Cleavage of Pro-X and Glu-X Bonds Catalyzed by the Branched Chain Amino Acid Preferring Activity of...

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 334, No. 1, October 1, pp. 113–120, 1996Article No. 0436

Cleavage of Pro-X and Glu-X Bonds Catalyzed by theBranched Chain Amino Acid Preferring Activityof the Bovine Pituitary Multicatalytic ProteinaseComplex (20S Proteasome)1

Christopher Cardozo,* Wei-Er Chen, and Sherwin Wilk2

Departments of Pharmacology and *Medicine, Mount Sinai School of Medicine of the City University of New York,New York, New York 10029

Received February 12, 1996, and in revised form June 10, 1996

The multicatalytic proteinase complex (MPC),3 alsoThe multicatalytic proteinase complex or 20S pro- known as the 20S proteasome, is a major extralyso-

teasome is involved in the extralysosomal degradation somal proteolytic molecule [for a recent compendiumof both long- and short-lived proteins. The eukaryotic of publications see (1)]. This proteinase of molecularenzyme is composed of 14 nonidentical subunits ar- weight of about 700,000 is composed of four rings ofranged as a complex dimer of the composition (a7b7)2. seven subunits each (2). The subunits fall into twoRecent studies identify N-terminal threonines present structural families termed a and b, with the a-subunitson some b-subunits as the active-site residues. It has occupying the outer rings (2, 3). As its name indicates,been proposed that the molecule contains three or four MPC contains multiple independent active sites cata-proteolytically active subunits [Seemuller et al., Sci- lyzing peptide hydrolysis (4). The crystal structure ofence 268, 579–582 (1995)]. Studies with synthetic sub- the enzyme from Thermoplasma acidophilum has re-strates, activators, and inhibitors, however, have iden- cently has been solved (5). Cocrystallization with a pep-tified at least five distinct catalytic activities. To fur- tidyl aldehyde inhibitor localized the active sites to thether characterize the specificity of the previously

central cavity of the complex at the amino termini ofdefined ‘‘peptidyl glutamyl peptide bond hydrolyzingthe b-subunits. Site-directed mutagenesis identified anactivity,’’ N-benzyloxycarbonyl-Leucyl-Leucyl-Gluta-N-terminal threonine as the active-site residue (6).mal was synthesized as a potential inhibitor. Surpris-This was further confirmed by covalent attachment ofingly, this aldehyde most potently inhibited thethe Streptomyces metabolite lactacystin to the N-termi-‘‘branched chain amino acid preferring activity’’nal threonine (7).(BrAAP). To further explore BrAAP specificity, novel

MPC also serves as the catalytic core of a much largersubstrates containing internal prolyl and glutamylmultiprotein complex commonly referred to as the 26Sresidues were synthesized. Their use established thatproteasome or 26S protease. The combination of MPCthe BrAAP activity catalyzed both a postproline and awith two 19S regulatory complexes to form the 26Spostglutamate cleavage and therefore has a broader

specificity than previously recognized. These results proteasome results in the formation of a molecule thathelp explain earlier observations on treatment of the can recognize and degrade ubiquitin–protein conju-multicatalytic proteinase complex with 3,4-dichloroi- gates in an ATP-requiring reaction (see 8 for a review).socoumarin. This reagent activates both the BrAAP ac- There is evidence that MPC either by itself or as parttivity and the degradation of b-casein and inhibits the of the 26S proteasome participates in a wide range ofother catalytic activities. q 1996 Academic Press, Inc.

3 Abbreviations used: MPC, multicatalytic proteinase complex;BrAAP, branched chain amino acid preferring, SnAAP, small neutral

1 This work was supported by Grant NS 29936 to S.W., Grant KO8 amino acid preferring; PGP, peptidylglutamyl peptide bond hydrolyz-ing; ONS, N-hydroxysuccinimide active ester; THF, tetrahydrofuran;HL02835 to C.C. and by Grant DK25377.

2 To whom correspondence should be addressed at Box 1215, De- DMF, dimethyl formamide; O-tBu, tert-butyl ester; Cbz, N-benyzlox-ycarbonyl; pAB, para-aminobenzoate; Boc, N-t-butoxycarbonyl; TFA,partment of Pharmacology, Mount Sinai School of Medicine, 1 Gus-

tave L. Levy Place, New York, NY 10029. Fax: (212) 831-0114. trifluoroacetic acid; pNA, p-nitroanilide; DCI, 3,4-dichloroisocoum-arin; DMSO, dimethyl sulfoxide; SC, semicarbazone.E-mail: [email protected].

