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PubMed1989

The activities of 'Pz-peptidase' and 'endopeptidase 24.15' are due to a single enzyme.

It was found that Pz-peptidase (assayed with 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys) and endopeptidase 24.15 (assayed with benzoyl-Gly-Ala-Ala-Phe-p-aminobenzoate) were co-purified from rat skeletal muscle, were co-eluted in high-resolution gel chromatography and co-existed in a homogeneous preparation of rat testis endopeptidase 24.15. The action of partially purified Pz-peptidase from rat testis on 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg was blocked by an inhibitor of endopeptidase 24.15, and also by a substrate of this enzyme. The partially purified enzyme hydrolysed two substrates of endopeptidase 24.15 with Km values similar to those published previously, and its action on 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys was inhibited by compounds that are considered specific for endopeptidase 24.15. We conclude that the activities previously attributed to two distinct enzymes are due to only one, and that the merging of the two literatures may lead to new lines of research.

Animals↗
PubMed1991

Accumulation of acyl-enzyme in DD-peptidase-catalysed reactions with analogues of peptide substrates.

Thioester substrates can be used to study the hydrolysis and transfer reactions catalysed by beta-lactamases and DD-peptidases. With the latter enzymes, accumulation of the acyl-enzyme can be detected directly. The efficiency of various amines as acceptor substrates was in excellent agreement with previous results obtained with peptide substrates of the DD-peptidases. The results indicated the presence of a specific binding site for the acceptor substrates. Although most of the results agreed well with a simple partition model, more elaborate hypotheses will be needed to account for all the data presented.

Acylation↗
PubMed1992

Importance of the two tryptophan residues in the Streptomyces R61 exocellular DD-peptidase.

Modification of the Streptomyces R61 DD-peptidase by N-bromosuccinimide resulted in a rapid loss of enzyme activity. In consequence, the role of the enzyme's two tryptophan residues was investigated by site-directed mutagenesis. Trp271 was replaced by Leu. The modification yielded a stable enzyme whose structural and catalytic properties were similar to those of the wild-type protein. Thus the Trp271 residue, though almost invariant among the beta-lactamases of classes A and C and the low-Mr penicillin-binding proteins, did not appear to be essential for enzyme activity. Mutations of the Trp233 into Leu and Ser strongly decreased the enzymic activity, the affinity for beta-lactams and the protein stability. Surprisingly, the benzylpenicilloyl-(W233L)enzyme deacylated at least 300-fold more quickly than the corresponding acyl-enzyme formed with the wild-type protein and gave rise to benzylpenicilloate instead of phenylacetylglycine. This mutant DD-peptidase thus behaved as a weak beta-lactamase.

Base Sequence↗
PubMed1992

Importance of the His-298 residue in the catalytic mechanism of the Streptomyces R61 extracellular DD-peptidase.

Among the active-site-serine penicillin-recognizing proteins, the Streptomyces R61 extracellular DD-peptidase is the only one to have a His-Thr-Gly sequence [instead of Lys-Thr(Ser)-Gly] in 'box' VII. The His residue was replaced by Gln or Lys. Both mutations induced a marked decrease in the rates of both tripeptide substrate hydrolysis and acylation by benzylpenicillin and cephalosporin C. The rate of hydrolysis of the thioester hippuryl thioglycollate was less affected. The most striking result was the disproportionate loss of transpeptidation properties by both mutants, indicating an important role of His-298 in this reaction. We believe that this result represents the first modification of a DD-peptidase leading to a specific decrease of the transpeptidation-to-hydrolysis ratio.

Amino Acid Sequence↗
PubMed1995

Breakdown of the stereospecificity of DD-peptidases and beta-lactamases with thiolester substrates.

With peptide analogues of their natural substrates (the glycopeptide units of nascent peptidoglycan), the DD-peptidases exhibit a strict preference for D-Ala-D-Xaa C-termini. Gly is tolerated as the C-terminal residue, but with a significantly decreased activity. These enzymes were also known to hydrolyse various ester and thiolester analogues of their natural substrates. Some thiolesters with a C-terminal leaving group that exhibited L stereochemistry were significantly hydrolysed by some of the enzymes, particularly the Actinomadura R39 DD-peptidase, but the strict specificity for a D residue in the penultimate position was fully retained. These esters and thiolesters also behave as substrates for beta-lactamases. In this case, thiolesters exhibiting L stereochemistry in the ultimate position could also be hydrolysed, mainly by the class-C and class-D enzymes. However, more surprisingly, the class-C Enterobacter cloacae P99 beta-lactamase also hydrolysed thiolesters containing an L residue in the penultimate position, sometimes with a higher efficiency than the D isomer.

