[A test system based on the lux-regulon from Vibrio fischeri for studying the functional activity of mutant forms of Escherichia coli lon-proteinase]. / Test-sistema na osnove lux-regulona Vibrio fischeri pri issledovanii funktsional'noi aktivnosti mutantnykh form lon-proteinazy Escherichia coli.

The possibility of application of the bioluminescence method (Lux-test) for studying in vivo functional activity of Escherichia coli protease Lon and its mutants was demonstrated. This assay is based on the capacity of protease Lon and its mutant forms for specific degradation of the LuxR protein, a positive transcriptional activator of the right operon luxICDABE from the marine bacterium Vibrio fischeri, and thus to affect the level of AB luciferase in the cells. A correlation between in vitro activity of the protease Lon mutants and the intensity of bioluminescence measured by the Lux-test was revealed.

[Suc-Phe-Leu-Phe-SBzl--a new substrate for functional study of Escherichia coli ATP-dependent Lon-proteinase and its modified forms]. / Suc-Phe-Leu-Phe-SBzl--novyi substrat dlia issledovaniia funktsionirovaniia ATP-zavisimoi Lon-proteinazy Escherichia coli i ee modifitsirovannykh form.

A new efficient substrate, Suc-Phe-Leu-Phe-SBzl, was proposed for studying the function of the Escherichia coli ATP-dependent Lon protease and its modified forms. The kinetic parameters of hydrolysis of the substrate were determined. The esterase activity of protease Lon was found to be nucleotide-regulated.

The isolated proteolytic domain of Escherichia coli ATP-dependent protease Lon exhibits the peptidase activity.

Selective protein degradation is an energy-dependent process performed by high-molecular-weight proteases. The activity of proteolytic components of these enzymes is coupled to the ATPase activity of their regulatory subunits or domains. Here, we obtained the proteolytic domain of Escherichia coli protease Lon by cloning the corresponding fragment of the lon gene in pGEX-KG, expression of the hybrid protein, and isolation of the proteolytic domain after hydrolysis of the hybrid protein with thrombin. The isolated proteolytic domain exhibited almost no activity toward protein substrates (casein) but hydrolyzed peptide substrates (melittin), thereby confirming the importance of the ATPase component for protein hydrolysis. Protease Lon and its proteolytic domain differed in the efficiency and specificity of melittin hydrolysis.

[Synthesis and characterisation of ATP-dependent forms of Lon-proteinase with modified N-terminal domain from Escherichia coli]. / Polucheniie i issledovanie modifitsirovannykh v oblasti N-kontsevogo domena form ATP-zavisimoi Lon-proteinazy iz Escherichia coli.

The functional domain boundaries of the ATP-dependent Lon proteases were identified by comparative analysis of the amino acid sequences of the enzymes from evolutionarily distant organisms. Modified forms of the Escherichia coli Lon protease with the elongated or substituted N-terminal domain and a truncated enzyme lacking the N-terminal domain were obtained through genetic engineering methods. Analysis of the enzymatic properties of the resulting modified forms of Lon protease revealed the importance of the N-terminal domain in its function.

[In vitro coupling of ATP hydrolysis to proteolysis of ATP site mutant forms of Lon-proteinase from E.coli]. / Sopriazhenie gidroliza ATP i proteoliza pri funktsionirovanii in vitro mutagennykh po ATP-asnomy tsentru form Lon-proteinazy E.coli.

In order to identify amino acid residues involved in ATP hydrolysis by Escherichia coli protease Lon or participating in the signal transduction from the ATPase domain to the proteolytic one, potentially important residues of the ATPase domain were substituted using site-directed mutagenesis, and the properties of the resulting mutant enzymes were studied. It was found that residues K362, T363 (Walker's motif A), and D423 (motif B) are involved in the catalysis of ATP hydrolysis. K362 and T363 also participate in the system of domain-domain coupling, whereas D423 does not play a significant role in this process. Residue D387 is important for ATPase activity; however, it is not a catalytically active residue, as was earlier postulated in the literature. Residue Y493 is also involved in the signal transduction from the ATPase domain to the proteolytic one.