1130003-9861/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

AID ARCH 9625 / 6b21$$$101 08-28-96 12:53:06 arcal AP: Archives

114 CARDOZO, CHEN, AND WILK

fundamental cellular processes. In addition to its role MATERIALS AND METHODSin general extralysosomal protein turnover, MPC is be- MPC was isolated in an apparently homogeneous form from bovinelieved to catalyze the generation of antigens from intra- pituitaries as previously described (23). Membrane alanine amino-

peptidase (EC 3.4.11.2) was purified from rabbit kidney cortex tocellular proteins for presentation by the MHC-1 path-apparent homogeneity as described (24). N-blocked amino acid inter-way (9, 10) and to catalyze the degradation of short-mediates were purchased from Bachem Bioscience Inc. (King of Prus-lived molecules such as ornithine decarboxylase (11), sia, PA). Silica gel, Merck grade 9385, and all other chemicals were

oncoproteins (12, 13), transcription factors (14), and the obtained from Aldrich Chemical Co. (St. Louis, MO). The inhibitorsCbz-Ile-Glu(O-tBu)-Ala-Leucinal, Cbz-Gly-Pro-Phe-Leucinal, Cbz-cyclin-dependent kinase inhibitor p27 (15). The recentPro-Prolinal, and 9-fluorenylmethoxycarbonyl-Pro-Pyrrololidine-2-development of active-site-specific inhibitors has pro-nitrile were synthesized as previously described (25–28).vided a means for exploring the role of MPC in cellular

function. Inhibitors of the ‘‘chymotrypsin-like activity’’Amino Acid Analysisof MPC impair the turnover of both long-lived and

Peptides were hydrolyzed in evacuated tubes with HCl:propionicshort-lived proteins, inhibit antigen presentation byacid at 1457C for 30 min. The contents were then evaporated tothe MHC-1 pathway (16), and block the activation ofdryness under a stream of nitrogen. Amino acid composition was

the transcription factor NF-kB (17, 18). When a neu- analyzed after derivatization with o-phthaldialdehyde (29) as pre-ronal cell line was exposed to a specific inhibitor of the viously described (30).chymotrypsin-like activity, an accumulation of ubiqui-tin–protein conjugates was observed (19). HPLC Analysis

MPC was originally defined as a proteinase con- HPLC was performed on a Waters 600E liquid chromatographtaining three active sites capable of cleaving peptide equipped with a Vydac C4 protein column protected by a Vydac C4

5-mm guard column. The column was equilibrated with 15% acetoni-bonds after hydrophobic, basic, and acidic amino acidtrile, 0.05% TFA at a flow rate of 1 ml/min. Elution was carried outresidues (4). These activities were termed chymotryp-by linearly increasing the acetonitrile concentration to 70% over asin-like, trypsin-like, and peptidylglutamyl peptideperiod of 34 min. The products were monitored by measurement of

bond hydrolyzing (PGP), respectively. Evidence from absorbance at 210 nm.studies of substrate hydrolysis and the effect of inhibi-tors has led Orlowski et al. (20) to expand the number Mass Spectrometryof independent active sites from the original three to

Ion spray mass spectrometric analysis was performed by Peptido-five. The newer activities were termed ‘‘branched chain Genic Research (Livermore, CA).amino acid preferring’’ or BrAAP and ‘‘small neutralamino acid preferring’’ or SnAAP. In addition Pereira Measurement of Enzymatic Activitieset al. (21) defined a distinct catalytic activity responsi-

The chymotrypsin-like activity was measured with the substratesble for the initial degradation of b-casein termed ‘‘ca- Cbz-Gly-Gly-Leu-p-nitroanilide (Cbz-Gly-Gly-Leu-pNA) and Cbz-seinolytic.’’ A catalytic activity optimally active at Glu(O-tBu)-Ala-Leu-pNA. The trypsin-like and peptidylglutamyl

peptide bond hydrolyzing activities were measured with the sub-acidic pH and termed ‘‘acidic chymotrypsin-like’’ hasstrates Cbz-D-Ala-Leu-Arg-2-naphthylamide (Cbz-D-Ala-Leu-Arg-also been described (22). The exact number of proteolyt-NA) and Cbz-Leu-Leu-Glu-NA, respectively. The released chromogenically active subunits contained within MPC is not was determined by a colorimetric procedure essentially as described

known, but three of the seven eukaryotic b-subunits (4). The acidic chymotrypsin-like activity was measured with thelack N-terminal threonine residues. Moreover, subunit substrate Boc-Val-Glu-Ala-Leu-NA at pH 5.0 as described (22). The

BrAAP and SnAAP activities were measured in the presence of ex-HsN3, although containing an N-terminal threoninecess membrane alanyl aminopeptidase as described (20). The hydro-residue, lacks a conserved proton acceptor–donor. Thislysis of other substrates was determined spectrophotometrically andhas led to the proposal that at least three of the seven by HPLC analysis. Substrates were dissolved in DMSO. Incubation

b-type subunits are proteolytically inactive (6). Since mixtures contained 3.5 mg MPC, 4 ml substrate, and Tris–HCl (0.05the detailed subsite specificities of each of the defined M, pH 7.5) in a final volume of 100 ml. One unit of enzymatic activity

is defined as the amount of enzyme catalyzing the degradation of 1activities remain unknown, the possibility that one ormmol of substrate/h.more are identical must be considered. For example, it

has been proposed that the subunits responsible forMeasurement of Enzyme Inhibitioncasein hydrolysis and for the ‘‘BrAAP activity’’ are iden-

