Interaction of tyrosine phenol-lyase with phosphoroorganic analogues of substrate amino acids.

The phosphinic analogues of tyrosine and pyruvate were first demonstrated to be substrates in the reactions of elimination and synthesis catalyzed by tyrosine phenol-lyase. Kinetic parameters of the enzymatic process were determined, and the first enzymic synthesis of an aminophosphinic acid was carried out. Replacement of the planar HOOC-group by the tetrahedral (HO)(O)PH-group in the substrate slightly affected its affinity for the enzyme but substantially diminished the conversion rate. For phosphonic analogues, containing (HO)2(O)P group, the affinity to the enzyme was decreased considerably while the conversion was completely prevented. Thus, the structural parameters of the acid group are important not only for the affinity for the enzyme, but also for the formation of the catalytically competent conformation of the active site.

[Initial kinetics of milk protein hydrolysis by chymotrypsin]. / Issledovanie nachal'noi kinetiki gidroliza belkov moloka khimotripsinom.

The kinetics of the initial stages of hydrolysis of alpha- and beta-caseins, alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin was studied quantitatively by electrophoresis. The hydrolysis rates of caseins (demasked polypeptide chains) exceeded the rates of the degradation of milk whey proteins limited by the protein globule demasking by more than two orders of magnitude. Even relatively accessible for hydrolysis globular proteins (beta-lactoglobulin and BSA) were hydrolyzed by the one-by-one mechanism. The maximum value of the ratio between the rates of the peptide chain demasking and the protein globule hydrolysis was 0.0035.

2-Methyl-L-tryptophan is a substrate of tryptophanase.

Tryptophanase was generally considered to be inactive towards tryptophan derivatives substituted at 2-position of the indole ring. We have shown that cells containing tryptophanase catalyze the formation of 2-methyl-L-tryptophan from 2-methylindole and L-serine, and from 2-methylindole, pyruvate and ammonium ion. The kinetics of pyruvate formation from 2-methyl-L-tryptophan and its alpha-deuterated analogue catalyzed by homogeneous tryptophanase were examined. The primary deuterium isotope effect (kH/kD = 4.0) as well as the absorption spectrum of tryptophanase complex with 2-methyl-L-tryptophan indicate that the rate of enzymatic reaction of 2-methyl-L-tryptophan is in a considerable degree determined by the stage of removal of alpha-proton.

Tryptophanase from Escherichia coli: catalytic and spectral properties in water-organic solvents.

In water-methanol and water-dimethylformamide (DMF) (1:1 v/v) solutions tryptophanase from E.coli retains its abilities to form a quinonoid complex with quasisubstrates and to catalyze the decomposition of S-o-nitrophenyl-L-cysteine (SOPC). Both the KM and Vmax values decrease in water-organic media. The affinities of tryptophanase for L-alanine, L-tryptophan, oxindolyl-L-alanine and indole in aqueous methanol are decreased, the effect being stronger for the more hydrophobic substances. In a water solution tryptophanase catalizes the reaction of SOPC with indole to form L-tryptophan while in water-organic solvents only decomposition of SOPC is observed.

[Factors, determining the effectiveness of tryptophanase interaction with amino acids]. / Faktory, opredeliaiushchie éffektivnost' vzaimodeistviia triptofanazys aminokislotami.

Inhibition of tryptophanase-catalyzed decomposition of S-(o-nitrophenyl)-L-cysteine by a variety of amino acids has been investigated. For amino acids similar to the natural substrate and for those having minimal steric requirements for the side chain, the linear correlation exists between-RTlnKi and side chain hydrophobicity. L-ornithine and L-arginine are anomalously potent inhibitors taking into account low hydrophobicity of their side chains. This can be explained by an interaction between a positively charged group of the side chain of L-arginine or L-ornithine and a nucleophilic group of the active site. The comparison of affinity of tryptophanase for L-phenylalanine and L-homophenylalanine indicates that there is a special locus in the active site where aromatic groups are bound and oriented approximately parallel to the cofactor plane experiencing no steric hindrance. For a large number of amino acids the rates of the enzymic alpha-proton exchange in 2H2O are comparable with the rate of the reaction with L-tryptophan. Very low rate of alpha-proton exchange observed with L-alanine is an exception.

[Catalytic properties of E. coli tryptophanase in a 50% aqueous solution of methanol and dimethylformamide]. / Kataliticheskie svoistva triptofanazy iz E. coli v 50% vodnykh rastvorakh metanola i dimetilformamida.

Tryptophanase from E. coli retains its ability to form quinonoid intermediate with L-alanine in water--methanol and water--dimethylformamide (1:1 v/v) solutions. Under these conditions the enzyme catalyzes decomposition of S-o-nitrophenyl-L-cysteine (SOPC) to o-nitrophenylthiol, pyruvate and ammonium ion. The enzyme's affinity for this substrate increases on going from water to water-organic solvents whereas the reaction rate decreases. In 50% methanol tryptophanase catalyzes the formation of L-tryptophan from indole and SOPC; in the mixture of 2H2O and C2H3O2H (1:1) the enzymatic isotope exchange of alpha-proton of L-phenylalanine with complete retention of configuration was observed.

