Transport characteristics of peptides and peptidomimetics: I. N-methylated peptides as substrates for the oligopeptide transporter and P-glycoprotein in the intestinal mucosa.

Peptides and peptidomimetics often exhibit poor oral bioavailability due to their metabolic instability and low permeation across the intestinal mucosa. N-Methylation has been used successfully in peptide-based drug design in an attempt to improve the metabolic stability of a peptide-based lead compound. However, the effect of N-methylation on the absorption of peptides through the intestinal mucosa is not well understood, particularly when transporters, i.e. the oligopeptide transporter (OPT) and P-glycoprotein (P-gp), modulate the passive diffusion of these types of molecules. To examine this, terminally free and terminally modified (N-acetylated and C-amidated) analogs of H-Ala-Phe-Ala-OH with N-methyl groups on either the Ala-Phe or Phe-Ala peptide bond were synthesized. Transport studies using Caco-2 cell monolayers, an in vitro model of the intestinal mucosa, showed that N-methylation of the Ala-Phe peptide bond of H-Ala-Phe-Ala-OH stabilized the molecule to protease degradation, and the resulting analog exhibited significant substrate activity for OPT. However, N-methylation of the Phe-Ala peptide bond of H-Ala-Phe-Ala-OH did not stabilize the molecule to protease degradation, and the substrate activity of the resulting molecule for OPT could not be determined. Interestingly, N-methylation of the Phe-Ala peptide bond of the terminally modified tripeptide Ac-Ala-Phe-Ala-NH2 decreased the substrate activity of the molecule for the efflux transporter P-gp. In contrast, N-methylation of the Ala-Phe peptide bond of the terminally modified tripeptide Ac-Ala-Phe-Ala-NH2 increased the substrate activity of the molecule for P-gp.     

Convergent synthesis of aminomethylene peptidomimetics

This protocol describes a convergent synthesis of reduced amide bond peptidomimetics using thioacid-terminated peptides and aziridine-containing peptide conjugates. This approach could be used to produce peptides and proteins with modified backbones. The peptide conjugates are made using readily available aziridine aldehydes. The convergent synthesis of peptidomimetics is demonstrated through the preparation of long and short peptide fragments with an aminomethylene group incorporated within them. This transformation is amenable to the synthesis of peptides with reduced amide bonds at cysteine and alanine. The procedure describes the preparation of each component used and highlights the ease of synthesis of aminomethylene peptidomimetics, and takes about 3 d to complete.     

Heterocyclic-based peptidomimetics

Summary Heterocyclic-based peptidomimetics possess properties that are dictated by the chemical and physical properties of the constituent heterocycle. Here we review two general classes of peptidomimetic, one in which the conformation of a peptide is confined into a particular geometry, and a second where chemical reactivity is masked and released in a controlled manner. We have used the aromatic heterocycles, pyrrole and tetrazole, to mimic the geometry ofcis-peptide bonds and peptide bond isosteres. A tetrazole-based hydroxyethylamine isostere, which mimics a bioactivecis-like geometry of JG365 as bound to HIV protease, has been developed and shown to provide the basis of conformationally constrained inhibitors of this enzyme. A number of other simple tetrazole-based molecular scaffolds are also discussed. The second section of this review discusses how the inherent reactivity of a hydroxymethylpyrrole can be masked with the introduction of an electron withdrawing group on nitrogen. The selective removal of this group then releases a highly reactive species. Compounds of this type have been shown to inhibit α-chymotrypsin.     

Heterocyclic-based peptidomimetics

Heterocyclic-based peptidomimetics possess properties that are dictated by the chemical and physical properties of the constituent heterocycle. Here we review two general classes of peptidomimetic, one in which the conformation of a peptide is confined into a particular geometry, and a second, where chemical reactivity is masked and released in a controlled manner. We have used the aromatic heterocycles, pyrrole and tetrazole, to mimic the geometry of cis-peptide bonds and peptide bond isosteres. A tetrazole-based hydroxyethylamine isostere, which mimics a bioactive cis-like geometry of JG365 as bound to HIV protease, has been developed and shown to provide the basis of conformationally constrained inhibitors of this enzyme. A number of other simple tetrazole-based molecular scaffolds are also discussed. The second section of this review discusses how the inherent reactivity of a hydroxymethylpyrrole can be masked with the introduction of an electron withdrawing group on nitrogen. The selective removal of this group then releases a highly reactive species. Compounds of this type have been shown to inhibit -chymotrypsin.     