Enzymatic reactions were initiated by addition of substrate to solu-tical (20). The study reported here was prompted bytions containing enzyme plus inhibitor (dissolved in DMSO). Reac-the lack of a specific inhibitor of the PGP activity. Totion mixtures were incubated at 377C. In some cases, enzyme wasthis end we designed and synthesized the peptidyl alde- preincubated with inhibitor for 15 min at 377C prior to addition of

hyde N-benzyloxycarbonyl-Leucyl-Leucyl-Glutamal substrate. The volume of inhibitor added was 1.5 ml. Controls (noinhibitor) contained 1.5 ml DMSO.(Cbz-Leu-Leu-Glutamal). These studies document that

this aldehyde more potently inhibits the BrAAP activ-Synthesis of Cbz-Gly-Pro-Ala-Glu-Gly-pAB and Cbz-ity than the PGP activity. They further reveal that the

Gly-Pro-Glu-Leu-Gly-pABBrAAP activity can in some cases catalyze postprolineand postglutamate cleavages, thus expanding its N-Hydroxysuccinimide esters (ONS) of N-Butoxycarbonyl (Boc)-

amino acids were prepared by the method of Anderson et al. (31).known specificity.


115PRO-X AND GLU-X BOND CLEAVAGE BY MULTICATALYTIC PROTEINASE

Cbz-Gly-Pro-ONS and Cbz-Gly-Pro-Ala-ONS were synthesized es- column developed with 97.5% CHCl3/2.5% ethanol. Evaporationyielded 587 mg (2.1 mmol) product which gave a single peak uponsentially as described previously (26). TFA salts of Gly-p-aminoben-

zoate (Gly-pAB) and Leu-Gly-pAB were synthesized by stepwise analysis by HPLC.elongation from the amino terminus of pAB as described previously C. Cbz-Glu(O-tBu)-semicarbazone (Cbz-Glu(O-tBu)-SC) (VII). A(20). Reactions were followed by HPLC. solution of (VI) in 10 ml 70% ethanol was cooled to 07C. A 10% molar

excess each of sodium acetate and semicarbazide.HCl was added toA. Glu-Leu-Gly-pAB.TFA (I). Leu-Gly-pAB.TFA (2 mmol) wasthe stirred solution and the reaction allowed to proceed for 18 h atdissolved in 12 ml tetrahydrofuran (THF) / 2.5 ml dimethyl for-47C. The mixture was then filtered, the filtrate evaporated to dryness,mamide (DMF); 2 mmol Boc-Glu(O-tBu)-ONS and 1.05 equivalentsand the residue dissolved in CHCl3. The CHCl3 solution was sequen-triethylamine were then added. The mixture was stirred for 24 h attially washed with saturated NaHCO3, H2O, 10% citrate, and H2Oroom temperature, and the solvent was then removed by evaporation.and dried over Na2SO4. The residue after evaporation of the CHCl3The residue was dissolved in ethyl acetate and washed with 0.5 M

was purified by silica gel chromatography as described for (B). Evapo-HCl. The ethyl acetate extract was dried over sodium sulfate, filtered,ration of solvent yielded 510 mg (1.3 mmol) white crystalline mate-and evaporated to yield a white foam. Reaction with TFA for 20 minrial, which gave a single peak when analyzed by HPLC.at room temperature followed by evaporation and trituration with

ethyl ether yielded Glu-Leu-Gly-pAB.TFA (I). D. Glu(O-tBu)-SC (VIII). To a solution of (VII) in 10 ml absoluteethanol, 140 mg 10% Pd/C was added. After stirring for 4 h, anB. Cbz-Gly-Pro-Glu-Leu-Gly-pAB (II). To 1 mmol (I) dissolved inadditional 70 mg Pd/C was added and stirring was allowed to proceed10 ml THF was added 1.05 mmol Z-Gly-Pro-ONS and 1.05 equiva-for an additional 2 h. After filtration and evaporation, 340 mg (0.94lents triethylamine. After stirring at room temperature for 24 h themmol) of a white sticky solid was obtained which gave a single peaksolvent was evaporated, the residue dissolved in 0.5 M Na2CO3, andby HPLC.the product precipitated by acidification with 0.5 M HCl. The precipi-

tate was dissolved in ethyl acetate and the solution dried over Na2SO4 E. Cbz-Leu-Leu-Glu(O-tBu)-SC (IX). An equimolar amount ofand evaporated to yield (II) as a white foam. Analysis by mass spec- (VIII) in 3 ml THF was added to a solution of 360 mg (0.76 mmol)troscopy yielded a value for M / 1 of 725.3. Z-Leu-Leu-ONS in 10 ml THF. The solution was stirred at room

