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Biomedical subjects

A Aaviksaar

Publications and source records attributed to A Aaviksaar.

At least 19 recordsLinked to original sources
PubMed1995

Aminolysis of acyl-chymotrypsins by amino acids. Kinetic appearance of concentration effect in peptide yield enhancement by freezing.

The effects of reagent concentrations, various added substances, pH and temperature on the yield of peptide synthesis by chymotrypsin in frozen and liquid solutions at subzero temperatures have been studied. Increased nucleophile concentration in the liquid microinclusions of ice has been shown to be sufficient for explaining the peptide yield improvement found at freezing conditions.

Amino Acids↗
PubMed1994

Electrostatic effects in trypsin reactions. Influence of salts.

The influence of inorganic salts on trypsin-catalyzed reactions has been studied. It is shown that: (a) monovalent cations are reversible competitive inhibitors of tryptic hydrolysis of cationic substrates, whereas their binding has no effect on the reaction of neutral substrates; (b) a nonelectrostatic salt effect on the binding of both cationic and non-ionic substrates is caused by changes in the thermodynamic activity coefficient of the substrate; (c) the rate of trypsin active-site acylation is not affected by inorganic salts with monovalent cations. The data suggest that low-molecular-mass substrates are extracted into the enzyme microphase during substrate binding and further chemical transformations proceed without an access from surrounding medium. It is proposed that formation of a properly oriented dipole in the trypsin binding pocket by the cationic group of the substrate and Asp189 carboxyl is responsible for the elevated acylation rate of trypsin active site by substrates containing lysine and arginine. Introduction of additional negative charges into the enzyme molecule by chemical modification of lysyl residues by pyromellitic anhydride increased the specificity of trypsin towards cationic substrates and inhibitors. Lysine residues are therefore considered as suitable targets for site-directed mutagenesis aimed at the improvement of selectivity and catalytic properties of trypsin.

Animals↗
PubMed1993

Peptide synthesis by chymotrypsin in frozen solutions. Free amino acids as nucleophiles.

Nucleophilic efficiency of the free amino acids in chymotrypsin-catalyzed acyl transfer in ice at -18 degrees C using ethyl esters of N-maleyl-L-tyrosine and L-tyrosine as the acyl group donors has been studied. Although the amino acids did not act as acyl acceptors in liquid water, the high yields of peptides were obtained in frozen solutions at pH 10.5 (before freezing). The efficiency of amino acids in the formation of the corresponding dipeptides depended on the substrate used, and decreased in the order Ser,Thr,Gln > Lys > Cit > Ala > Ala > Gly > Asn > Arg > Glu > Val > Orn > Asp with no peptide formed with His, Leu, Ile and Pro) for N-maleyl-L-tyrosine ethyl ester and Ser > Lys > Orn > Arg,Cit > Gln > Thr > Asn > Ala > Gly (with no peptide formed with Glu, Val, Asp, His, Leu, Ile and Pro) for L-tyrosine ethyl ester.

Amino Acids↗
PubMed1991

Anionic subsites of the catalytic center of acetylcholinesterase from Torpedo and from cobra venom.

A peptide of acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7) from the venom of the cobra Naja naja oxiana labeled by the affinity reagent N,N-dimethyl-2-phenylaziridinium (DPA) has been identified. The sequence is Gly-Ala-Glu-Met-Trp-Asn-Pro-Asn. In AcChoEase from Torpedo californica, a homologous peptide was labeled and isolated. Its sequence is Ser-Gly-Ser-Glu-Met-Trp-Asn-Pro-Asn, representing positions 79 through 87. In both cases labeling can be prevented by 0.1 mM edrophonium, indicating that the respective peptides form part of the anionic subsite of the catalytic center. The modified residue was tryptophan (Trp-84 in Torpedo AcChoEase) in both enzymes. In contrast to AcChoEase from Torpedo, the enzyme from cobra venom does not contain a peripheral anionic binding site.