Interactions of racemic mefloquine and its enantiomers with P-glycoprotein in an immortalised rat brain capillary endothelial cell line, GPNT

The brain distribution of the enantiomers of the antimalarial drug mefloquine is stereoselective according to the species. This stereoselectivity may be related to species-specific differences in the properties of some membrane-bound transport proteins, such as P-glycoprotein (P-gp). The interactions of racemic mefloquine and its individual enantiomers with the P-glycoprotein efflux transport system have been analysed in immortalised rat brain capillary endothelial GPNT cells. Parallel studies were carried out for comparison in human colon carcinoma Caco-2 cells. The cellular accumulation of the P-glycoprotein substrate, [(3)H]vinblastine, was significantly increased both in GPNT cells and in Caco-2 cells when treated with racemic mefloquine and the individual enantiomers. In GPNT cells, the (+)-stereoisomer of mefloquine was up to 8-fold more effective than its antipode in increasing cellular accumulation of [(3)H]vinblastine, while in Caco-2 cells, both enantiomers were equally effective. These results suggest that racemic mefloquine and its enantiomers are effective inhibitors of P-gp. Furthermore, a stereoselective P-glycoprotein inhibition is observed in rat cells but not in human cells. The efflux of [(14)C]mefloquine from GPNT cells was decreased when the cells were incubated with the P-gp modulators, verapamil, cyclosporin A or chlorpromazine, suggesting that MQ could be a P-gp substrate.     

Stereoselective regulations of P-glycoprotein by ginsenoside Rh2 epimers and the potential mechanisms from the view of pharmacokinetics

Chirality is an interesting topic and it is meaningful to explore the interactions between chiral small molecules and stereoselective biomacromolecules, with pre-clinical and clinical significances. We have previously demonstrated that 20(S)-ginsenoside Rh2 is an effective P-glycoprotein (P-gp) inhibitor in vitro and in vivo. Considering the stereochemistry of ginsenoside Rh2, in our present study, the regulatory effects of 20(R)-Rh2 on P-gp were assayed in vivo, and the differential regulations of P-gp by ginsenoside Rh2 epimers in vivo were compared and studied. Results showed that 20(S)-Rh2 enhanced the oral absorption of digoxin in rats in a dose-dependent manner; 20(R)-Rh2 at low dosage increased the oral absorption of digoxin, but this effect diminished with elevated dosage of 20(R)-Rh2. Further studies indicated stereoselective pharmacokinetic profiles and intestinal biotransformations of Rh2 epimers. In vitro studies showed that Rh2 epimers and their corresponding deglycosylation metabolites protopanaxadiol (Ppd) epimers all exhibited stereoselective regulations of P-gp. In conclusion, in view of the in vitro and in vivo dispositions of Rh2 and the regulations of P-gp by Rh2 and Ppd, it is suggested that the P-gp regulatory effect of Rh2 in vivo actually is a double actions of both Rh2 and Ppd, and the net effect is determined by the relative balance between Rh2 and Ppd with the same configuration. Our study provides new evidence of the chiral characteristics of P-gp, and is helpful to elucidate the stereoselective P-gp regulation mechanisms of ginsenoside Rh2 epimers in vivo from a pharmacokinetic view.     

Enhanced daunomycin accumulation in human intestinal Caco-2 cells from non-ionic food emulsifiers unrelated to the p-glycoprotein inhibitory mechanism

Some of the non-ionic surfactants used in pharmaceutical formulations inhibit P-glycoprotein (P-gp), the multi-drug transporter. The effect of such food emulsifiers as polyglycerol esters (PGE) and sugar esters (SE) of fatty acids on the P-gp activity was studied by using human intestinal Caco-2 cells. The cellular accumulation of [(3)H]-daunomycin, a P-gp substrate, was markedly enhanced by PGE and SE. This accumulation-enhancing activity varied among the emulsifiers, but was correlated with their surface activity. The uptake of soluble nutrients such as amino acids was only slightly reduced by PGE and SE. These results suggest that these emulsifiers specifically inhibited P-gp. When the basal-to-apical transport of daunomycin across the Caco-2 monolayers was measured, however, the emulsifiers did not decrease the efflux of daunomycin to the apical chamber. The enhanced accumulation of daunomycin would therefore not have been due to P-gp inhibition, but instead to the increased daunomycin permeability of cell membranes caused by the emulsifiers.     