temperature for 3 h and the solvent then removed in vacuo. TheC. Glu-Gly-pAB.TFA (III). Gly-pAB.TFA (1 mmol) was dissolvedresidue was dissolved in CHCl3 and the solution washed sequentiallyin 5 ml THF / 1 ml DMF. To the solution was added 1 mmol Boc-with saturating amounts of NaHCO3, H2O, 10% citrate, and H2O.Glu(O-tBu)-ONS and 1.05 mmol triethylamine. After stirring for 24After drying over Na2SO4 and evaporation of solvent, 420 mg (0.68h at room temperature, the solvent was removed by evaporation.mmol) white crystalline material was obtained.The resulting solid was washed with 0.5 M HCl and water and then

dried. The product was treated with TFA for 20 min at room tempera- F. Cbz-Leu-Leu-Glu-SC (X). The O-tBu protecting group was re-ture, and the residue after evaporation was triturated with ether to moved by a 1-h treatment of (IX) with 2.5 ml 25% TFA in CH2Cl2

yield Glu-Gly-pAB.TFA as a white solid. immediately followed by addition of 2 ml H2O. After evaporation,ethyl ether was added to the residue and 215 mg white crystallineD. Cbz-Gly-Pro-Ala-Glu-Gly-pAB (IV). To 0. 4 mmol (III) in 2 mlmaterial (0.38 mmol) was obtained.THF, 0.404 mmol Z-Gly-Pro-Ala-ONS and 0.404 mmol triethylamine

were added. Upon stirring at room temperature, the product was G. Cbz-Leu-Leu-Glutamal (XI). To a solution of (X) in 10 mlcrystallized out of the reaction mixture. Mass spectroscopic analysis methanol, 2 ml 37% formaldehyde and 2 ml glacial acetic acid weregave a value for M / 1 of 683.3. added. The mixture was stirred for 18 h at room temperature. After

removal of the methanol by evaporation, 50 ml H2O was added tothe residue, and the product was extracted into CHCl3. After washing

Synthesis of Cbz-Leu-Leu-Glutamal with 0.3 N NaHSO4 and brine and drying over Na2SO4, the solventwas removed under N2. Ethyl ether was added to obtain 125 mgCbz-Leu-Leu-Glutamal was synthesized by a modification of the(0.25 mmol) white crystalline material.procedure of Graybill et al. (32) for the preparation of peptidyl aspar-

The aldehyde content was 97% of the theoretically predicted value.tyl aldehydes.Minor impurities revealed by HPLC were removed by preparative

A. Cbz-Glu(O-tBu)N,O-dimethylhydroxylamine (V). A solution of HPLC of aliquots of the product. Analysis by mass spectroscopy gaveCbz-Glu(O-tBu)-OH (3.1 mmol; 1.04 g) in 15 ml dichloromethane was a value for M / 1 of 492.3.cooled to0207C under nitrogen. N-Methylmorpholine (6.6 mmol) wasadded and the solution stirred for 5 min. This was followed by the

RESULTSaddition of 3.3 mmol isobutyl chloroformate in two equal portionsand 3.5 mmol N,O-dimethylhydroxylamine–HCl. The mixture was Effect of Cbz-Leu-Leu-Glutamal on Catalyticstirred for 5 min at 0207C and then at room temperature for 4 h.

Activities of MPCThe mixture was then filtered, concentrated in vacuo, and washedin a separatory funnel with H2O. The aqueous phase was washed Cbz-Leu-Leu-Glutamal was synthesized by a modi-three times with ethyl ether. The organic extracts were combined

fication of the procedure of Graybill et al. (32) for theand dried over Na2SO4. Evaporation yielded 1.24 g clear oil whichsynthesis of peptidyl aspartyl aldehydes. This com-was dissolved in anhydrous toluene and azeotroped.pound was designed as a specific inhibitor of the PGPB. Cbz-Glu(O-tBu)-CHO (VI). After azeotroping, (V) was trans-

ferred to a three-necked flask equipped with a drying tube, thermom- activity of MPC. However Cbz-Leu-Leu-Glutamal waseter, and septum connected to a N2 inlet. Ethyl ether (20 ml) was found to have only moderate potency as a PGP inhibitoradded and the solution cooled to 07C. Next, 4 ml of 1.0 M LiAlH4 in (IC50 Å 50 mM). When tested at a concentration of 160ethyl ether was added dropwise by a syringe to the stirred solution.

mM, inhibition only reached 71% (Fig. 1). Inhibition wasThe temperature was maintained below 57C. The solution was stirredsimilar for basal or for SDS-stimulated PGP activityfor 1 h at 07C and the reaction then quenched with 12 ml 0.3 M

NaHSO4. The mixture was transferred to a separatory funnel con- (Fig. 1). Inhibition was not enhanced by preincubationtaining 30 ml ethyl ether. Additional 0.2 M NaHSO4 was added to of Cbz-Leu-Leu-Glutamal with enzyme (data notclarify the layers. The product was extracted into ethyl ether, the shown).aqueous phase was reextracted with ethyl ether, and the ether