Enantiomeric and diastereoisomeric analysis of nonproteinogenic methyl and hydroxy amino acids.

The enantiomeric and diastereoisomeric analysis of nonproteinogenic methyl and hydroxy amino acids, obtained by asymmetric synthesis with the aid of a chiral regenerable reagent, has been carried out by liquid chromatography.

[The composition of amino acid-peptide mixtures obtained during hydrolysis of proteins]. / Issledovanie sostava aminokislotnykh-peptidnykh smesei, poluchaemykh pri gidrolize belkov.

The composition of the amino acid and peptide fraction of the protein hydrolyzate and its dependence upon the spray-drying process at elevated temperatures were investigated by GLC and GLC-MS. Spray-drying leads to decrease in the Glu and Met contents, to transformation of Cys into cystine and to formation of lactames (pyroglutamic acid and pyrrolidone) and diketopiperazines. Some dipeptides, acetylproline, paraffines, fatty acid pyridinecarboxylic and lactic acids, ethylene glycol were identified in the fraction. The incidental occurrence of these compounds should be taken into account when investigating the biological activity of protein hydrolyzates.

[Effect of enteral feeding on protein metabolism in patients with peritonitis]. / Vliianie énteral'nogo zondovogo pitaniia na belkovyi obmen u bol'nykh peritonitom.

Thirty patients with peritonitis of different etiology were examined. Deep alterations of protein metabolism were revealed resulting from dyscoordination of amino acid metabolism. Efficiency of unbalanced amino acid mixtures was shown and on the contrary, the absence of a therapeutic effect when using balanced mixtures.

Tyrosine phenol-lyase from Citrobacter intermedius. Factors controlling substrate specificity.

L-Amino acids are competitive inhibitors of tyrosine phenol-lyase from Citrobacter intermedius. For non-branched amino acids the correlation exists between -RTlnKi and side-chain hydrophobicity. Aspartic and glutamic acids are anomalously potent inhibitors taking into account low hydrophobicity of their side chains. This suggests the presence of an electrophilic group in the active site which interacts with the terminal carboxylic group of aspartic or glutamic acids. Tyramine, beta-phenylethylamine and tryptamine do not display detectable inhibition. The esters and amides of aromatic L-amino acids, D-phenylalanine and D-tryptophan are competitive inhibitors. The enzymatic isotope exchange of the alpha-proton in 2H2O was observed only in the case of L-amino acids. For L-phenylalanine and L-tryptophan it was shown to proceed with complete retention of configuration. The substrate specificity of tyrosine phenol-lyase is controlled during the stage of phenol elimination. The OH group in the para position of the ring is necessary for this stage to proceed. The same stage is also sensitive to the steric parameters of the substituent in the ring which ensures the second factor of control. When all the requirements of substrate specificity are fulfilled (L-tyrosine, 3-fluoro-L-tyrosine), the 'key' phenol-elimination step is not the rate-limiting one, the reaction velocity being determined by the preceding alpha-proton abstraction.

Acyl group transfer by proteases forming acyl-enzyme intermediate: kinetic model analysis.

A kinetic model of peptide synthesis via transfer of the acyl moiety from activated derivatives of amino acids (S) to nucleophiles (N) catalyzed by proteases forming an acyl-enzyme intermediate has been analyzed. The kinetic model takes into account the subsequent enzymatic hydrolysis of synthesized peptide (P), and so the kinetic curve for this compound shows a maximum (denoted as p(max)). Particular stress is placed on analyzing the effects of initial concentrations and of kinetic constants on the value of p(max).The analysis has demonstrated that at a given ratio of initial S and N concentrations, p(max) is affected only by (i) the ratio of the second-order rate constants for enzymatic hydrolysis of S and P(alpha) and (ii) the ratio of rate constants for an attack of the acyl-enzyme intermediate by the nucleophile and water (beta). These conclusions apply regardless of the existence of linear inhibition by the components of the reaction mixture. Thus, the kinetically controlled maximum yield of peptide (p(max)) can be calculated a priori from values of alpha and beta that can be estimated experimentally or from reference data. Simple analytical expressions were obtained, allowing a fairly accurate prediction of p(max) for a broad spectrum of S and N initial concentrations.

Kinetic description of proteolysis. Part 3. Total kinetics of peptide bonds hydrolysis in peptide mixtures.

A theoretical analysis of the general kinetics of peptide bond hydrolysis in peptide mixtures has been carried out. It is shown that the equation for the hydrolysis rate must include ensemble-averaged values which change as functions during hydrolysis. Thus the problem of describing the hydrolysis is reduced to finding these functions. As an example of theoretical prediction for the behavior of the averaged functions the procedure of calculating the averaged constant of chymotrypsin substrate specificity as a function of the degree of hydrolysis is presented. The analysis performed enables to find factors responsible for the decrease of hydrolysis rate, in particular, the role of the substrate specificity range and the S/E ratio. The design of specific experiments making it possible to measure the averaged values in the course of hydrolysis has been considered.