Mutations in the proteolytic domain of Escherichia coli protease Lon impair the ATPase activity of the enzyme.

Conserved residues of the proteolytic domain of Escherichia coli protease Lon, putative members of the classic catalytic triad (H665, H667, D676, and D743) were identified by comparison of amino acid sequences of Lon proteases. Mutant enzymes containing substitutions D676N, D743N, H665Y, and H667Y were obtained by site-directed mutagenesis. The mutant D743N retained the adenosine triphosphate (ATP)-dependent proteolytic activity, thereby indicating that D743 does not belong to the catalytic site. Simultaneously, the mutants D676N, H665Y, and H667Y lost the capacity for hydrolysis of protein substrates. The ATPase activity of these three mutants was decreased by more than an order of magnitude, which suggests a close spatial location of the ATPase and proteolytic active sites and their tight interaction in the process of protein degradation.

[ATP-dependent proteinase La from Escherichia coli]. / ATP-zavisimaia proteinaza La iz Escherichia coli.

Homogeneous preparations of the ATP-dependent La proteinase from E. coli and two its mutant forms, containing an alanine residue instead of Ser679 or Ser368, were isolated. Ser679 was shown to be catalytically active rather than Ser368 as suggested in the literature. To choose between the alternative structures of the gene lon La proteinase fragments within the controversial regions were analysed and the gene structure established at the Laboratory of Proteolytic Enzymes (Institute of Bioorganic Chemistry) was confirmed. Inactivity of La proteinase in some in vitro systems suggests its functioning in vivo to be not autonomous, requiring additional factors.

[Comparative characteristics of soluble and membrane brain aminopeptidases. I. Isolation, physico-chemical properties, catalytic activity]. / Sravnitel'naia kharakteristika rastvorimoi i membrannoi aminopeptidamozga. I. Vydelenie, fiziko-khimicheskie svoistva, kataliticheskaia aktivnost'.

The methods were worked out for isolating of the bovine brain soluble and membrane-bound aminopeptidases in highly purified state. One of the stages involved a biospecific-type chromatography on aminohexyl-Sepharose. Both enzymes were found to have equal molecular masses (ca. 100-107 kD) and isoelectric points (pI 4,6). None of the enzymes possessed a subunit structure. Both aminopeptidases were inactivated by omicron-phenanthroline and by an SH-reagent, p-hydroxymercuribenzoate. The catalytic constants for the hydrolysis of a specific substrate, L-leucine p-nitroanilide, were identical for the two enzymes. So far no differences in the physico-chemical or enzymatic properties of the soluble and membrane-bound enzymes were disclosed.

[Comparative characteristics of soluble and membrane brain aminopeptidases. II. Substrate specificity]. / Sravnitel'naia kharakteristika rastvorimoi i membrannoi aminopeptidamozga. II. Substratnaia spetsifichnost'.

Comparative studies on substrate specificity of the soluble and membrane-bound aminopeptidases from bovine brain were carried out. A series of p-nitroanilides and beta-naphthylamides of amino acids, di- and tripeptides with the aminoterminal phenylalanine residue, as well as a biologically active pentapeptide--[Leu5]enkephalin--were used as substrates. The soluble and membrane-bound aminopeptidases manifested identical specificity towards the employed substrates. The aminopeptidases were equally effective towards the p-nitroanilides of amino acids and peptides, whereas beta-naphthylamides were more susceptible to hydrolysis by both aminopeptidases than p-nitroanilides and peptides. Taking into account physico-chemical characteristics of these enzymes, it was concluded that the soluble and membrane-bound aminopeptidases are quite similar or perhaps identical. Their role in the regulation of nervous system functioning was discussed. A comparison of specificities for brain aminopeptidases and leucine aminopeptidase from bovine lens led to the conclusion that they belong to different groups. This feature allows planning the synthesis of selective inhibitors.

[Mechanisms of proteolysis]. / Mekhanizmy proteoliza.