When tested at a concentration of 400 mM, Cbz-Leu-phases were combined and then dried over Na2SO4. The crude prod-uct obtained after removal of solvent was purified on a silica gel Leu-Glutamal did not inhibit the trypsin-like activity



nal prolyl and glutamyl residues were synthesized.Their design was based in part on the observation thatcleavage by the BrAAP and SnAAP activities is facili-tated by the presence of a prolyl residue in the P3 posi-tion (20). To facilitate solubility, these substrates con-tained the pAB group as chromogen. Hydrolysis of bothsubstrates by MPC was totally dependent on the pres-ence of alanyl aminopeptidase in the incubation mix-ture. In control experiments alanyl aminopeptidase byitself had no effect on substrate hydrolysis (not shown).These experiments demonstrated that cleavage oc-curred at an internal site and not at the glycyl–pABbond. Cbz-Leu-Leu-Glutamal strongly inhibited thehydrolysis of both substrates. Similar IC50 values werefound (12 mM for Cbz-Gly-Pro-Glu-Leu-Gly-pAB and 15mM for Cbz-Gly-Pro-Ala-Glu-Gly-pAB) (Fig. 3).

Determination of the Site of Peptide Bond Cleavage inFIG. 1. Inhibition of the hydrolysis of Cbz-Leu-Leu-Glu-NA (pepti- the Substrates Cbz-Gly-Pro-Glu-Leu-Gly-pAB anddylglutamyl peptide hydrolyzing activity){SDS (0.04%, final concen-

Cbz-Gly-Pro-Ala-Glu-Gly-pABtration) by Cbz-Leu-Leu-Glutamal. Inhibition was determined with-out preincubation. Substrate concentration, 0.4 mM. 0SDS (open Since internal peptide bond cleavage of the new sub-circles), /SDS (solid circles). Basal PGP specific activity, 2 U/mg.

strates was established, HPLC coupled with amino acidSDS-stimulated PGP activity, 30.8 U/mg.analysis of products was used to establish the site ofpeptide bond cleavage. Although both substrates con-

and only slightly inhibited the chymotrypsin-like activ- tain multiple potential scissile peptide bonds, HPLCity. The extent of inhibition was dependent on the sub- analysis of reaction mixtures revealed only a singlestrate used to measure the chymotrypsin-like activity. cleavage site in each (Fig. 4A). The products were man-Thus, 400 mM Cbz-Leu-Leu-Glutamal inhibited the hy- ually collected, the solvent was evaporated under adrolysis of Cbz-Glu(O-tBu)Ala-Leu-pNA by 57% and stream of nitrogen, and the residue was subjected toCbz-Gly-Gly-Leu-pNA by 11%. The acidic chymotryp- analysis for amino acids and for pAB. Results indicatedsin-like activity was also minimally affected (29% inhi- that Cbz-Gly-Pro-Ala-Glu-Gly-pAB was cleaved at thebition at an inhibitor concentration of 200 mM). Morerecently Orlowski et al. (20) described the presence oftwo additional catalytic activities in pituitary MPC.These have been termed BrAAP and SnAAP. Surpris-ingly Cbz-Leu-Leu-Glutamal inhibited the BrAAP ac-tivity more potently than the PGP activity (IC50 Å 12.5mM) (Fig. 2). Inhibitions of the SnAAP activity (IC50 Å65 mM) and PGP activity were similar (Fig. 2). Sincemeasurement of the BrAAP and SnAAP activities re-quires the action of excess alanyl aminopeptidase toliberate the chromogen, it was first necessary to demon-strate that the aminopeptidase was unaffected by Cbz-Leu-Leu-Glutamal. Indeed, the aldehyde tested at 400mM had no effect on alanyl aminopeptidase.

Inhibition of the BrAAP and SnAAP activities byCbz-Leu-Leu-Glutamal suggested that they both mayunder certain circumstances cleave peptide bonds aftera glutamyl residue. To further explore this possibility,potential BrAAP and SnAAP substrates containing in-ternal glutamyl residues were synthesized.

Effect of Cbz-Leu-Leu-Glutamal on the Hydrolysis of FIG. 2. Inhibition of the hydrolysis of Cbz-Gly-Pro-Ala-Leu-Gly-pAB (BrAAP component) (open circles) and Cbz-Gly-Pro-Ala-Gly-Cbz-Gly-Pro-Glu-Leu-Gly-pAB and Cbz-Gly-Pro-Gly-pAB (SnAAP component) (closed circles) by Cbz-Leu-Leu-Gluta-Ala-Glu-Gly-pABmal. Inhibition was determined without preincubation. Substrate

