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At least 19 recordsLinked to original sources
PubMed1983

Studies of the protein encoded by the lon mutation, capR9, in Escherichia coli. A labile form of the ATP-dependent protease La that inhibits the wild type protease.

The product of the lon (capR or deg) gene in Escherichia coli is protease La, an ATP-dependent protease with a linked ATPase activity. Unlike most lon mutations, capR9 is dominant over the wild type under certain conditions. When protease La was isolated from R9 cells and from a recessive capR- strain using DEAE-cellulose chromatography, the mutant enzymes showed about 50% of the wild type activity. Unlike the wild type, the R9 and R- proteases were inhibited by addition of NaCl (less than 0.1 M). In addition, the R9, but not the R-, material inhibited protelysis by normal protease La, and this effect may account for its dominant phenotype. When isolated by phosphocellulose chromatography, the R9 protein lost proteolytic activity but still inhibited the wild type enzyme. This inhibitory activity was purified to near homogeneity using DEAE-cellulose and heparin-agarose chromatography, and corresponded to the 94,000-dalton R9 gene product. At different concentrations, it inhibited ATP-dependent casein degradation and casein-stimulated ATP hydrolysis to a similar extent. Thus, rates of ATP and protein cleavage remained proportional. Similar inhibition of the wild type protease was observed in the presence of DNA which stimulates both protein and ATP hydrolysis. Half-maximal inhibition was observed with approximately a 1:1 ratio of the R9 to the wild type protein. The subunit sizes of the R9 and the wild type protease were indistinguishable but they differed in isoelectric points. Upon gel filtration, both eluted as tetramers (450,000 daltons) in the absence of salt. However, with 0.1 M NaCl, the wild type protease La remained as a tetramer, but the R9 protein dissociated into dimers and monomers and became a more effective inhibitor. After mixing with R9 protein, 3H-labeled protease La remained tetrameric, though it had lost activity. These findings suggest that tetramer formation between the wild type and defective R9 subunits is responsible for the inhibition of the proteolytic and ATPase activities.

ATP-Dependent Proteases↗
PubMed1989

Location of enzymatic and DNA-binding domains on E. coli protease La.

Escherichia coli protease La is an ATP-dependent enzyme that has a DNA-binding site. The locations of the enzymatic and DNA-binding sites are not known. We report that a 75-residue segment at the carboxy-terminus of the protease La is similar to part of Bacillus licheniformis beta-lactamase, a serine enzyme. The comparison score is 8.2 standard deviations higher than that obtained with 10,000 comparisons of randomized sequences of these segments. The probability of obtaining such a score by chance is 1.2 x 10(-16). We also find that a 107-residue segment in the amino-terminus half of protease La is similar to part of the sopB protein, a DNA-binding protein of the plasmid F of E. coli. The comparison score for these segments is 8 standard deviations (P = 6 x 10(-16)). These strong amino acid sequence similarities suggest the locations of the catalytic serine and the DNA-binding domains of protease La.

ATP-Dependent Proteases↗
PubMed1993

ATP hydrolysis is not stoichiometrically linked with proteolysis in the ATP-dependent protease La from Escherichia coli.

Protein degradation in Escherichia coli involves the ATP-dependent serine protease La. Protease La is a homotetramer with one proteolytic and one ATP binding site per monomer. Its proteolytic activity has been shown to be highly increased by simultaneous hydrolysis of ATP, which is essential for the degradation of protein substrates by this enzyme. We have cloned and purified a proteolytically inactive La mutant, in which the catalytically active serine residue at position 679 was replaced by alanine. Fluorescence and circular dichroism spectra of the purified wild type and mutant enzyme revealed identical conformations of the proteins. Based on this observation, the catalytic properties of the wild type enzyme and the S679A mutant were compared. Although the S679A mutant lacks proteolytic activity toward both peptide and protein substrates under all conditions investigated, its ATPase activity is completely unaffected by the removal of the protease activity. Since protein substrates stimulate the ATP-dependent hydrolysis of peptides by protease La, it has been argued that this stimulation is due to interactions with a regulatory binding site on the enzyme. In accordance with this model, protein substrates such as alpha-casein and denatured bovine serum albumin stimulate the ATPase activity of the S679A mutant to the same degree as in the active protease. Therefore, the intrinsic ATPase activity of protease La as well as its stimulation is not dependent on the simultaneous hydrolysis of the protein substrate.