Acetylcholinesterase↗
PubMed1991

Electrostatic effects in the alpha-chymotrypsin-catalyzed acyl transfer. I. Influence of different inorganic salts.

We investigated the deacylation of two acyl-alpha-chymotrypsins by added nucleophiles. The nucleophile binding site of the enzyme shows a strong preference for positively charged compounds. Most of our data can be explained by direct electrostatic interaction between the ionic nucleophiles and two negatively charged residues which are located close to the active site of the enzyme molecule. The influence of inorganic salts on the acyl transfer includes the following effects: (1) reduction of electrostatic interactions between the acyl-enzyme and the nucleophile by addition of salts; (2) binding of divalent cations to the nucleophile binding site of the acyl-enzyme leading to a significantly changed specificity; and (3) linear dependence of the activity coefficients of the added nucleophiles on salt concentration.

Acylation↗
PubMed1991

Protease-catalyzed peptide synthesis in frozen aqueous systems: the "freeze-concentration model".

Freezing of the aqueous reaction mixtures can cause dramatically increased peptide yields in protease-catalyzed kinetically controlled peptide synthesis reactions. The "Freeze-concentration model" which implies that the enzymic reaction within a macroscopically frozen system takes place in an unfrozen liquid phase is presented as a reasonable basis for the understanding of the effect of freezing. It is shown that L-amino acid esters are effective nucleophiles in alpha-chymotrypsin-catalyzed acyl transfer reactions when these are performed in frozen state.

Amino Acid Sequence↗
PubMed1991

S'-subsite mapping of endoproteinase Glu/Asp-C from Actinomyces sp.

The S'-subsite specificity of the endoproteinase Glu/Asp-C from Actinomyces sp. was studied by acyl transfer reactions using amino-acid- and peptide-derived nucleophilic amino components. The following results were obtained: 1. The enzyme prefers amino acid amides with hydrophobic side chains in P'i position. In addition, positively charged functions in this position favour S'-P' interactions significantly. 2. Stereospecific binding is a prerequisite for nucleophilic efficiency. 3. Dipeptide amides are more efficient amino components in comparison to free dipeptides whereas oligoglycines show a poor nucleophilic behaviour independent of chain length.

Actinomyces↗
PubMed1990

Characterization of the S'-subsite specificity of V8 proteinase via acyl transfer to added nucleophiles.

The S'-subsite specificity of endoproteinase Glu-C (V8 proteinase) was studied by acyl transfer reactions using Z-Glu-OMe as acyl donor and a series of amino acid- and peptide-derived nucleophiles. The partition constant, which characterizes specificity, was determined by a method based on the integrated rate equation. V8 proteinase prefers amino acid residues with hydrophobic side chains in the P'1 position. Di- and tripeptide amides are more efficient nucleophilic amino components than amino acid amides.

Binding Sites↗
PubMed1990

Anionic subsites of the acetylcholinesterase from Torpedo californica: affinity labelling with the cationic reagent N,N-dimethyl-2-phenyl-aziridinium.

Several peptides of acetylcholinesterase of Torpedo californica labelled with the alkylating reagent [3H]N,N-dimethyl-2-phenyl-aziridinium (DPA) were localized within the primary structure. One peptide had the sequence KPQELIDVE (positions 270-278); the incorporation of DPA into this peptide could be specifically suppressed by propidium, which suggests that it is part of the peripheral anionic site. The incorporation of DPA into two other peptides was insensitive to propidium but could be prevented by edrophonium; the sequence of one of the peptides assumed to be part of the anionic site in the catalytic centre was found to be DLFR (positions 217-220). Decamethonium efficiently blocked alkylation by DPA in all three investigated peptides.

Acetylcholinesterase↗
PubMed1990

The active site and partial sequence of cobra venom acetylcholinesterase.