Two substrates, Cbz-Gly-Pro-Ala-Glu-Gly-pAB and concentration, 1 mM in each case. BrAAP specific activity, 2.2 U/mg.SnAAP specific activity, 0.8 U/mg.Cbz-Gly-Pro-Glu-Leu-Gly-pAB, containing both inter-



fairly selective inhibitor of the BrAAP activity (26).Cbz-Gly-Pro-Phe-Leucinal effectively inhibited the hy-drolysis of both Cbz-Gly-Pro-Glu-Leu-Gly-pAB andCbz-Gly-Pro-Ala-Glu-Gly-pAB. Inhibition of both sub-strates was virtually identical with an IC50 of 8 mM

(Fig. 5).A competition experiment was designed in which the

standard BrAAP substrate Cbz-Gly-Pro-Ala-Leu-Gly-pAB and the new substrate Cbz-Gly-Pro-Ala-Glu-Gly-pAB were incubated with MPC either separately or incombination. Each substrate was present at a final con-centration of 1 mM. The products were analyzed byHPLC. In the combined mixture the hydrolysis of theBrAAP substrate was reduced by 30% and the hydrolysisof Cbz-Gly-Pro-Ala-Glu-Gly-pAB was reduced by 38%.

Of the catalytic activities of MPC defined with theaid of chromogenic substrates, only the BrAAP activityis stimulated by 3,4-dichloroisocoumarin (DCI) (20).FIG. 3. Inhibition of the hydrolysis of Cbz-Gly-Pro-Glu-Leu-Gly-The other activities are inactivated, each at a differentpAB (open circles) and Cbz-Gly-Pro-Ala-Glu-Gly-pAB (solid circles)rate. The hydrolysis of casein by MPC is also stimu-by Cbz-Leu-Leu-Glutamal. Inhibition was determined without prein-

cubation. Substrate concentration, 1 mM in each case. lated by DCI (21). To determine the effect of DCI on

Glu–Gly bond consistent with the inhibition by Cbz-Leu-Leu-Glutamal (Table I). Further confirmation wasobtained by determination that the more rapidly elut-ing peak and authentic Gly-pAB have identical reten-tion times (not shown). Unexpectedly, amino acid anal-ysis indicated that Cbz-Gly-Pro-Glu-Leu-Gly-pAB wascleaved after a prolyl residue (Table I). Since prolineis not determined by this method of amino acid analy-sis, it was necessary to rule out cleavage at the iminobond of proline. Accordingly, the retention times of au-thentic Cbz-Gly-Pro and product peaks were compared.The retention time of Cbz-Gly-Pro was identical to theretention time of peak 1 (Fig. 4A). Thus, the Pro–Glubond was cleaved in the substrate Cbz-Gly-Pro-Glu-Leu-Gly-pAB.

Nature of the Catalytic Component Cleaving Cbz-Gly-Pro-Glu-Leu-Gly-pAB and Cbz-Gly-Pro-Ala-Glu-Gly-pAB

Since the hydrolysis of the new substrates was inhib-ited by Cbz-Leu-Leu-Glutamal, and since this inhibitorwas most effective against the BrAAP activity, the mostlikely candidate responsible for substrate hydrolysiswas the BrAAP activity. To probe the identity of theactivity responsible for the cleavage of Cbz-Gly-Pro-Glu-Leu-Gly-pAB, two additional peptidyl aldehyde in- FIG. 4. HPLC analysis of the cleavage of Cbz-Gly-Pro-Glu-Leu-hibitors were tested. Cbz-Ile-Glu(O-tBu)-Ala-Leucinal Gly-pAB by MPC in the absence (A) and (B) presence of 3,4-dichloroi-is a fairly specific inhibitor of the chymotrypsin-like socoumarin. Incubation mixtures contained 1 mM substrate, 3 mg

MPC, and 0.05 M Tris–HCl buffer, pH 7.5, in a total volume of 100activity of MPC (25). This compound, while potentlyml. In each case 25 ml was injected. A, 90 min incubation at 377C. B,inhibiting the hydrolysis of Cbz-Ile-Glu(O-tBu)-Ala-MPC was preincubated for 30 min at 257C with 10 mM DCI, prior toLeu-pNA, failed to inhibit the hydrolysis of Cbz-Gly- addition of substrate, followed by 30 min incubation at 377C. Peak

Pro-Glu-Leu-Gly-pAB even at a concentration of 100 1, Cbz-Gly-Pro; peak 2, Glu-Leu-Gly-pAB; peak 3, Cbz-Gly-Pro-Glu-Leu-Gly-pAB.mM. On the other hand, Cbz-Gly-Pro-Phe-Leucinal is a



TABLE I

Amino Acid Composition of Major Cleavage Products of Cbz-Gly-Pro-Glu-Leu-Gly-pAB and Cbz-Gly-Pro-Ala-Glu-Gly-pAB

Amino acid ratio of major cleavage products

Substrate Gly Pro Glu Leu Ala pAB

Cbz-Gly-Pro-Glu-Leu-Gly-pAB 1.0 1.05 0.85 PresentCbz-Gly-Pro-Ala-Glu-Gly-pAB 1.0 0.89 0.92 Absent