Amino Acid Sequence↗
PubMed1997

Site-directed mutagenesis of the Actinomadura R39 DD-peptidase.

The role of various residues in the conserved structural elements of the Actinomadura R39 penicillin-sensitive dd-peptidase has been studied by site-directed mutagenesis. Replacement of Ser-298 of the 'SDN loop' by Ala or Gly significantly decreased the kcat/Km value for the peptide substrate, but only by a factor of 15 and had little effect on the other catalytic properties. Mutations of Asn-300 of the same loop and of Lys-410 of the KTG triad yielded very unstable proteins. However, the N300S mutant could be purified as a fusion protein with thioredoxin that exhibited decreased rates of acylation by the peptide substrate and various cephalosporins. Similar fusion proteins obtained with the N300A, K410H and K410N mutants were unstable and their catalytic and penicillin-binding properties were very strongly affected. In transpeptidation reactions, the presence of the acceptor influenced the kcat/Km values, which suggested a catalytic pathway more complex than a simple partition of the acyl-enzyme between hydrolysis and aminolysis. These results are compared with those obtained with two other penicillin-sensitive enzymes, the Streptomyces R61 dd-peptidase and Escherichia coli penicillin-binding protein (PBP) 5.

Actinomycetales↗
PubMed1991

Plasma exudation into airways induced by inhaled platelet-activating factor: effect of peptidase inhibition.

1. To evaluate whether endogenous peptide release is involved in the airway responses to inhaled platelet-activating factor, we measured lung resistance and airway microvascular leakage in anaesthetized guinea pigs pretreated with inhalation of either saline or a combination of the peptidase inhibitors phosphoramidon (0.1 mmol/l: 60 breaths; 7.5 nmol), to inhibit neutral endopeptidase, and captopril (4.6 mmmol/l: 60 breaths; 350 nmol), to inhibit angiotensin-converting enzyme. 2. Airway microvascular leakage was determined by the albumin marker Evans Blue dye injected intravenously (20 mg/kg) before platelet-activating factor or sham challenge. 3. Inhaled platelet-activating factor induced a maximum increase in lung resistance (1.43 +/- 0.33 cmH2O s-1 ml-1) which was not significantly different after pretreatment with phosphoramidon and captopril (1.44 +/- 0.21 cmH2O s-1 ml-1). 4. Inhalation of platelet-activating factor caused a significant increase in extravasated Evans Blue dye at all airway levels, an effect which was not potentiated by peptidase inhibition. Similar results were obtained with dye extravasated into the airway lumen and absorbed by a filter paper placed on the tracheal mucosa. Approximately 11% of the total tracheal dye was found in the lumen. There was a high correlation between tracheal tissue and tracheal lumen Evans Blue dye (r = 0.91; P less than 0.001). 5. We found a significantly lower dry to wet weight ratio in proximal intrapulmonary airways of animals exposed to platelet-activating factor, suggesting that platelet-activating factor caused airway oedema at this airway level.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Inhalation↗
PubMed1999

Role of peptidase and cyclooxygenase inhibitors in the guinea-pig bronchial response to the synthetic endothelin ET(B) agonist IRL 1620 and antagonist BQ-788.

In isolated guinea-pig bronchial preparations the selective endothelin ETB agonist, IRL 1620 caused a concentration-dependent contraction. The pD2 value (7.16 +/- 0.09, n = 6) was significantly increased in the presence of peptidase inhibitors (thiorfan 1 microM, captopril 1 microM, bestatin 1 microM) (pD2 = 7.75 +/- 0.09, n = 6). Indomethacin (5 microM) did not appear to influence the ETB-agonist pD2 value (6.92 + 0.11, n = 6) but potentiated its maximal response significantly (67.23 +/- 4.81% vs. 53.37 +/- 4.80%). The concentration-response curve for the contractile response to IRL 1620 (pD2=7.83 +/- 0.01, n=16); was reproducible, although not completely, since the second curve to this selective ETB agonist was shifted significantly to the right (pD2 = 7.34 +/- 0.09, n = 16) and a decrease in the maximal response was observed (20.0 +/- 2.0%). BQ 788, a selective antagonist for ETB receptors, employed in concentrations ranging from 1.5 to 150 nM, caused a dose-dependent shift to the right of the concentration-response curve to IRL 1620, with a pIC50 value of 8.11 +/- 0.03; this action was not influenced by adding enzyme inhibitors (pIC50 = 8.17 +/- 0.29). Our data show that IRL 1620 undergoes a hydrolytic metabolism in guinea-pig bronchial preparations, which could influence the calculation of the pD2. Pretreatment of the tissue with peptidase inhibitors and indomethacin is consequently significant in the evaluation of IRL 1620 activity, while it does not influence the action of the antagonist, BQ 788.

