein molecules.

Amidohydrolases↗

PubMed1977

[Physiochemical properties of 7-aminoacetoxycephalosporanic acid].

Potentiometric titration of the zwitter-ion of 7-aminoacetoxycephalosporanic acid (7-ACA) was performed and the constants of its ionization were estimated. The minimum solubility of the 7-ACA zwitter-ion (20 degrees C, 0.1 M NaCl) was determined and the solubility curve of 7-ACA at wide pH ranges was calculated. Equilibrium of the cationic, zwitter-ionic and anionic forms of 7-ACA was estimated as dependent on pH.

Cephalosporins↗

PubMed1977

[Penicillin amidase from E. coli. A comparative study of the stability of penicillin amidase immobilized by various means].

The properties of immobilized penicillinamidases prepared by different methods were studied. Immobilization of penicillinamidase was achieved by using the covalend binding ion exchange sorption, incorporation into gel and other procedures. The effect of the carrier type, purification level of the native enzyme and other factors on stability of the immobilized preparations was studied. In 4 cases significant stabilization of the enzyme in the immobilized state was observed, while in 3 cases destabilization was registered.

Amidohydrolases↗

PubMed1977

[Acid-base equilibrium of D-(--)-alpha-aminophenylacetic acid].

Potentiometric titration was performed and the ioniziation macroconstants of D-(--)-alpha-aminophenylacetic acid and its methyl ether were determined. The constants were used for estimation of 4 microconstants of D-(--)-alpha-aminophenylacetic acid and the constants of the tautomeric equilibrium of the zwitter-ionic and neutral forms of the amino acid. Minimum solubility of D-(--)-alpha-aminophenylacetic acid was determined and pH-dependence of its solubility was estimated.

Acid-Base Equilibrium↗

PubMed1976

[Physico-chemical properties of 7-aminodesacetoxycephalosporanic acid].

7-Aminodesacetoxycephalosporanic acid (7-ADCA) of 99 per cent purity was prepared by enzymatic hydrolysis of 7-phenylacetamidodesacetoxycephalosporanic acid. Its isoelecti point was determined by the electrophoretic method. Potentiometric titration of zwitterion of 7-ADCA was performed and the constants of its ionization were calculated. Minimum solubility of 7-ADCA zwitterion was determined and the curve of 7-ADCA solubility at wide pH ranges was estimated. Equilibrium of 3 forms of 7-ADCA was estimated.

Cephalosporins↗

PubMed1976

[Study of penicillin amidase from E. coli. pH-dependence of kinetic parameters of enzymatic hydrolysis of benzylpenicillin].

The authors studies pH-dependencies of the kinetic parameters (Vm, KM, Vm/KM) and constants of competitive inhibition by phenylacetic acid of penicillinamidase-catalyzed hydrolysis of benzylpenicillin. The experimental data are in agreement with the assumption according to which there are 3 equilibrium ionogenic forms of the enzyme and enzyme-substrate (or enzyme-inhibitor) complexes, i.e. acidic, neutral and alkaline, the neutral form being the only active form of the Michaelis complex. Values of pK in the ionogenic groups controlling interconversions of both the free enzyme (pK1 6.1 and pK2 7.6) and of the enzyme-substrate complex (pKa 6.1 and pK2 10.2 or the enzyzme-inhibitor complex (pK''1 6.1 and pK''2 9.5) were determined. From this and the previously published results it was concluded that the group with pK 6.1 was involved in the catalysis and the group with pK 10.2 in the maintenance of the active conformation of the active centre of penicillinamidase. The ionogenic group with pK 7.6 was apparently involved in the enzyme-substrate binding.

Amidohydrolases↗

PubMed1976

[Study of penicillin amidase for E. coli. Kinetics of enzymatic hydrolysis of 7-phenylacetamidodeacetoxycephalosporanic acid].

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.

Amidohydrolases↗