Decisive structural determinants for the interaction of proline derivatives with the intestinal H+/peptide symporter.

To elucidate the decisive structural factors relevant for dipeptide-carrier interaction, the affinity of short amide and imide derivatives for the intestinal H+/peptide symporter (PEPT1) was investigated by measuring their ability to inhibit Gly-Sar transport in Caco-2 cells. Dipeptides with proline or alanine in the C-terminal position displayed affinity constants (Ki) of 0.15-1.2 mM and 0.08-9.5 mM, respectively. There was no clear relationship between hydrophobicity, size or ionization status of the N-terminal amino acid and the affinity of the dipeptides. However, analyzing the individual peptide bond conformations of Xaa-Pro dipeptides, a striking correlation between the cis/trans ratios (trans contents 24-70%) and the affinity constants was observed. After correcting the Ki values for the incompetent cis isomers, the Ki corr values of most dipeptides were in a small range of 0.1-0.16 mM. This result revealed the decisive role of peptide bond conformation even for a transport protein that is quite promiscuous in substrate translocation. When measuring affinity constants of Xaa-Pro and Xaa-Sar dipeptides, the cis/trans ratios cannot be ignored. Lower affinities of Lys-Pro, Arg-Pro and Pro-Pro indicate that additional molecular factors affect their binding at PEPT1. The Ki values obtained for the corresponding Xaa-Ala dipeptides support this conclusion. Potential substrates or inhibitors of peptide transport were found among Xaa-piperidides and Xaa-thiazolidides. Dipeptides with N-terminal proline displayed a very diverse affinity profile. However, in contrast to current knowledge, several Pro-Xaa dipeptides such as Pro-Leu, Pro-Tyr and Pro-Pro are recognized by PEPT1 with appreciable affinities. Binding seems mainly determined by the hydrophobicity of the C-terminal amino acid and the rigidity of the structure.     

Aza-β-amino acid containing peptidomimetics as cAMP-dependent protein kinase substrates

Peptidomimetic analogs of the peptide RRASVA, known as the “minimal substrate” of the catalytic subunit of the cAMP-dependent protein kinase (PKA), were synthesized by consecutive replacement of natural amino acids by their aza-β³ analogs. The peptidomimetics were tested as PKA substrates and the kinetic parameters of the phosphorylation reaction were determined. It was found that the interaction of these peptidomimetics with the enzyme active center was sensitive to the location of the backbone modification, while the maximal rate of the reaction was practically not affected by the structure of substrates. The pattern of molecular recognition of peptidomimetics was in agreement with the results of structure modeling and also with the results of computational docking study of peptide and peptidomimetic substrates with the active center of PKA. It was concluded that the specificity determining factors which govern substrate recognition by the enzyme should be grouped along the phosphorylatable substrate, and such clustering might open new perspectives for pharmacophore design of peptides and peptide-like ligands.     

The advantages of the Ussing chamber in drug absorption studies

By adding high concentrations of test drugs to an Ussing chamber with rat jejunum, we established a system that yields very high correlations between the rat absorption percentage and the membrane permeability, and that can accurately predict the absorption percentage for rats. An advantage of this technique is that, unlike the results obtained using Caco-2, the slope of the absorption/membrane-permeability curve is gentle, which facilitates a more exact prediction of the absorption percentage. In addition, the results obtained with this technique demonstrated that it could be used to evaluate the absorption percentage of drugs with an affinity for P-glycoprotein (P-gp), which cannot be assessed using Caco-2. This method also allows for cassette screening, which would facilitate evaluation of the contribution of P-gp to absorption in the small intestine. Cassette screening showed that absorption of fexofenadine was unaffected by combination with the P-gp substrate ketoconazole. Consistent with this finding, in vivo studies showed that ketoconazole did not affect the Fa Fg for fexofenadine, a pharmacokinetic parameter that reflects absorption and bioavailability in the small intestine. This confirms the usefulness of the Ussing chamber for cassette screening and also suggests that intestinal P-gp has a minimal contribution to drug absorption.     

