Proteolysis Coupled to ATP Hydrolysis: Regulation of the Activity of Proteolytic Sites of Lon Protease from Escherichia coli

Regulation of activity of the proteolytic sites of Lon protease was studied. It was found that ATP–Mg has the properties of a noncompetitive activator of peptidase sites. The processive mechanism of the hydrolysis of protein substrates by Lon protease was experimentally confirmed under the conditions of ATP hydrolysis. It was shown that the oligomeric state of the enzyme is the necessary prerequisite for the processive proteolysis by native Lon protease. The study of the properties of the mixed mutant Lon-K362Q/S679A confirmed the existence of intra- and intersubunit pathways of signal transduction from the ATPase to proteolytic sites. The mutual influence of substrates of Lon protease was studied, and the existence of cooperative interactions between the peptidase sites in the oligomeric enzyme was suggested.     

Forms of LonB protease from Archaeoglobus fulgidus devoid of the transmembrane domain: the contribution of the quaternary structure to the regulation of enzyme proteolytic activity

Deletion of the transmembrane domain (TM-domain) of Archaeoglobus flggidus LonB protease (AfLon) was shown to result in uncontrollable activation of the enzyme proteolytic site and in vivo autolysis yielding a stable and functionally inactive fragment consisting of both alpha-helical and proteolytic domains (alphaP). The deltaTM-AfLonTM-S590A enzyme form, obtained by site-directed mutagenesis of the catalytic Ser residue, is capable of recombination with the alphaP fragment. The mixed oligomers were shown to be proteolytically active, which indicates a crucial role of subunit interactions in the activation of the AfLon proteolytic site. The thermophilic nature of AfLon protease was found to be due to the special features of the enzyme activity regulation, the structure of ATPase domain, and the quaternary structure.     

Forms of LonB protease from Archaeoglobus fulgidus devoid of the transmembrane domain: The contribution of the quaternary structure to the regulation of enzyme proteolytic activity

Deletion of the transmembrane domain (TM-domain) of Archaeoglobus fulgidus LonB protease (Archaeoglobus fulgidus (AfLon)) was shown to result in uncontrollable activation of the enzyme proteolytic site and in vivo autolysis yielding a stable and functionally inactive fragment consisting of both α-helical and proteolytic domains (αP). The ΔTM-AfLon-S509A enzyme form, obtained by site-directed mutagenesis of the catalytic Ser residue, is capable of recombination with the αP fragment. The mixed oligomers were shown to be proteolytically active, which indicates a crucial role of subunit interactions in the activation of the AfLon proteolytic site. The thermophilic nature of AfLon protease was found to be due to the special features of the enzyme activity regulation, the structure of ATPase domain, and the quaternary structure.     

The catalytic domain of Escherichia coli Lon protease has a unique fold and a Ser-Lys dyad in the active site

ATP-dependent Lon protease degrades specific short-lived regulatory proteins as well as defective and abnormal proteins in the cell. The crystal structure of the proteolytic domain (P domain) of the Escherichia coli Lon has been solved by single-wavelength anomalous dispersion and refined at 1.75-A resolution. The P domain was obtained by chymotrypsin digestion of the full-length, proteolytically inactive Lon mutant (S679A) or by expression of a recombinant construct encoding only this domain. The P domain has a unique fold and assembles into hexameric rings that likely mimic the oligomerization state of the holoenzyme. The hexamer is dome-shaped, with the six N termini oriented toward the narrower ring surface, which is thus identified as the interface with the ATPase domain in full-length Lon. The catalytic sites lie in a shallow concavity on the wider distal surface of the hexameric ring and are connected to the proximal surface by a narrow axial channel with a diameter of approximately 18 A. Within the active site, the proximity of Lys(722) to the side chain of the mutated Ala(679) and the absence of other potential catalytic side chains establish that Lon employs a Ser(679)-Lys(722) dyad for catalysis. Alignment of the P domain catalytic pocket with those of several Ser-Lys dyad peptide hydrolases provides a model of substrate binding, suggesting that polypeptides are oriented in the Lon active site to allow nucleophilic attack by the serine hydroxyl on the si-face of the peptide bond.     

The ATP-dependent proteases and proteolytic complexes involved into intracellular protein degradation

The review characterizes the main enzymatic systems of selective proteolysis responsible for maintenance of intracellular proteome in prokaryotes, eukaryotes and archaea. The features of proteolytic components of the ATP-dependent proteases as well as similarity and diversity of their regulatory components belonging to AAA⁺ ATPases are discussed.     