Animals↗
PubMed1996

Bacillus subtilis can modulate its capacity and specificity for protein secretion through temporally controlled expression of the sipS gene for signal peptidase I.

Bacillus subtilis contains three chromosomally encoded type I signal peptidases (SipS, SipT and SipU), which remove signal peptides from secretory precursor proteins. In the present study the biological function of SipS and the regulation of its synthesis were analysed. Unlike the type I signal peptidase of Escherichia coli, SipS was essential neither for protein secretion nor viability of the cell. However, in the absence of SipS the rate of processing of several preproteins was reduced, and four of the seven major secreted proteins of B. subtilis were hardly detectable in the growth medium. Surprisingly, the processing of Bacillus amyloliquefaciens alpha-amylase and the secretion of at least two endogenous B. subtilis proteins was improved in the absence of SipS. These findings indicate that the substrate preference of SipS differs from that of SipT and SipU, and that SipS is an important factor determining the efficiency of protein secretion in B. subtilis. SipS is transcribed in a growth phase- and medium-dependent manner. In minimal medium, the growth phase-dependent transcription of sipS is controlled by the DegS-DegU two-component regulatory system, indicating that the expression of sipS is regulated by the same factors that control the expression of most genes for secreted degradative enzymes. Our observations suggest that B. subtilis can modulate its capacity and specificity for protein secretion through the controlled expression of sipS.

Bacillus subtilis↗
PubMed1999

The prepilin peptidase is required for protein secretion by and the virulence of the intracellular pathogen Legionella pneumophila.

Prepilin peptidases cleave, among other substrates, the leader sequences from prepilin-like proteins that are required for type II protein secretion in Gram-negative bacteria. To begin to assess the importance of type II secretion for the virulence of an intracellular pathogen, we examined the effect of inactivating the prepilin peptidase (pilD) gene of Legionella pneumophila. Although the pilD mutant and its parent grew similarly in bacteriological media, they did differ in colony attributes and recoverability from late stationary phase. Moreover, at least three proteins were absent from the mutant's supernatant, indicating that PilD is necessary for the secretion of Legionella proteins. The absence of both the major secreted protein and a haemolytic activity from the mutant signalled that the L. pneumophila zinc metalloprotease is excreted via type II secretion. Most interestingly, the pilD mutant was greatly impaired in its ability to grow within Hartmannella vermiformis amoebae and the human macrophage-like U937 cells. As reintroduction of pilD into the mutant restored inefectivity and as a mutant lacking type IV pilin replicated like wild type, these data suggested that the intracellular growth of L. pneumophila is promoted by proteins secreted via a type II pathway. Intratracheal inoculation of guinea pigs revealed that the LD50 for the pilD mutant is at least 100-fold greater than that for its parent, and the culturing of bacteria from infected animals showed a rapid clearance of the mutant from the lungs. This is the first study to indicate a role for PilD and type II secretion in intracellular parasitism.

Animals↗
PubMed2003

Peptidase activities of the 20/26S proteasome and a novel protease in human brain.

Many neurodegenerative diseases are characterized by ubiquitin-positive protein aggregates or inclusion bodies. Ubiquitin-conjugated proteins are degraded by the 20/26S proteasome, and reduced proteasome peptidase activities in brain homogenates have been reported in pathologic lesions of Parkinson's and Alzheimer's diseases. However, it is unknown whether crude extracts of human brain contain other proteases having peptidase activities. We found a novel protease of molecular weight of approximately 105 kDa in normal human brain, which exhibited trypsin-like (T-L) and chymotrypsin-like (ChT-L) activities (corresponding to 52% and 21% of the total activities in crude extracts) but not peptidyl glutamyl peptide hydrolase activity. Both T-L and ChT-L activities of this protease were partially inhibited by proteasome inhibitors (MG132, lactacystin) and, in contrast to those of the proteasome, also by sodium dodecyl sulfate. A simple method to obtain a brain fraction specific to the 20/26S proteasome was developed. Our human brain data suggest that T-L and ChT-L activity levels of the proteasome reported previously may include those of the 105 kDa protease, an enzyme of as yet unknown biological significance, and that it is necessary to separate the proteasome from this protease to evaluate the actual status of the ubiquitin-proteasome system in neurodegenerative disorders.