Transport kinetics of leucine enkephalin across Caco-2 monolayers: quantitative analysis for contribution of enzymatic and transport barrier

In this study, we determined the activities of four aminopeptidases such as aminopeptidase B (APB), M (APM), N (APN) and dipeptidylpeptidase IV (DPP IV) in Caco-2 cells and compared with those in the rat intestinal mucosae. The activities of APB, APM and APN appeared to be highest in rat small intestinal mucosa, while DPP IV activity was much higher in Caco-2 cells than that in the rat intestinal mucosa. Next the inhibitory effects of various protease inhibitors were examined in Caco-2 homogenate. Three tested inhibitors, bacitracin, amastatin and puromycin, effectively inhibited the activities of APM, APN and DPP IV except for APB. Further, we quantitatively evaluated the permeation and degradation properties of leucine enkephalin (Leu-Enk) in the presence or absence of inhibitors in Caco-2 monolayer system. Leu-Enk had a high degradation clearance (CLd) and a low permeation clearance (CLp) in Caco-2 monolayers. This finding indicates that the very rapid degradation of Leu-Enk on the apical side of Caco-2 monolayers was due to aminopeptidases. However, these protease inhibitors besides sodium glycocholate were able to reduce the CLd values markedly, thereby increasing the permeation amount of Leu-Enk across Caco-2 monolayers. In particular, amastatin significantly decreased the CLd value and increased the CLp value. This enhanced CLp value was further increased by the coadministration with an absorption enhancer, EDTA or laurylmaltoside. These findings are relevant to the oral administration of peptide drugs and to developing an efficient oral delivery system.     

Carbonic anhydrase inhibitors: transepithelial transport of thioureido sulfonamide inhibitors of the cancer-associated isozyme IX is dependent on efflux transporters

Sulfonamides and their derivatives inhibit the catalytic activity of carbonic anhydrases (CA, EC 4.2.1.1). Isozyme IX (CA IX) is a transmembrane isoform with the active site oriented toward the extracellular space. CA IX was recently shown to be a drug target, and it is highly overexpressed in hypoxic tumors with limited distribution in normal tissues. The present report deals with the drug design, synthesis, and biological investigation of a group of thioureido sulfonamides, which have been obtained by reaction of isothiocyanate-substituted aromatic sulfonamides with amines. These compounds have potent inhibitory properties against CA IX with K(I) values in the range of 10-37 nM and P(app)values > 0.34 x 10(-6) cm/s for the absorptive transepithelial transport in Caco-2 cells. In Caco-2 cells, one of these compounds (A6) was shown to be a substrate for efflux transporters such as P-glycoprotein (P-gp). P-gp activity is not likely to be rate-limiting for intestinal absorption, but might be useful when targeting hypoxic tumors expressing both P-gp and CA IX.     

Relationship between structure and permeability of dipeptide derivatives containing tryptophan and related compounds across human intestinal epithelial (Caco-2) cells

The permeability of dipeptide derivatives containing tryptophans and indole derivatives through Caco-2 cells was used as an in vitro intestinal absorption model in order to clarify structural factors which influence their intestinal epithelial permeation and metabolism. Most peptide derivatives were hydrolysed not only by the cytosolic enzymes in Caco-2 cells during permeation but also by enzymes released to the apical solution before cell permeation. The N-terminal blocked dipeptides were more resistant to hydrolases expressed in the Caco-2 cells and indole derivatives were not entirely degraded. Based on compound concentration dependency and comparison of permeability coefficients in apical-to-basolateral and basolateral-to-apical directions, the main absorption mechanism of compounds were determined. Compounds were then classified into three groups; (1) passively transported compounds, (2) actively transported compounds and (3) compounds excreted by P-glycoprotein.     

Poly-lysine peptidomimetics having potent antimicrobial activity without hemolytic activity

Diversity of sequence and structure in naturally occurring antimicrobial peptides (AMPs) limits their intensive structure–activity relationship (SAR) study. In contrast, peptidomimetics have several advantages compared to naturally occurring peptide in terms of simple structure, convenient to analog synthesis, rapid elucidation of optimal physiochemical properties and low-cost synthesis. In search of short antimicrobial peptides using peptidomimetics, which provide facile access to identify the key factors involving in the destruction of pathogens through SAR study, a series of simple and short peptidomimetics consisting of multi-Lys residues and lipophilic moiety have been prepared and found to be active against several Gram-negative and Gram-positive bacteria containing methicillin-resistant Staphylococcus aureus (MRSA) without hemolytic activity. Based on the SAR studies, we found that hydrophobicity, +5 charges of multiple Lys residues, hydrocarbon tail lengths and cyclohexyl group were crucial for antimicrobial activity. Furthermore, membrane depolarization, dye leakage, inner membrane permeability and time-killing kinetics revealed that bacterial-killing mechanism of our peptidomimetics is different from the membrane-targeting AMPs (e. g. melittin and SMAP-29) and implied our peptidomimetics might kill bacteria via the intracellular-targeting mechanism as done by buforin-2.     

