Subject(s)
Escherichia coli Proteins , Escherichia coli/genetics , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Protease La , Regulon , Repressor Proteins/genetics , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Trans-Activators/genetics , Vibrio/genetics , ATP-Dependent Proteases , Escherichia coli/enzymology , Hydrolysis , Mutation , PlasmidsABSTRACT
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.
Subject(s)
Escherichia coli Proteins , Escherichia coli/enzymology , Heat-Shock Proteins/metabolism , Mutation , Protease La , Regulon , Repressor Proteins/genetics , Serine Endopeptidases/metabolism , Trans-Activators/genetics , Vibrio/genetics , ATP-Dependent Proteases , Bacterial Proteins/genetics , Heat-Shock Proteins/genetics , Luciferases/genetics , Luminescent Measurements , Operon , Serine Endopeptidases/geneticsABSTRACT
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.
Subject(s)
Adenosine Triphosphate/chemistry , Escherichia coli Proteins , Escherichia coli/enzymology , Heat-Shock Proteins/chemistry , Oligopeptides/chemistry , Protease La , Serine Endopeptidases/chemistry , ATP-Dependent Proteases , Heat-Shock Proteins/genetics , Hydrolysis , Kinetics , Mutation , Serine Endopeptidases/genetics , Substrate SpecificityABSTRACT
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.
Subject(s)
Endopeptidases/metabolism , Escherichia coli Proteins , Escherichia coli/enzymology , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/metabolism , Protease La , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , ATP-Dependent Proteases , Amino Acid Sequence , Catalysis , Escherichia coli/chemistry , Heat-Shock Proteins/isolation & purification , Hydrolysis , Melitten/metabolism , Molecular Sequence Data , Protein Structure, Tertiary , Serine Endopeptidases/isolation & purificationABSTRACT
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.
Subject(s)
Adenosine Triphosphate/metabolism , Escherichia coli Proteins , Escherichia coli/enzymology , Heat-Shock Proteins/biosynthesis , Heat-Shock Proteins/genetics , Protease La , Serine Endopeptidases/biosynthesis , Serine Endopeptidases/genetics , ATP-Dependent Proteases , Amino Acids/analysis , Cloning, Molecular , DNA Primers , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Gene Expression , Heat-Shock Proteins/isolation & purification , Mutation , Polymerase Chain Reaction , Restriction Mapping , Sequence Deletion , Serine Endopeptidases/isolation & purificationABSTRACT
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.
Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphate/chemistry , Escherichia coli Proteins , Escherichia coli/enzymology , Heat-Shock Proteins/chemistry , Protease La , Serine Endopeptidases/chemistry , ATP-Dependent Proteases , Adenosine Triphosphatases/genetics , Allosteric Regulation , Amino Acid Substitution , Amino Acids/analysis , Catalysis , DNA Primers , Heat-Shock Proteins/genetics , Hydrolysis , Mutagenesis, Site-Directed , Mutation/genetics , Polymerase Chain Reaction , Serine Endopeptidases/genetics , Signal Transduction/genetics , Substrate SpecificityABSTRACT
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.
Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Escherichia coli Proteins , Escherichia coli/enzymology , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/metabolism , Protease La , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , ATP-Dependent Proteases , Amino Acid Sequence , Binding Sites , Brain/enzymology , Humans , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
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.
Subject(s)
Adenosine Triphosphatases/metabolism , Escherichia coli Proteins , Escherichia coli/enzymology , Heat-Shock Proteins/metabolism , Protease La , Serine Endopeptidases/metabolism , ATP-Dependent Proteases , Amino Acid Sequence , Binding Sites , Catalysis , Chromatography, High Pressure Liquid , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Heat-Shock Proteins/genetics , Heat-Shock Proteins/isolation & purification , Molecular Sequence Data , Mutation , Serine Endopeptidases/genetics , Serine Endopeptidases/isolation & purificationSubject(s)
Aminopeptidases/antagonists & inhibitors , Aminopeptidases/metabolism , Aminopeptidases/isolation & purification , Animals , Brain/drug effects , Brain/enzymology , Cattle , Cell Membrane/enzymology , Chemical Phenomena , Chemistry, Physical , Enkephalins/metabolism , Hydrolysis , Kinetics , Male , Neuropeptides/metabolism , Rats , Solubility , Substrate SpecificityABSTRACT
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.
Subject(s)
Aminopeptidases/isolation & purification , Brain/enzymology , Aminopeptidases/analysis , Animals , Catalysis , Cattle , Humans , Hydrolysis , Kinetics , Membranes/enzymology , Mice , Molecular Weight , Rats , Solubility , Species SpecificityABSTRACT
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.
Subject(s)
Aminopeptidases/analysis , Brain/enzymology , Amino Acids/analysis , Animals , Cattle , Hydrolysis , Kinetics , Lens, Crystalline/enzymology , Leucyl Aminopeptidase/analysis , Membranes/enzymology , Naphthalenes/analysis , Solubility , Substrate SpecificityABSTRACT
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.
Subject(s)
Peptide Hydrolases/metabolism , Proteins/metabolism , Binding Sites , Chemical Phenomena , Chemistry, Physical , Hydrolysis , Models, Molecular , Peptides/metabolism , Protease Inhibitors/metabolism , Substrate SpecificityABSTRACT
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.
Subject(s)
Amides/metabolism , Leucyl Aminopeptidase/pharmacology , Oxygen/metabolism , Pepsin A/pharmacology , Animals , Cattle , Chemical Phenomena , Chemistry , Kinetics , Lens, Crystalline/enzymology , Substrate SpecificityABSTRACT
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.
Subject(s)
Chymotrypsin/metabolism , Leucyl Aminopeptidase/metabolism , Pepsin A/metabolism , Thermolysin/metabolism , Animals , Cattle , Isotope Labeling/methods , Kinetics , Lens, Crystalline/enzymology , Oxygen Isotopes , SwineSubject(s)
Pepsin A/metabolism , Animals , Aspartic Acid/metabolism , Binding Sites , Catalysis , Dipeptides/metabolism , Oxygen , SwineABSTRACT
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.
Subject(s)
Chymotrypsin , Catalysis , Esters , Glycine , Hydrogen-Ion Concentration , Kinetics , Peptides , Substrate Specificity , ThermodynamicsABSTRACT
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.