ATP-Dependent Proteases↗
PubMed1993

Yeast mitochondrial ATP-dependent protease: purification and comparison with the homologous rat enzyme and the bacterial ATP-dependent protease La.

Homogenous ATP-dependent protease has been isolated for the first time from mitochondria of yeast Saccharomyces cerevisiae. The enzyme molecule consists of six 120 kDa subunits. It is a serine protease with an absolute ATP requirement for its activity. Basic enzymatic characteristics of the yeast protease are similar to those of the corresponding rat mitochondrial enzyme and of the E. coli protease La. The yeast enzyme immunochemically cross-reacts with the bacterial protease La.

ATP-Dependent Proteases↗
PubMed1985

Protease La, the lon gene product, cleaves specific fluorogenic peptides in an ATP-dependent reaction.

Protease La is an ATP-dependent protease that catalyzes the rapid degradation of abnormal proteins and certain normal polypeptides in Escherichia coli. In order to learn more about its specificity and the role of ATP, we tested whether small fluorogenic peptides might serve as substrates. In the presence of ATP and Mg2+, protease La hydrolyzes two oligopeptides that are also substrates for chymotrypsin, glutaryl-Ala-Ala-Phe-methoxynaphthylamine (MNA) and succinyl-Phe-Leu-Phe-MNA. Methylation or removal of the acidic blocking group prevented hydrolysis. Closely related peptides (glutaryl-Gly-Gly-Phe-MNA and glutaryl-Ala-Ala-Ala-MNA) are cleaved only slightly, and substrates of trypsin-like proteases are not hydrolyzed. Furthermore, several peptide chloromethyl ketone derivatives that inhibit chymotrypsin and cathepsin G (especially benzyloxycarbonyl-Gly-Leu-Phe-chloro-methyl ketone), inhibited protease La. Thus its active site prefers peptides containing large hydrophobic residues, and amino acids beyond the cleavage site influence rates of hydrolysis. Peptide hydrolysis resembles protein breakdown by protease La in many respects: 1) ADP inhibits this process rapidly, 2) DNA stimulates it, 3) heparin, diisopropyl fluorophosphate, and benzoyl-Arg-Gly-Phe-Phe-Leu-MNA inhibit hydrolysis, 4) the reaction is maximal at pH 9.0-9.5, 5) the protein purified from lon- E. coli or Salmonella typhymurium showed no activity against the peptide, and that from lonR9 inhibited peptide hydrolysis by the wild-type enzyme. With partially purified enzyme, peptide hydrolysis was completely dependent on ATP. The pure protease hydrolyzed the peptide slowly when only Mg2+, Ca2+, or Mn2+ were present, and ATP enhanced this activity 6-15-fold (Km = 3 microM). Since these peptides cannot undergo phosphorylation, adenylylation, modification of amino groups, or denaturation, these mechanisms cannot account for the stimulation by ATP. Most likely, ATP and Mg2+ affect the conformation of the enzyme, rather than that of the substrate.

ATP-Dependent Proteases↗
PubMed1987

An increased content of protease La, the lon gene product, increases protein degradation and blocks growth in Escherichia coli.