A bitter melon extract inhibits the P-glycoprotein activity in intestinal Caco-2 cells: monoglyceride as an active compound

P-glycoprotein (P-gp) is a 170 kDa membrane protein that belongs to the ATP-binding cassette (ABC) transporter superfamily. In normal tissues, P-gp functions as an ATP-dependent efflux pump that excretes highly hydrophobic xenobiotic compounds, playing an important role in protecting the cells/tissues from xenobiotics. In the present study, chemical substances that could directly modulate the intestinal P-gp activity were searched in vegetables and fruits. By using human intestinal epithelial Caco-2 cells as a model of the small intestinal cells, we observed that a bitter melon fraction extracted from 40% methanol showed the greatest increase of the rhodamine-123 accumulation by Caco-2 cells. Inhibitory compounds in the bitter melon fraction were then isolated by HPLC using Pegasil C4 and Pegasil ODS columns. The HPLC fraction having the highest activity was analyzed by (1)H-NMR and FAB-MS, and the active compound was identified as 1-monopalmitin. It is interesting that certain types of monoglyceride might be involved in the drug bioavailability by specifically inhibiting the efflux mediated by P-gp.     

p-Nitrophenoxycarbonyl derivatives of Boc-protected diaminoalkanes in the synthesis of enkephalin peptidomimetics.

The synthesis of p-nitrophenoxycarbonyl derivatives of 1-Boc-1,2-diaminoethane, 1-Boc-1,3-diaminopropane and 1-Boc-1,4-diaminobutane is described. These derivatives were used to synthesize five peptidomimetics, analogues of enkephalin, containing alkylurea units inside the peptide chain and at the C-terminal. All syntheses were carried out in solid phase on MBHA resin. Peptidomimetics with alkylurea units inserted into the peptide chain were synthesized using the standard method employing the Boc-strategy, with TFA deprotection and HF cleavage. The analogue containing a C-terminal alkylurea unit was synthesized using the Boc-strategy, with HCl/dioxane deprotection and TFA cleavage. All of the analogues were examined for opioid activity in GPI and MVD assays. The activity of the analogue containing a C-terminal alkylurea unit was comparable to that of [Leu5]-enkephalin, while the other analogues were less active.     

Challenges in the design of insulin and relaxin/insulin-like peptide mimetics

Peptidomimetics are designed to overcome the poor pharmacokinetics and pharmacodynamics associated with the native peptide or protein on which they are based. The design of peptidomimetics starts from developing structure-activity relationships of the native ligand-target pair that identify the key residues that are responsible for the biological effect of the native peptide or protein. Then minimization of the structure and introduction of constraints are applied to create the core active site that can interact with the target with high affinity and selectivity. Developing peptidomimetics is not trivial and often challenging, particularly when peptides’ interaction mechanism with their target is complex. This review will discuss the challenges of developing peptidomimetics of therapeutically important insulin superfamily peptides, particularly those which have two chains (A and B) and three disulfide bonds and whose receptors are known, namely insulin, H2 relaxin, H3 relaxin, INSL3 and INSL5.     

Upregulation of P-glycoprotein by probiotics in intestinal epithelial cells and in the dextran sulfate sodium model of colitis in mice