Aged↗
PubMed1988

Sex-linked peptidase-1 patterns in Pleurodeles waltlii Michah (Urodele Amphibian): genetic evidence for a new codominant allele on the W sex chromosome and identification of ZZ, ZW and WW sexual genotypes.

In Pleurodeles waltlii, peptidase-1 is a dimeric enzyme which has been shown to be controlled by two codominant alleles, Pep-1A and Pep-1B (Ferrier et al., 1980, 1983), linked respectively to the Z and W sex chromosomes. The enzymatic patterns obtained after starch gel electrophoresis were used routinely to identify the sexual genotypes of animals reared in our laboratory. We describe here three new patterns that were encountered in standard females, in sex-reversed animals and in thelygenous females. The inheritance data indicated the occurrence of a third codominant allele, Pep-1 beta, linked to the W sex chromosome. The discovery of this new allele does not impair peptidase-1 polymorphism as a reliable tool for ZZ, ZW and WW genotype identification.

Alleles↗
PubMed1979

Translational and post-translational cleavage of M13 procoat protein: extracts of both the cytoplasmic and outer membranes of Escherichia coli contain leader peptidase activity.

The coat protein of coliphage M13 is an integral protein of the host cytoplasmic membrane at all stages of the infectious cycle. Both in in vivo and DNA-directed in vitro synthesis, it is initially made with an NH2-terminal "leader peptide" of 23 amino acids and is termed procoat. We now report that leader peptidase, and activity which removes the leader peptide and converts procoat to coat, is found in both the inner (cytoplasmic) and outer membrane of Escherichia coli. However, only cytoplasmic membranes will catalyze cleavage of procoat in the absence of detergent. Leader peptidase will cleave procoat either during translation or after protein synthesis is complete.

Cell Membrane↗
PubMed1981

A mutation downstream from the signal peptidase cleavage site affects cleavage but not membrane insertion of phage coat protein.

Morphogenesis of filamentous phage includes synthesis of the phage major coat protein in precursor form, its insertion into the host cell plasma membrane, its cleavage to the mature form of the protein, and its assembly there into virions. The M13 mutant am8H1R6 encodes a coat protein in which leucine replaces glutamic acid as residue 2 of the mature protein [Boeke, J. D., Russel, M. & Model, P. (1980) J. Mol. Biol. 144, 103-116]. The coat protein precursor produced by this variant is a poor substrate for the Escherichia coli signal peptidase both in vivo and in vitro. This pre-coat protein, which is eventually processed and assembled into viable phage particles, is associated with the membrane fraction of the infected cell. We conclude that the domain recognized by the signal peptidase extends beyond the signal peptide itself. Furthermore, membrane association and signal peptide cleavage can be separated temporally under conditions that permit membrane insertion, cleavage, and phage assembly.

Coliphages↗
PubMed1990

Albumin Redhill (-1 Arg, 320 Ala----Thr): a glycoprotein variant of human serum albumin whose precursor has an aberrant signal peptidase cleavage site.

Albumin Redhill is an electrophoretically slow genetic variant of human serum albumin that does not bind 63Ni2+ and has a molecular mass 2.5 kDa higher than normal albumin. Its inability to bind Ni2+ was explained by the finding of an additional residue of Arg at position -1. This did not explain the molecular basis of the genetic variation (since proalbumin contains adjacent Arg residues at -1 and -2) or the increase in apparent molecular mass. Fractionation of tryptic digests on concanavalin A-Sepharose followed by peptide mapping of the bound and unbound fractions and sequence analysis of the glycopeptides identified a mutation of 320 Ala----Thr. This introduces an Asn-Tyr-Thr oligosaccharide attachment sequence centered on Asn-318 and explains the increase in molecular mass. This, however, did not satisfactorily explain the presence of the additional Arg residue at position -1. DNA sequencing of polymerase chain reaction-amplified genomic DNA encoding the prepro sequence of albumin indicated an additional mutation of -2 Arg----Cys. This introduces a prepro sequence, Met-Lys-Trp-Val-Thr-Phe-Ile-Ser-Leu-Leu-Phe-Leu-Phe-Ser-Ser-Ala-Tyr- Ser-Arg-Gly-Val-Phe-Cys-Arg (cf.-Tyr-Ser-Arg-Gly-Val-Phe-Arg-Arg- in normal human pre-proalbumin). We propose that the new Phe-Cys-Arg sequence in the propeptide is an aberrant signal peptidase cleavage site and that the signal peptidase cleaves the propeptide of albumin Redhill in the lumen of the endoplasmic reticulum before it reaches the Golgi vesicles, the site of the diarginyl-specific proalbumin convertase.