The lon gene product in Escherichia coli is an ATP-dependent protease (La) that plays an important role in the breakdown of abnormal proteins and certain normal polypeptides. Since transcription of the lon gene rises as part of the heat-shock response, we studied the physiological effects of increased levels of protease La. In cells carrying additional copies of the lon gene under the control of the lac or tac promoter, induction of the protease resulted in a rapid cessation of cell growth and in a loss of viability at stationary phase. Similarly, cells carrying a multicopy plasmid encoding the lon gene contained 2-5-fold more protease La and grew much more slowly than did control cells. In such cells, insertion sequences appeared spontaneously in the lon gene on the plasmid and prevented the excess protease production and allowed more rapid growth. The cells with increased content of protease La (due to the lon plasmid or induction of the lon gene) exhibited severalfold higher rates of degradation of abnormal proteins containing amino acid analogs and of incomplete polypeptides containing puromycin. Also, a beta-galactosidase fusion protein with enzymatic activity was relatively stable in control cells but unstable in the cells with high protease La content. In these cells, the overall degradation of normal proteins increased 2-fold, and certain cellular polypeptides appeared particularly sensitive to proteolysis. Thus, rates of protein degradation in vivo are limited in part by the cellular content of the ATP-dependent protease, and increases in transcription of the lon gene enhance proteolysis and can be deleterious to the cell.

ATP-Dependent Proteases↗
PubMed1987

Binding of nucleotides to the ATP-dependent protease La from Escherichia coli.

A critical enzyme in protein breakdown in Escherichia coli is the ATP-hydrolyzing protease La, the lon gene product. In order to clarify the role of ATP in proteolysis, we studied ATP and ADP binding to this enzyme using rapid gel filtration to separate free from bound ligands. In the presence of Mg2+ or Mn2+ and 10 microM ATP, two molecules of ATP were bound to the tetrameric enzyme, while at 100 microM ATP (or higher), four ATP molecules were bound, both at 0 and 37 degrees C. Protease La thus has two high affinity sites (S0.5 less than 10(-7) M) for ATP and two lower affinity sites (S0.5 = 12-15 microM). Binding was reversible. In the absence of a divalent ion, ATP bound to only two sites. However, much lower Mg2+ concentrations (50 microM) were required for maximal ATPase binding than for maximal proteolytic and ATPase activity (2 mM). Decavanadate, which is a potent inhibitor of proteolysis, also blocked ATP binding, but orthovanadate had neither effect. Different ATP analogs bind to these sites in distinct ways. Adenyl-5'-yl imidodiphosphate binds to only one high affinity site, while adenyl-5'-yl methylene monophosphonate binds to two. Nevertheless, both non-metabolizable analogs can activate oligopeptide hydrolysis as well as ATP. Although binding of a single nucleotide can activate peptide hydrolysis, occupancy of all four sites appears necessary for maximal protein breakdown. The ATP molecules on all four sites are hydrolyzed rapidly. The Pi is released, but ADP remains on the enzyme. ADP binds to the same four sites, but this process does not require divalent ions. Protease La shows higher affinity for ADP than for ATP. Therefore, in vivo, ADP should inhibit ATP binding and protease La function.

ATP-Dependent Proteases↗
PubMed1998

LonR9 carrying a single Glu614 to Lys mutation inhibits the ATP-dependent protease La (Lon) by forming mixed oligomeric complexes.

An unusual lon mutation (called lonR9) is dominant over the wild-type gene, which encodes the ATP-dependent protease La (Lon) in Escherichia coli, when present in multicopy plasmids. Here, we cloned and sequenced lonR9, and showed that the mutant gene carries a single point mutation in its open reading frame, which leads to replacement of Glu614 by Lys. The LonR9 protein and its poly-His-tagged form were purified to apparent homogeneity. Both of the purified proteins were capable of inhibiting the ATP-dependent proteolysis and the protein-activated ATP hydrolysis by protease La. Furthermore, the His-tagged LonR9 protein was found to form mixed oligomeric complexes with protease La, upon analysis by chromatography on a metal-chelating column. These results suggest that the phenotypic dominance of the lonR9 mutant is due to the formation of mixed oligomeric complexes between LonR9 and protease La, in which the defective components prevent the function of the wild-type subunits.

ATP-Dependent Proteases↗
PubMed1987

Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La.

The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. We have partially purified another ATP-dependent protease from lon-cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes [3H]methyl-casein to acid-soluble products in the presence of ATP and Mg2+. ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. We propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show proteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. [Katayama-Fujimura, Y., Gottesman, S. & Maurizi, M. R. (1987) J. Biol. Chem. 262, 4477-4485] indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles.