Mechanisms of the amide bond hydrolysis catalyzed by proteolytic enzymes have been analyzed using mainly our own experimental data. The rationale for the efficiency and specificity of proteinases has been proposed that relates these features to the state of the substrate in the productive enzyme-substrate complex.

[Mechanism of oxygen exchange in the amide group of substrates during their hydrolysis catalyzed by leucine aminopeptidase and pepsin]. / Mekhanizm obmena kisloroda v amidnoi gruppe substratov v khode ikh gidroliza, kataliziruemogo leitsinaminopeptidazoi i pepsinom.

Oxygen exchange in the amide group of leucine amide catalyzed by leucine aminopeptidase, and in leucyltyrosine amide catalyzed by porcine pepsin, was found to proceed mainly by the transfer of the leucyl residue onto the ammonia or tyrosine amide which are formed during the hydrolysis. Thus oxygen exchange in the non-hydrolyzed substrate can not be a proof of the tetrahedral intermediate formation in the course of the catalysis by proteolytic enzymes.

Studies on the mechanisms of action of proteolytic enzymes using heavy oxygen exchange.

Transpeptidation reactions catalyzed by chymotrypsin, pepsin, leucine aminopeptidase and thermolysin have been studied in heavy oxygen water (H2 18O). The 18O incorporation into the peptide bond of transpeptidation products and into the non-hydrolyzed substrate has been measured. The rates of 18O exchange in the carboxylic groups of N-acetylphenylalanine and leucine, catalyzed by pepsin and leucine aminopeptidase, respectively, have also been determined. These rates have been compared with that of the exchange in the presence of amino compounds which reversibly form amide bonds with the above carboxyl-containing substances. The data obtained show that, in contrast to chymotrypsin, other enzymes studied do not form 'acyl-enzymes' but function by the mechanism of general-base catalysis. In other words, their catalytically active groups promote the abstraction of a proton from the water molecule, which attacks the susceptible bond of the substrate. The structure of intermediate compounds in this type of catalysis and the mechanism of the transpeptidation reaction are discussed.

[Kinetics of alpha-chymotrypsin catalyzed hydrolysis in equilibrium. III. Rate constants for individual stages and thermodynamic parameters at different pH's]. / Kinetika kataliziruemogo alpha-khimotripsinom gidroliza v sostoianii ravnovesiia. III. Konstanty skorosti individual 'nykh stadii i termodinamicheskie parametry pri razlichnykh pH.

The rate constants of the individual steps of the reversible chymotryptic hydrolysis of N-acetyl-L-phenylalanylglycinamide and methyl N-acetyl-L-phenylalaninate have been calculated on the basis of data on the velocity of the exchange between these substrates and products of their hydrolysis at equilibrium and also on the basis of steady-state kinetics of their cleavage. This was done for peptide substrate at pH 5.5, 7.3 and 8.2 and for ester substrate at pH 5.5. The free energy of the formation of intermediate complexes and free energy of activation were calculated. Thus a complete kinetic and thermodynamic description of chymotryptic catalysis of various pH is performed.

[Kinetics of alpha-chymotrypsin catalyzed hydrolysis in equilibrium. II. Comparison of ester and amide substrates]. / Kinetika kataliziruemogo alpha-khimotripsinom gidroliza v sostoianii ravnovesiia. II. Sravnenie éfirnykh i amidnykh substratov.

Kinetic of the alpha-chymotrypsin catalyzed reversible hydrolytic reaction of methyl N-acetyl-L-phenylalaninate and N-acetyl-L-phenylalanylglycinamide at pH 5.5 and equilibrium conditions has been studied. The rates of the labeled reaction products incorporated into the substrate a different methanol concentrations shows that the reaction proceeds by a compulsory mechanism with the formation of N-acetyl-L-phenylalanine-alpha-chymotrypsin complex. For the amide substrate the data obtained are also in agreement with the compulsory mechanism of its hydrolysis. Equilibrium kinetics of ester and amide substrates hydrolysis has been compared.