Note. Products were separated by HPLC and subjected to amino acid analysis as described under Materials and Methods. Mass ratiosare expressed relative to Gly which is assigned a value of 1.0.

the hydrolysis of the new substrates, MPC was preincu- Effect of Prolyl Oligopeptidase Inhibitors on the NovelPostproline Cleaving Activitybated for 30 min with 10 mM DCI. Under these condi-

tions, the chymotrypsin-like activity was totally inhib- Prolyl oligopeptidase (EC 3.4.21.26) cleaves peptidyl-ited and the PGP activity only moderately inhibited prolyl peptide and peptidylprolyl amino acid bonds(Table II). These results are consistent with previous (33). Specific and potent active-site-directed inhibitorsfindings from our laboratory on the effect of DCI. The of this serine proteinase have been synthesized. It washydrolysis of both Cbz-Gly-Pro-Glu-Leu-Gly-pAB and of interest to determine whether they could inhibit theCbz-Gly-Pro-Ala-Glu-Gly-pAB was stimulated more postproline cleavage catalyzed by the BrAAP activitythan sixfold by DCI (Table II). This marked stimulation of MPC. Z-Pro-Prolinal, a peptidyl aldehyde and potenttogether with the results of the peptidyl aldehyde inhi- prolyl oligopeptidase inhibitor (Ki Å 14 nM) (27), didbition experiments and the competition experiment not inhibit the hydrolysis of Cbz-Gly-Pro-Glu-Leu-Gly-confirms that cleavage of the new substrates was in- pAB by MPC when tested at a concentration of 10 mM.deed catalyzed by the BrAAP activity. To determine A new compound, N-Fluorenylmethoxycarbonyl-Pro-that DCI did not modify the cleavage specificity, the Pyrrolidine-2-CN which inhibits prolyl oligopeptidasereaction mixtures were subjected to HPLC analysis. with a Ki of 5 nM (28), similarly failed at a concentrationIdentical retention times were found for degradation of 10 mM to block the novel postproline cleavage.products in incubation mixtures of MPC and Cbz-Gly-Pro-Glu-Leu-Gly-pAB (Fig. 4B) or Cbz-Gly-Pro-Ala-

DISCUSSIONGlu-Gly-pAB (not shown) in the presence and absence

The primary specificities of the catalytic activities ofof DCI, thus demonstrating that DCI did not alterMPC have been defined in earlier studies with a limitedcleavage sites in these substrates.number of chromogenic substrates. More detailed sub-site specificity characterization of an individual activ-

TABLE II

Effect of 3,4-Dichloroisocoumarin onSubstrate Hydrolysis by MPC

Substrate % control

f

Cbz-Glu(O-tBu)-Ala-Leu-pNA 0f

Cbz-Leu-Leu-Glu-NA 90f

Cbz-Gly-Pro-Glu-Leu-Gly-pAB 623f

Cbz-Gly-Pro-Ala-Glu-Gly-pAB 628

Note. MPC was preincubated for 30 min with 10 mM DCI andenzymatic activity compared to MPC preincubated with vehicle(DMSO) only as control. Control activity was assigned a value of100%. Arrows designate site of peptide bond cleavage. Control spe-FIG. 5. Inhibition of the hydrolysis of Cbz-Gly-Pro-Glu-Leu-Gly-

pAB (open circles) and Cbz-Gly-Pro-Ala-Glu-Gly-pAB (solid circles) cific activities (U/mg): Cbz-Glu(O-t-Bu)-Ala-Leu-pNA, 3.1; Cbz-Leu-Leu-Glu-NA, 2.2; Cbz-Gly-Pro-Glu-Leu-Gly-pAB, 1.7; Cbz-Gly-Pro-by Cbz-Gly-Pro-Phe-Leucinal. Inhibition was determined without

preincubation. Substrate concentration, 1 mM in each case. Ala-Glu-Gly-pAB, 1.4.



ity is complicated by the presence of other potentially to the BrAAP activity, the PGP activity is extremelysensitive to inhibition by various proteins (34).competing activities. In the absence of inhibitors, it is