P-glycoprotein (P-gp) mediates efflux of xenobiotics and bacterial toxins from the intestinal mucosa into the lumen. Dysregulation of P-gp has been implicated in inflammatory bowel disease. Certain probiotics have been shown to be effective in treating inflammatory bowel disease. However, direct effects of probiotics on P-gp are not known. Current studies examined the effects of Lactobacilli on P-gp function and expression in intestinal epithelial cells. Caco-2 monolayers and a mouse model of dextran sulfate sodium-induced colitis were utilized. P-gp activity was measured as verapamil-sensitive [(3)H]digoxin transepithelial flux. Multidrug resistant 1 (MDR1)/P-gp expression was measured by real-time quantitative PCR and immunoblotting. Culture supernatant (CS; 1:10 or 1:50, 24 h) of Lactobacillus acidophilus or Lactobacillus rhamnosus treatment of differentiated Caco-2 monolayers (21 days postplating) increased (～3-fold) MDR1/P-gp mRNA and protein levels. L. acidophilus or L. rhamnosus CS stimulated P-gp activity (～2-fold, P < 0.05) via phosphoinositide 3-kinase and ERK1/2 MAPK pathways. In mice, L. acidophilus or L. rhamnosus treatment (3 × 10(9) colony-forming units) increased mdr1a/P-gp mRNA and protein expression in the ileum and colon (2- to 3-fold). In the dextran sulfate sodium (DSS)-induced colitis model (3% DSS in drinking water for 7 days), the degree of colitis as judged by histological damage and myeloperoxidase activity was reduced by L. acidophilus. L. acidophilus treatment to DSS-treated mice blocked the reduced expression of mdr1a/P-gp mRNA and protein in the distal colon. These findings suggest that Lactobacilli or their soluble factors stimulate P-gp expression and function under normal and inflammatory conditions. These data provide insights into a novel mechanism involving P-gp upregulation in beneficial effects of probiotics in intestinal inflammatory disorders.     

Inhibitory effect of phospholipids on P-glycoprotein: Cellular studies in Caco-2, MDCKII mdr1 and MDCKII wildtype cells and P-gp ATPase activity measurements

Phospholipids are widely used excipients for pharmaceutical formulations, such as for preparing biphasic systems or to solubilize or encapsulate poorly soluble drugs. The present study investigates a new property of this class of substance: its ability to inhibit the efflux transporter Pglycoprotein (P-gp). P-gp is expressed in the intestinal epithelium, thereby significantly impairing the systemic absorption of various pharmaceutically active substances. The phospholipid screening performed in this study involved derivatives with different headgroups and fatty acid residues and a number of experimental parameters. For in vitro studies we carried out transport experiments and calcein accumulation assays in Caco-2- and MDCKII mdr1 and wildtype cell lines. The three compounds which displayed significant P-gp inhibition in both assays and in Caco-2 as well as in MDCKII mdr1, consisted of phosphatidylcholine (PC) and either two saturated fatty acid residues of eight (8:0 PC) or ten carbon atoms (10:0 PC), or of two unsaturated docosahexaeonic acid residues (cis-22:6 PC).Supported by P-gp ATPase activity measurements, 8:0 and 10:0 PC were assumed to function as direct P-gp inhibitors interacting with the transporter probably in their monomeric state, whereas a different, as yet unknown mechanism of action applied for cis-22:6 PC.Because of their proven ability to significantly inhibit P-gp in vitro, these phospholipids shall further be elucidated in vivo, whether they may truly serve to increase the bioavailability of orally applied drugs with a P-gp substrate character.     

Degradation of oxidized insulin B chain by the multiproteinase complex macropain (proteasome)

The peptides generated from the degradation of the oxidized B chain of bovine insulin by the multiproteinase complex macropain (proteasome) have been analyzed by reverse-phase peptide mapping and identified by N-terminal amino acid sequencing and composition analysis. Six of the 29 peptide bonds in the insulin B chain were found to be rapidly cleaved by macropain. The catalytic center that cleaves the Gln4-His5 bond could be distinguished from the center or centers that cleave the other preferred bonds by its specific susceptibility to inhibition by leupeptin, antipain, chymostatin, and pentamidine, suggesting that macropain utilizes at least two distinct catalytic centers for the degradation of this model polypeptide. The same effectors simultaneously enhance the rate of cleavage at the other susceptible sites in insulin B. The quantitative characteristics of this effect indicate that different catalytic centers of the complex may be functionally coupled, possibly by an allosteric mechanism or possibly by a mechanism in which binding to the catalytic centers is preceded by a rate-limiting binding of the substrate to a site or sites on the enzyme distinct from the catalytic centers. The kinetics of insulin B chain degradation indicate that macropain can catalyze sequential hydrolysis of peptide bonds in a single substrate molecule via a reaction pathway that involves channeling of peptide intermediates between different catalytic centers within the multienzyme complex. This capacity for channeling may confer potential physiological advantages of increasing the efficiency of amino acid recycling and reducing the pool sizes of peptide intermediates that are generated during the degradation of polypeptides in the intracellular milieu.