ATP-Dependent Proteases↗
PubMed1991

Induction of protease La under stress and its effect on intracellular proteolysis in Escherichia coli.

Induction of protease La was found to increase to higher extent in E. coli that had been treated with canavanine for longer period. However, hydrolysis of canavanine-containing proteins occurred rapidly but at nearly an identical rate regardless of the period of canavanine-treatment. Exposure of E. coli to heat also raised the level of protease La but showed little effect on overall rate of proteolysis. These results suggest that induction of protease La under stress occurs as a part of heat shock response but not necessarily for elimination of denatured or abnormal proteins.

ATP-Dependent Proteases↗
PubMed2001

Mg2+-linked oligomerization modulates the catalytic activity of the Lon (La) protease from Mycobacterium smegmatis.

Lon (La) proteases are multimeric enzymes that are activated by ATP and Mg(2+) ions and stimulated by unfolded proteins such as alpha-casein. The peptidase activity of the Lon protease from Mycobacterium smegmatis (Ms-Lon) is dependent upon both its concentration and that of Mg(2+). Addition of alpha-casein partially substitutes for Mg(2+) in activating the enzyme. In chemical dissociation experiments, higher concentrations of urea were required to inhibit Ms-Lon's catalytic activities after an addition of alpha-casein. Analytical ultracentrifugation was used to directly probe the effect of activators of peptidase activity on Ms-Lon self-association. Sedimentation velocity experiments reveal that Ms-Lon monomers are in a reversible equilibrium with oligomeric forms of the protein and that the self-association reaction is facilitated by Mg(2+) ions but not by AMP-PNP or ATP gamma S. NaCl at 100 mM facilitates oligomerization and stimulates peptidase activity at suboptimal concentrations of MgCl(2). Sedimentation equilibrium analysis shows that Ms-Lon associates to a hexamer at 50 mM Tris and 10 mM MgCl(2), at pH 8.0 and 20 degrees C, and that the assembly reaction is Mg(2+) dependent; the mole fraction of hexamer decreases with decreasing MgCl(2) to undetectable levels in 10 mM EDTA. The analysis of experiments conducted at a series of initial protein and MgCl(2) concentrations yields two assembly models: dimer <--> tetramer <--> hexamer and timer <--> hexamer, equally consistent with the data. Limited trypsin digestion, CD, and tryptophan fluorescence suggest only minor changes in secondary and tertiary structure upon Mg(2+)-linked oligomerization. These results show that activation of Ms-Lon peptidase activity requires oligomerization and that Ms-Lon self-association reaction is facilitated by its activator, Mg(2+), and stimulator, unfolded protein.

ATP-Dependent Proteases↗
PubMed1986

Selectivity of intracellular proteolysis: protein substrates activate the ATP-dependent protease (La).

A critical enzyme in protein breakdown in Escherichia coli is protease La (the lon gene product), which hydrolyzes proteins and adenosine triphosphate (ATP) in a coupled process. The mechanism of this process was studied with fluorogenic tripeptides. Although proteins and peptides are degraded at the same active site, protein substrates enhance the ability of the enzyme to degrade these peptides two- to tenfold. Proteins that are not substrates had little or no effect. Thus, protein substrates must bind to protease La at two sites, the active site and an allosteric site whose occupancy enhances proteolytic activity. This effect did not require that the proteins themselves be degraded. Proteins could induce peptide breakdown even in the absence of ATP, and proteins and ATP had additive effects in stimulating peptidase activity. A multistep cyclical mechanism is proposed in which the binding of the substrate and ATP activates the protease. The enzyme can then cleave a peptide bond, but is inactivated through ATP hydrolysis. Such a mechanism may help account for the selectivity of protein breakdown and prevent inappropriate or excessive proteolysis in vivo.

ATP-Dependent Proteases↗
PubMed1987

Protein substrates activate the ATP-dependent protease La by promoting nucleotide binding and release of bound ADP.