Assignment of the postprolyl and postglutamyl cleav-difficult to predict which catalytic activity is responsi-ages to the BrAAP activity demonstrates that this activeble for the cleavage of a given peptide bond in an oligo-site can accommodate residues other than branchedpeptide substrate. The absence of a specific inhibitor ofchain amino acids in the P1 position. The postprolyl andthe PGP activity prompted the synthesis of Cbz-Leu-postglutamyl cleavages in the new substrates proceedLeu-Glutamal, a peptidyl aldehyde based on the struc-at about half the rate of the postleucyl cleavage in theture of the classical PGP substrate Cbz-Leu-Leu-Glu-standard BrAAP substrate Cbz-Gly-Pro-Ala-Leu-Gly-NA. The rather modest inhibition of the PGP activitypAB. Thus, the BrAAP activity may be able to cleaveby this aldehyde indicates that Cbz-Leu-Leu-Glu-NApeptidyl glutamyl peptide and peptidyl prolyl peptidepossesses suboptimal properties as a PGP substrate.bonds in some peptides or even proteins. This may helpThe finding that both the BrAAP and SnAAP activitiesto explain the results of previous studies on the effect ofwere also inhibited by this aldehyde was unexpected.DCI on MPC. It has been shown that treatment of MPCThis suggested that both the BrAAP and SnAAP activi-with DCI accelerates casein hydrolysis to small peptidesties may under some conditions also cleave peptidedespite the fact that DCI inhibits the chymotrypsin-like,bonds after glutamyl residues. To explore this possibil-PGP, and trypsin-like activities (21). Since DCI activatesity additional experiments were designed.the BrAAP activity, this activity may by itself be capableIn earlier studies, detection of the BrAAP andof fully degrading casein (20). In this respect it shouldSnAAP activities utilized substrates containing an in-also be noted that Mykles has shown that brief heatingternal proline residue which results in selective degra-activates both the BrAAP and proteolytic activities of thedation of substrates by these activities (20). Accord-lobster muscle enzyme (35).ingly, we synthesized two new substrates containing

These studies demonstrate for the first time thatboth internal prolyl and glutamyl residues. Release ofMPC can cleave peptidyl prolyl peptide bonds in chro-the chromogenic group, pAB, from both Cbz-Gly-Pro-mogenic substrates. Early studies from this laboratoryAla-Glu-Gly-pAB and Cbz-Gly-Pro-Glu-Leu-Gly-pABhave shown minor postproline cleavages in the natu-by MPC required the presence of alanyl aminopepti-rally occurring peptides angiotensin II and neurotensindase, thereby precluding direct cleavage of the Gly–(36). The ability of MPC to cleave after a prolyl residuepAB bond. Amino acid analysis of HPLC separatedindicates that under some circumstances the presenceproducts revealed in the case of Cbz-Gly-Pro-Glu-Leu-of a prolyl residue in the P1 position does not limit itsGly-pAB a postproline cleavage, while cleavage of Cbz- action. Cleavage of peptidyl prolyl peptide bonds is notGly-Pro-Ala-Glu-Gly-pAB occurred after glutamate. a common feature of endopeptidases. Only one enzyme,

Assignment of these cleavages to the BrAAP activity prolyl oligopeptidase (EC 3.4.21.26), has a known speci-was made on the basis of (i) inhibition by the BrAAP ficity exclusively directed toward such bonds (33). Po-inhibitor Cbz-Gly-Pro-Phe-Leucinal, (ii) competition by tent active-site-directed inhibitors of prolyl oligopepti-a BrAAP substrate, and (iii) activation by DCI. dase however were unable to inhibit the postproline

Earlier specificity studies have indicated a prefer- cleavage catalyzed by MPC. Interestingly, prolyl oligo-ence of the BrAAP activity for cleavages after branched peptidase is also a cytosolic enzyme and may partici-chain amino acids such as Leu, Ile, or Val (20, 26, 30). pate in the terminal degradation of oligopeptides gen-Cleavages after the small neutral amino acid alanine erated by MPC (37).are also catalyzed by the BrAAP activity albeit slowly This report is not the first to document that peptide(20). This activity might therefore have been expected cleavage assigned to a specific catalytic activity of MPCto cleave the Leu–Gly bond of Cbz-Gly-Pro-Glu-Leu- differs from its defined primary specificity. EvidenceGly-pAB or possibly, based on inhibition of the BrAAP has been presented that the Gln4–His5 bond of the oxi-activity by Cbz-Leu-Leu-Glutamal, the Glu–Leu bond. dized insulin B chain is cleaved by the trypsin-like ac-Surprisingly, a Pro–Glu cleavage occurred. Apparently tivity of MPC (38). Factors governing cleavage specific-the glutamyl residue of this novel substrate directs the ity of the catalytic components of MPC therefore re-BrAAP component toward a postproline cleavage. quire further study. In this respect it should be notedTherefore, primary cleavages are greatly influenced by that Stein et al. recently proposed that substrates ofsubsite substituents and are not readily predictable. MPC can bind to and be hydrolyzed by more than oneAlthough these studies might suggest that the BrAAP active site and that each active site can bind substratesand PGP activities are identical, it should be stressed possessing a variety of residues in the P1 position (39).that the BrAAP activity is markedly and rapidly acti-vated by DCI (20), whereas the PGP activity is slowly REFERENCESinhibited by this reagent (23). Moreover, in contrast to 1. Enzyme Protein (1993) 47, 187–369.the PGP activity, the BrAAP activity is unable to cleave 2. Grziwa, A., Baumeister, W., Dahlmann, B., and Kopp, F. (1991)

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Cleavage of Pro-X and Glu-X Bonds Catalyzed by the Branched Chain Amino Acid Preferring Activity of...

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