Coupling of Proteolysis to ATP Hydrolysis upon Escherichia coliLon Protease Functioning: II. Hydrolysis of ATP and Activity of the Enzyme Peptide Hydrolase Sites

The absence of direct correlation between the efficiency of functioning of ATPase and peptide hydrolase sites of Lon protease was revealed. It was shown that Lon protease is an allosteric enzyme, in which the catalytic activity of peptide hydrolase sites is provided by the binding of nucleotides, their magnesium complexes, and free magnesium ions in the enzyme ATPase sites. It was revealed that the ADP–Mg complex, an inhibitor of the native enzyme, is an activator of the Lon-K362Q (the Lon protease mutant in the ATPase site). Variants of functional contacts between different sites of the enzyme are considered. It was established that two ways of signal transduction from the ATPase sites to peptide hydrolase ones exist in the Lon protease oligomer--intra- and intersubunit ways. The enzyme ATPase sites are suggested to be located in the areas of the complementary surfaces of subunits. It is hypothesized that upon degradation of protein substrates by the E. coliLon protease in vivoATP hydrolysis acts as a factor of limitation of the enzyme degrading activity.     

Intracellular proteolysis: Signals of selective protein degradation

Selective proteolysis is one of the mechanisms for the maintenance of cell homeostasis via rapid degradation of defective polypeptides and certain short-lived regulatory proteins. In prokaryotic cells, high-molecular-mass oligomeric ATP-dependent proteases are responsible for selective protein degradation. In eukaryotes, most polypeptides are attacked by the multicatalytic 26S proteasome, and the degradation of the majority of substrates involves their preliminary modification with the protein ubiquitin. The proteins undergoing the selective proteolysis often contain specific degradation signals necessary for their recognition by the corresponding proteases. This article is dedicated to the 25th Anniversary of the journal Bioorganicheskaya Khimiya     

Influence of the (1–106) fragment of <Emphasis Type="Italic">Escherichia coli</Emphasis> Lon protease on the enzyme function and DNA binding

Bifunctional Escherichia coli LonA protease (Ec-Lon) belongs to the superfamily of AAA⁺ proteins. It is a key member of the quality control system of the cell proteome. The enzyme degrades abnormal and defective polypeptides, as well as a number of regulatory proteins, by the processive mechanism. In addition to the ATPase module and the proteolytic domain, Ec-Lon subunit includes a two-domain N-terminal noncatalytic region. A comparative study of the enzyme properties and the DNA-binding ability of full-size Ec-Lon and its form with a deletion of 106 amino acid residues at the N-end has been carried out to reveal the role of the missing fragment in the Ec-Lon function. It has been shown that the fragment does not affect the enzyme peptidase site function or the hydrolysis of the protein substrate by the processive mechanism. However, it is essential for the manifestation of proper ATPase activity and for the implementation of the conformational rearrangements in the ATPase domain, stemming from the coordination of different nucleotides or their complexes by magnesium ions. The loss of the (1–106) fragment destabilizes the active Ec-Lon structure and results in intense Ec-Lon autolysis.     

Comparative study of the regulation of catalytic activity of soluble and membrane forms of enzymes in reverse micellar systems. Gamma-glutamyltransferase and aminopeptidase]

The regulations of functioning of water soluble and membrane forms of enzymes in the systems of reversed micelles of surfactants in organic solvents are compared. By an examples of gamma-glutamyltransferase (in AOT reversed micelles in octane) and amino-peptidase (in Brij 96 reversed micelles in cyclohexane) the principal difference in the catalytic activity regulation of water soluble and membrane forms is demonstrated. The catalytic activity of the membrane form depends largely on the surfactant concentration at the constant hydration degree, whereas the activity of the water soluble form is constant under these conditions. The catalytic activity dependence on the surfactant concentration is regarded as a "test for the enzyme's membrane activity".     

Conformational states of pancreatic lipase

Spin-label method was applied to the studies of conformation properties of pancreatic lipase. Spin-labelled derivatives of the enzyme in SH- and NH2-groups were obtained. ESR-spectra of both samples belong to the immobilized type, in the first case the ESR-spectrum corresponding to strong immobilization of the spin-label, and in the second--to the average one. In both cases the rotation correlation time of the enzyme molecule was measured. The time proved the same independent of the site of the label attachment; it corresponded to the rotation of macromolecule with molecular weight 50000. This fact points to the absence of both intramolecular flexibility of the enzyme molecule and of the association of lipase molecules in solution. It has been shown that introduction of substrates and inhibitors of the enzyme and the interface as well, induces no changes in the ESR spectra, which points to the absence of local conformation changes of protein near the spin-labels introduced.