The interaction of protein substrates with protease La from Escherichia coli enhances its ability to hydrolyze ATP and peptide bonds. These studies were undertaken to clarify how unfolded proteins allosterically stimulate this ATPase activity. The tetrameric protease can bind four molecules of ATP, which activates proteolysis, or four molecules of ADP, which inhibits enzymatic activity. Protein substrates stimulate binding of the nonhydrolyzable ATP analog [3H] adenyl-5'yl imidodiphosphate, although they do not increase the net binding of [3H]ATP or [3H]ADP. Once bound, ATP is quickly hydrolyzed to ADP, which remains noncovalently associated with protease La even through repeated gel filtrations. Exposure to protein substrates (e.g. denatured bovine serum albumin at 37 degrees C) induces the release of all the bound ADP from the enzyme. Nonhydrolyzable ATP analogs bound to the enzyme were not released by these substrates. Proteins that are not degraded (e.g. native bovine serum albumin) and oligopeptides that only bind to the catalytic site do not induce ADP release. Thus, polypeptide substrates have to interact with an allosteric site to induce this effect. The protein-induced ADP release is inhibited by high concentrations of Mg2+ and is highly temperature-dependent. Protein substrates promoted [3H]ATP binding in the presence of ADP and Mg2+ (i.e. ATP-ADP exchange) and reduced the ability of ADP to inhibit the enzyme's peptidase and ATPase activities. These results indicate that: 1) ADP release is a rate-limiting step in protease La function; 2) bound ADP molecules inhibit protein and ATP hydrolysis in vivo; 3) denatured proteins interact with the enzyme's regulatory site and promote ADP release, ATP binding, and their own hydrolysis.

ATP-Dependent Proteases↗
PubMed1988

Sequence of the lon gene in Escherichia coli. A heat-shock gene which encodes the ATP-dependent protease La.

To learn more about the mechanism and regulation of the ATP-dependent protease La in Escherichia coli, the lon gene was completely sequenced using the dideoxy method on fragments generated by Bal31 digestion. The predicted amino acid composition based on the DNA sequence agreed well with the composition of the acid-hydrolyzed protease. The predicted NH2-terminal amino acid sequence, tryptophan content, and the carboxyl terminus also agreed with experimental data. However, the molecular weight of 87,000 (783 amino acids) calculated from the DNA sequence was lower than prior estimates. The tetrameric enzyme contains four binding sites for ATP, a DNA-binding domain, a proteolytic site, and a regulatory site that binds unfolded polypeptides. An ATP-binding pocket exists on each subunit as shown by consensus sequences and elements of secondary structure resembling those on other nucleotide-binding proteins (e.g. adenylate kinase, RecA). For this purpose, improved consensus patterns for identifying ATP-binding domains were developed. Computer-assisted comparisons, however, failed to demonstrate any regions homologous to sequences in other polypeptides including proteases or DNA-binding proteins. This enzyme also contains an unusual highly acidic domain surrounded by very basic sequences. Protease La is the first ATP-dependent protease sequenced and seems to represent a new type of enzyme. The promoter sequence was similar to consensus sequences for other heat-shock promoters. Using site-directed mutagenesis, alterations were introduced into the putative promoter sequence. Mutations upstream of -35 had little effect, but alterations immediately upstream of -10 lowered basal transcription of a lon-lacZ operon fusion and reduced its response to inducers of the heat-shock response.

ATP-Dependent Proteases↗
PubMed1981

The product of the lon (capR) gene in Escherichia coli is the ATP-dependent protease, protease La.