About 30% of the primary structure of acetylcholinesterase (AchE) from the cobra Naja naja oxiana has been determined. The sequence around the serine residue labeled by diisopropylfluorophosphate (DFP) was found to be TVTLFGESAGAASVGM which is similar to the active sites of AChE from other tissues. The part of the primary structure determined shows 76% identity with AChE from Torpedo and 42% identity with the Drosophila enzyme. A surprisingly large identity (42% in the sequence determined) was found with lysophospholipase from rat.

Acetylcholinesterase↗
PubMed1987

Acetylcholinesterase as polyelectrolyte in reaction with cationic substrates.

It is shown that the salt effect in acetylcholinesterase-catalyzed hydrolysis of 2-(N-methylmorpholinium)-ethylacetate can be quantitatively described by the equation log(k2/KS) = log(k2/KS) degrees--psi log[M+Z] following from Manning's polyelectrolyte theory; the psi values for salts with univalent and bivalent cations at different pH values of the reaction medium were in accordance with the conclusions of the theory. Manning's polyelectrolyte theory seems to be a useful framework for studying salt effects in the reactions of charged substrates with enzymes as globular polyions.

Acetylcholinesterase↗
PubMed1985

Irreversible inhibition of pancreatic lipase by bis-p-nitrophenyl methylphosphonate.

The reaction of porcine pancreatic lipase with an organophosphorus compound bis-p-nitrophenyl methylphosphonate (BNMP) resulted in the complete and irreversible inhibition of lipase activity on tributyrin emulsion (25 degrees C, pH 7.5, 40 mM Na-veronal-HCl buffer) whereas the activity of the enzyme on p-nitrophenyl acetate solution remained unchanged. The BNMP-modified enzyme did not bind on hydrophobic interfaces (siliconized glass beads). Tyr 49 was presumed to be the modification site, and the conclusion has been made that this residue is implicated in the interface recognition site of pancreatic lipase.

Animals↗
PubMed1984

Salting effects in cinnamoyl-chymotrypsin deacylation.

The influence of inorganic salts on the trans-cinnamoyl-chymotrypsin deacylation kinetics at 25 degrees C and various pH values has been studied: KCl (pH 6.0-13.4), CsCl (pH 9.8 and 13.1), NaCl and Na2SO4 (pH 9.8). Each salt was found to accelerate the reaction with different effectiveness which depended upon pH. The data show that the acyl-enzyme occurs in two forms, which are seen kinetically by different salting effects in deacylation; the equilibrium between the forms is controlled by an ionizing group in the enzyme with pKa about 12.

Acylation↗
PubMed1982

Cobra venom acetylcholinesterase: nature of charge isoforms.

Charge isoforms of cobra (Naja naja oxiana) venom acetylcholinesterase, separated by isoelectric focusing, differ only by the number of free carboxyl groups of glutamic and/or aspartic acid side-chains in the enzyme molecule. The isoforms appear to be produced by a post-translational deamidation of accessible glutamin and/or asparagine residues. The isoforms have identical catalytic specificities towards characteristic acetylcholinesterase substrates.

Acetylcholinesterase↗
PubMed1979

Cobra venom acetylcholinesterase. Purification and molecular properties.

Acetylcholinesterase from cobra (Naja naja oxiana) venom has been purified by affinity chromatography to an homogeneous state, as ascertained by sodium dodecylsulfate/polyacrylamide gel electrophoresis and sedimentation analysis. The specific activity of the preparation was 5000 IU/mg with acetylcholine as substrate. Unlike acetylcholinesterases from insoluble cell structures, the native molecule of the cobra venom enzyme consists of a single polypeptide chain of molecular weight 67,000 +/- 2000. At high enzyme concentrations (greater than 0.2 mg/ml, greater than 1 microM) and ionic strength 0.1 M, it reversibly tends to form higher-molecular-weight 7.1-S aggregates. Despite the apparent structural simplicity of the venom acetylcholinesterase, the disc electrophoresis and isoelectric focusing experiments revealed that the enzyme exists in a number of forms with a common molecular weight but with different isoelectric points. Neuraminidase treatment did not reduce the number of the forms.

Acetylcholinesterase↗