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V Kasche

Publications and source records attributed to V Kasche.

At least 37 records · Page 2Linked to original sources
PubMed1991

Electrostatic effects in the alpha-chymotrypsin-catalyzed acyl transfer. II. Efficiency of nucleophiles bearing charged groups in various locations.

We investigated the alpha-chymotrypsin-catalyzed acyl transfer to a series of glycine oligomers. It could be established that the electrostatic interactions between the carboxylate group of the nucleophiles and the S'-subsites of the enzyme fall off with the length of the nucleophile molecule. Additional negatively charged residues in the nucleophile lead to a considerable reduction of the acyl transfer efficiency. An arginine residue in P'1- or P'3-position, but not in P'2-position, makes favourable interactions with the appropriate S'-subsites of the enzyme.

Acylation↗
PubMed1991

Nucleophile specificity in papain-catalyzed acyl transfer reactions.

The ratio of hydrolysis to aminolysis product in papain-catalyzed acyl transfer reactions using various nucleophiles was determined. The data are interpreted in terms of binding specificity. The acyl transfer reactions were performed using the acyl donor Mal-Phe-Ala-OEtCl. The analysis of the structure-activity relationships of the hydrophobic S1'-P1' contact indicates that the S1' subsite can accommodate maximally three methyl(ene) groups. Hydrophilic amino acid side chains are better bound to S1' than can be explained by their hydrophobicities. The S2' as well as the S3' binding subsite exhibits a preference for space-filling hydrophobic amino acid residues.

Acylation↗
PubMed1991

Stereo- and sequence specificity of serine proteases in peptide synthesis.

The sequence- and stereospecificity of the S1- and S' i-subsites (i = 1-3) of bovine alpha-chymotrypsin and trypsin, proteinase K and penicillin amidase from E. coli and A. viscosus has been determined by hydrolysis and kinetically controlled peptide synthesis using different substrates. The data are compared with results for other serine proteases and the thiol protease papain. The stereospecificities differ by orders of magnitude, decreased when the enzyme was immobilized and were influenced when organic solvent molecules were bound to the enzyme.

Amino Acid Sequence↗
PubMed1990

Rapid protein purification using phenylbutylamine-Eupergit: a novel method for large-scale procedures.

Electrophoretic desorption was used to compare the protein binding capacities of some hydrophobic adsorbents [the phenylbutylamine (PBA) derivatives of Eupergit C and agarose and Phenyl-Sepharose] for low-pressure chromatography. The highest capacity was observed for the bifunctional adsorbent PBA-Eupergit. The hydrophobically adsorbed proteins can be selectively desorbed by decreasing the pH of the eluent due to electrostatic repulsion between positive charges on the adsorbed proteins and positively charged secondary amines on the adsorbent. This was used to purify 1500 U penicillin amidase from E. coli homogenates per gram wet weight of PBA-Eupergit in 50 adsorption-desorption cycles without organic solvents (greater than 90% yield, purification factor = 5.3).

Adsorption↗
PubMed1989

A native, affinity-based protein blot for the analysis of streptavidin heterogeneity: consequences for the specificity of streptavidin mediated binding assays.

Commercial preparations of streptavidin, a bacterial biotin-binding protein, were analyzed by isoelectric focusing combined with an affinity-based protein blot using biotinylated, protein-saturated nitrocellulose. The colorimetrical detection of streptavidin with biotinylated alkaline phosphatase allows the selective visualization of streptavidin molecules with at least two active biotin-binding sites. Dependent on the preparation, seven to sixteen streptavidin forms were found with isoelectric points ranging from 5 to 8. Molecular weight analysis of the subunits of streptavidin showed that the observed heterogeneity was mainly due to limited proteolysis, which does not destroy the biotin-binding activity. The preparations differed also in the nonspecific reactivity of streptavidin with single-stranded DNA, bovine serum albumin and Tween 20. No relationship was observed between heterogeneity and non-specific binding activity. Data obtained from protein blots onto nitrocellulose saturated with single-stranded DNA showed that it cannot be excluded that streptavidin with only a single active biotin-binding site is mainly responsible for the nonspecific reactivity of some streptavidin preparations.

Bacterial Proteins↗
PubMed1986

Reaction mechanism, specificity and pH-dependence of peptide synthesis catalyzed by the metalloproteinase thermolysin.

The initial rates of peptide bond formation catalyzed by the metalloproteinase thermolysin were determined. The dependence of the formation rates on the concentration of the carboxyl donor and the acceptor can be explained by a rapid-equilibrium random bireactant mechanism, in which the binding of one substrate has a positive influence on the binding of the other (synergism). The specificity of the enzyme for the donor and acceptor in the condensation reaction was further investigated by determining the apparent kinetic parameters kcat and Km for various substrates. The pH-dependence of the initial rates of synthesis was found to be identical to the pH-dependence of the hydrolytic action of the enzyme. The rates are also shown to be independent of the pKa of the amino group of the acceptor, indicating that deprotonation of the attacking nucleophile in the synthetic reaction is not rate-limiting.