Kinetic description of proteolysis. Part 4. Hydrolysis kinetics of partial protein hydrolysates.

This report presents experimental data on kinetics of casein hydrolysate hydrolysis. The experiments were interpreted in the framework of a theory reported, which is guided by an experimentally measurable value--a total molar concentration of all peptides and amino acids (total amino nitrogen). The total amino nitrogen N was determined by photometry of N-trinitrophenyl (N-TNP) derivatives obtained by means of trinitrobenzenesulfonic acid (TNBS). Unlike the procedures reported previously, we stopped the proteolysis reaction by boiling, and performed the trinitrophenylation in a 20% V. aqueous solution of MeCN.

Plasteins. Their preparation, properties, and use in nutrition.

The theoretical and practical aspects of the plastein reaction, which consist in the formation of a gel following the addition of an endopeptidase to a concentrated solution of a partial protein hydrolysate, are examined and the properties and possibilities of using plasteins in nutrition are discussed. It is shown that valuable protein food products can be obtained with the aid of the plastein reaction from proteins with an unbalanced amino acid composition and from chemically synthesized amino acids. Other applications of plasteins in nutrition are discussed and the studies carried out hitherto on the mechanism and driving forces of plastein formation are considered.

Kinetic description of proteolysis. Part 1. Peptic hydrolysis of proteins isolated from chicken heart: optimization in terms of time and substrate concentration.

Simple equation for the total rate of proteolysis is proposed. The equation, based on the renal mechanism of proteolysis includes the substrate concentration and some functions of the degree of hydrolysis. These functions are experimentally determined in the case of peptic hydrolysis of chicken heart proteins. The integration of differential equation made it possible to describe the kinetics of the proteolysis in terms of substrate concentration, degree of proteolysis and reaction time. The kinetics of proteolysis does not obey the Michaelis-Menten law. The proposed way for mathematical modelling permits the optimization by productivity.

Kinetic description of proteolysis. Part 2. Substrate regulation of peptide bond demasking and hydrolysis. Liquid chromatography of hydrolyzates.

Lower peptides and amino acids in hydrolyzates of casein obtained with protosubtilin were determined using high pressure liquid chromatography (HPLC). The dependences of product concentrations in hydrolyzates produced at different substrate concentrations on the degree of hydrolysis were obtained. With an increase of the substrate concentration the real type of proteolysis changes from "zipper" to "one-by-one". This result supports the assumption that the substrate proteolysis regulation is realized through the change in the ratio of the rates of peptide bond demasking to those of peptide bond hydrolysis.

[Isolation and properties of tyrosine phenol-lyase from Citrobacter intermedius]. / Vydelenie i nekotorye svoistva tirozin--fenol-liazy iz Citrobacter intermedius.

A method for preparation of homogeneous tyrosine phenol lyase (EC 4.199.2) from Citrobacter intermedius has been developed. The cells were cultivated in the media with a view to obtain a cell culture with a high activity of tyrosine phenol lyase. The isoelectric point for the enzyme lies at pH 4.9. Tyrosine phenol lyase is strictly stereospecific: it catalyzes the formation of pyruvate only from L-tyrosine, but not from D-tyrosine. Kinetic studies showed that K+ and NH4+ cations are non-competitive activators of the enzyme (Ka = 3.57 X 10(-3) and 1.34 X 10(-4) M, respectively).

[Amino acid analysis of culture media and autolysates by capillary gas chromatography]. / Aminokislotnyi analiz kul'tural'nykh sred i avtolizatov metodom kapilliarnoi gazovoi khromatografii.

N-heptofluorbutyryl-O-propyl ethers from 20 natural amino acids were analysed on a capillary column Am-Ac by gas chromatography. A high reproducibility of the quantitative estimation of the compounds studied was achieved not only for the standard mixtures but when analysing the amino acid composition of the autolysates of the baker's yeast and cultural media.

[Coordination immobilization of fungal proteinases]. / Koordinatsionnaia immobilizatsiia gribnykh proteinaz.

The possibility of coordination immobilization of fungal enzymes from Aspergillus niger MB and Aspergillus flavipes on the styrene copolymer with maleic acid has been explored. Optimal conditions for preparing insoluble chromium-containing enzyme polymeric complexes have been studied. The synergistic effect of salts of different metals on stability of chromium-containing immobilized preparations has been demonstrated. Properties of the resultant immobilized preparations have been examined.

[Stabilization of proteases from Bacillus subtilis in solution]. / Stabilizatsiia proteaz Bacillus Subtilis v rastvore.

Experiments were carried out to study the effect of the styrene and maleinic acid copolymer on the activity of proteases produced by Bacillus subtilis--protosubtilin G15x. Unlike the hydrophilic ethylene--maleinic acid copolymer, styrenated maleinic acid produced a stabilizing effect on proteases via hydrophobic interaction. It is suggested that at least two forms of the enzyme differing in their activity and stability are involved in the process of inactivation. On the basis of the data obtained a scheme of protease inactivation is proposed.