Alanine↗
PubMed1991

Yeast signal peptidase contains a glycoprotein and the Sec11 gene product.

Partially purified yeast microsomal signal peptidase appears to be a complex of four polypeptides of 13, 18, 20, and 25 kDa. The 18-kDa chain is the product of the Sec11 gene, which is necessary for signal peptidase activity. The 25-kDa subunit is a glycoprotein that binds Con A. Two related methods for purification of the enzyme are presented; the first includes removal of peripheral membrane proteins from microsomes by alkali extraction, solubilization of the enzyme by nonionic detergent and high salt, and four different chromatographic procedures. An alternative method was developed based on lectin-affinity chromatography.

Chromatography↗
PubMed1992

Sequence analysis of rat mitochondrial intermediate peptidase: similarity to zinc metallopeptidases and to a putative yeast homologue.

Proteolytic removal of amino-terminal octapeptides from mitochondrial intermediate proteins is a required step for a subgroup of nuclear-encoded mitochondrial precursors and is specifically catalyzed by mitochondrial intermediate peptidase (MIP). We recently reported the purification of MIP from rat liver and showed that the enzyme is a monomer of 75 kDa. We now report the sequence of a full-length rat MIP cDNA. This cDNA codes for a protein of 710 amino acids, including an amino-terminal mitochondrial leader peptide of 33 residues. The region surrounding the mature MIP amino terminus shows a cleavage site typically recognized by the general mitochondrial processing peptidase (MPP). In vitro synthesized MIP precursor is cleaved to mature MIP by purified MPP, and thus MIP is not required for its own proteolytic maturation. Comparison of the deduced MIP sequence with other sequences in the GenBank data base reveals two important similarities. The first is to a sequence encoding a putative MIP homologue in the recently reported sequence of yeast chromosome III. The putative yeast protein is predicted to be 712 amino acids long and includes a putative 23-residue mitochondrial leader peptide also with a MPP processing site. It shows 47% similarity and 24% identity to rat MIP. The second similarity is to members of a subfamily of metallopeptidases that includes rat metalloendopeptidase EC 3.4.24.15 and two bacterial proteases, oligopeptidase A and dipeptidyl carboxypeptidase. A region of greater than 50% similarity over 400 residues between MIP and these proteins is centered around the sequence motif HEXXH, typical of zinc metallopeptidases.

Amino Acid Sequence↗
PubMed1992

Leukotriene A4 hydrolase: abrogation of the peptidase activity by mutation of glutamic acid-296.

The metal-binding motif in the sequence of leukotriene A4 (LTA4) (EC 3.3.2.6), a bifunctional zinc metalloenzyme, contains a glutamic acid that is conserved in several zinc hydrolases. To study its role for the two catalytic activities, Glu-296 in mouse leukotriene A4 hydrolase was replaced by a glutamine or alanine residue by site-directed mutagenesis. Wild-type and mutated cDNAs were expressed four or five times in Escherichia coli, and the resulting proteins were purified to apparent homogeneity. With respect to their epoxide hydrolase activities--i.e., the conversion of LTA4 into leukotriene B4--the mutated enzymes [Gln296]LTA4 hydrolase and [Ala296]LTA4 hydrolase exhibited specific activities of 1070 +/- 160 and 90 +/- 30 nmol of LTB4 per mg of protein per min (mean +/- SD; n = 4 or 5), respectively, corresponding to 150% and 15% of unmutated enzyme. In contrast, when the mutated proteins were assayed for peptidase activity toward alanine-4-nitroanilide, they were found to be virtually inactive (less than or equal to 0.2% of unmutated enzyme). To serve as a positive control, we also replaced Ser-298 with an alanine residue, which resulted in a protein ([Ala298]LTA4 hydrolase) with catalytic properties almost indistinguishable from the wild-type enzyme. Substitution of Glu-296 by glutamine or alanine was also carried out with human LTA4 hydrolase, and the mutated human enzymes displayed specific activities similar to the corresponding mouse proteins. Zinc analyses of the purified mouse and human proteins confirmed that the mutations did not significantly influence their zinc content. In conclusion, the results of the present study indicate a direct catalytic role for Glu-296 in the peptidase reaction of LTA4 hydrolase, where it presumably acts as a base to polarize water, whereas its function, if any, is apparently not essential in the epoxide hydrolase reaction.

Amino Acid Sequence↗