In Escherichia coli, degradation of abnormal proteins is an energy-requiring process; it is decreased in mutants in the lon (capR or deg) gene. We find that the protein encoded by the lon gene is an ATP-dependent protease and is identical to protease La, recently described in E. coli. Both proteins are serine proteases that hydrolyze casein and globin, but not insulin, in the presence of ATP and Mg2+. Both respond to ATP, less well to other nucleoside triphosphates, and not to nonhydrolyzable ATP analogs. The purified lon protein has an apparent Mr of 450,000 and appears to be composed of four identical subunits. Its size, chromatographic behavior, and sensitivity to various inhibitors and heat are indistinguishable from those of protease La. Moreover, in a strain that carries additional copies of the lon+ allele on a plasmid, the content of protease La, but not of other proteases, is 2- to 10-fold greater than in the lon+ parent strain. Strains carrying the nonsense mutations capR9 and capR- also contain this ATP-dependent proteolytic activity, but it is present in substantially lower amounts and is inactivated by phosphocellulose chromatography, unlike the wild-type enzyme. Degradation of abnormal proteins in these lon- strains, which is slower than in the wild type, still requires ATP. Alterations in the ATP-dependent protease in the lon- mutants can account for the defect in intracellular proteolysis and perhaps also for the other phenotypic effects of this pleiotropic gene.

Adenosine Triphosphatases↗
PubMed2006

Oligomeric structure of the ATP-dependent protease La (Lon) of Escherichia coli.

Lon, also known as protease La, belongs to a class of ATP-dependent serine protease. It plays an essential role in degradation of abnormal proteins and of certain short-lived regulatory proteins, and is thought to possess a Ser-Lys catalytic dyad. To examine the structural organization of Lon, we performed an electron microscope analysis. The averaged images of Lon with end-on orientation revealed a six-membered, ring-shaped structure with a central cavity. The side-on view showed a two-layered structure with an equal distribution of mass across the equatorial plane of the complex. Since a Lon subunit possesses two large regions containing nucleotide binding and proteolytic domains, each layer of the Lon hexamer appears to consist of the side projections of one of the major domains arranged in a ring. Lon showed a strong tendency to form hexamers in the presence of Mg(2+), but dissociated into monomers and/or dimers in its absence. Moreover, Mg(2+)-dependent hexamer formation was independent of ATP. These results indicate that Lon has a hexameric ring-shaped structure with a central cavity, and that the establishment of this configuration requires Mg(2+), but not ATP.

ATP-Dependent Proteases↗
PubMed1982

DNA stimulates ATP-dependent proteolysis and protein-dependent ATPase activity of protease La from Escherichia coli.

The product of the lon gene in Escherichia coli is an ATP-dependent protease, protease La, that also binds strongly to DNA. Addition of double-stranded or single-stranded DNA to the protease in the presence of ATP was found to stimulate the hydrolysis of casein or globin 2- to 7-fold, depending on the DNA concentration. Native DNA from several sources (plasmid pBR322, phage T7, or calf thymus) had similar effects, but after denaturation the DNA was 20-100% more effective than the native form. Although poly(rA), globin mRNA, and various tRNAs did not stimulate proteolysis, poly(rC) and poly(rU) were effective. Poly(dT) was stimulatory but (dT)10 was not. In the presence of DNA as in its absence, proteolysis required concomitant ATP hydrolysis, and the addition of DNA also enhance ATP hydrolysis by protease La 2-fold, but only in the presence of casein. At much higher concentrations, DNA inhibited proteolysis as well as ATP cleavage. Thus, association of this enzyme with DNA may regulate the degradation of cell proteins in vivo.

ATP-Dependent Proteases↗
PubMed1994

A point mutation within the ATP-binding site inactivates both catalytic functions of the ATP-dependent protease La (Lon) from Escherichia coli.

A point mutant in the ATP-binding motif (GPPGVGK362T) of the ATP-dependent protease La from Escherichia coli was investigated in which the lysine at position 362 was replaced by an alanine. The catalytic efficiency of the K362A mutant is at least two orders of magnitude lower than that of wild-type protease La due to a decreased Vmax and an increased KM for ATP. Simultaneously, the peptidase activity of La K362A is almost completely eliminated. Since selective inactivation of the peptidase activity of La does not affect its intrinsic ATPase activity, coupling of proteolysis with ATP hydrolysis is only uni-directional in this energy-dependent protease.

ATP-Dependent Proteases↗