Hydrogen-Ion Concentration↗
PubMed1985

Peptide synthesis catalyzed by the serine proteinases chymotrypsin and trypsin.

The ratio of the initial rates of aminolysis and hydrolysis in peptide semisynthesis catalyzed by chymotrypsin (EC 3.4.21.1) and trypsin (EC 3.4.21.4) was found to depend non-linearly on the concentration of the added nucleophile. This is in agreement with a mechanism for the peptide semisynthesis where nucleophile binding to the acyl-enzyme precedes the aminolysis reaction. The acyl-enzyme-nucleophile complex can still be deacylated by water. A temperature optimum was observed for peptide synthesis for valinamide as nucleophile. This and the similarity of the P'1 specificity in peptide hydrolysis and nucleophile specificity in peptide semisynthesis also support the mechanism including the nucleophile binding. The influence of added nucleophiles on the acylation step during peptide synthesis was studied by determining kcat and Km for the appearance of the leaving group from the acyl donor. Acceptor (= nucleophile) specificity was shown to be more important for high ratios of aminolysis: hydrolysis than donor specificity. The maximum product concentration during kinetically controlled peptide semisynthesis was found to be independent of the enzyme content.

Acylation↗
PubMed1984

Kinetic studies on the mechanism and the specificity of peptide semisynthesis catalyzed by the serine proteases alpha-chymotrypsin and beta-trypsin.

The mechanism of peptide semisynthesis catalyzed by alpha-chymotrypsin and beta-trypsin has been investigated. The dependence of the apparent ratio of the second order rate constants for the deacylation of the acyl-enzyme intermediate by water and other nucleophiles (amino acid amides) on the nucleophile concentration indicates a mechanism that involves two acyl-enzymes. One with and one without bound nucleophile that both can be deacylated by water. The nucleophile specificity in peptide semisynthesis catalyzed by the proteases was found to reflect the P1-specificity in the corresponding hydrolytic reaction.

Acylation↗
PubMed1984

Kinetic studies on the mechanism of the penicillin amidase-catalysed synthesis of ampicillin and benzylpenicillin.

Hydrophobic protein chromatography was used to prepare homogeneous fractions of penicillin amidase (EC 3.5.1.11) from E. coli. The apparent ratios of the rate constants for the deacylation of the acyl-penicillin amidase formed in the hydrolysis of phenylacetylglycine or D-phenylglycine methyl ester, by H2O and 6-aminopenicillanic acid (6-APA), were determined at different concentrations of the latter compound. The ratios were obtained from direct measurements of the initial rates of formation of phenylacetic acid and benzylpenicillin or D-phenylglycine and ampicillin. For the semisynthesis of ampicillin as well as of benzylpenicillin the ratio was found to depend on the concentration of 6-APA. This was observed for heterogeneous and homogeneous enzyme preparations. These results show that 6-APA must be bound to the acyl-enzyme before the deacylation, yielding ampicillin and benzylpenicillin, occurs. The dissociation constant KN for the formation of the complex was estimated to be approximately 10mM. This mechanism in which acyl-enzyme with and without bound nucleophile is involved, is in agreement with the principle of microscopic reversibility. Both acyl-enzymes can be deacylated by H2O. The finding that there is a specific binding site for 6-APA adjacent to the binding site for the phenylacetyl-(D-phenylglycyl-) group in the active site of the enzyme is supported by the observation that 6-APA acts as a mixed inhibitor in the hydrolysis of D-phenylglycine methyl ester. The ionic strength dependence indicates that the binding site for 6-APA of the acyl-enzyme is positively charged.

Amidohydrolases↗
PubMed1982

Tris(hydroxymethyl)methylamine is acylated when it reacts with acyl-chymotrypsin.

Tris [Tris(hydroxymethyl)methylamine] was found to participate directly in the deacylation of acyl-chymotrypsin formed when the enzymes hydrolyses specific substrates. The acyl-Tris compound formed in this reaction is a less specific substrate and may give rise to product inhibiton kinetics. Only uncharged Tris was found to be a (nucleophilic) deacylating reagent for the acyl-enzyme and as such better than H2O and methanol. More than 50% and 10% of the acyl groups of the initial substrate could be transferred to Tris at pH 9 (0.17M Tris) and pH 8 (0.06M Tris), respectively. This indicates that Tris may interfere in all enzyme mechanisms involving acyl-enzymes.

Acylation↗