Tài liệu Báo cáo Y học: The insert within the catalytic domain of tripeptidyl-peptidase II is important for the formation of the active complex potx

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The insert within the catalytic domain of tripeptidyl-peptidase IIis important for the formation of the active complexBirgitta Tomkinson, Bairbre Nı´Laoi and Kimberly WellingtonDepartment of Biochemistry, Uppsala University, Biomedical Center, Uppsala, SwedenTripeptidyl-peptidase II (TPP II) is a large (Mr>106)tripeptide-releasing enzyme with an active si te of the subtil-isin-type. Compared with other subtilases, TPP II has a 200amino-acid insertion b etween the catalytic Asp44 a ndHis264 residues, and is active as an oligomeric c omplex. Thisstudy demonstrates that the insert is important for theformation of the active high-molecular mass complex.A recombinant human TPP II and a murine TPP II werefound to display different c omplex-forming characteristicswhen over-expressed in human 293-cells; t he human enzymewas mainly in a nonassociated, inactive state whereas themurine enzyme formed active oligomers. This was s urprisingbecause native human TPP II is purified from erythrocytesas an active oligom eric complex, and t he amino-acidsequences of the human and murine enzymes were 96%identical. Using a combination of chimeras and a singlepoint mutant, the amino acid res ponsible for this differencewas identified as Arg252 in the recombinant humansequence, which c orresponds to a glycine in the murinesequence. As Gly252 is conserved in all sequenced variants ofTPP II, the recombinant enzyme with Arg252 is atypical.Nevertheless, as Arg252 evidently interferes with complexformation, and this residue is close to t he catalytic His264, itmay also e xplain why oligomerization influences enzymeactivity. The exact mechanism for how th e G252R substi-tution interf eres with complex formation remains to b edetermined, but will be of importance for the understandingof the unique properties of TPP II.Keywords: tripeptidyl-peptidase II; complex f ormation;association/ dissociation; exopeptidase; serine peptidase.Tripeptidyl-peptidase II (TPP II) (EC 3.4.14.10) is anenzyme with remarkable characteristics. It was discovered1983 as an extralysosoma l peptidase in rat liver [1] and hassince b een extensively characterized [2–6]. It is one of onlytwo known mammalian tripeptide-releasing enzymes(reviewed in [7]). Native TPP I I is a high-molecular massprotein where the s ubunit (138 kDa) forms a largeoligomeric complex (Mr>106) [2,8]. The enzyme has acatalytic domain o f the subtilisin-type [ 4], but in comparisonwith other sub tilases, it has a 200 a mino-acid insertionbetween the Asp and His of the catalytic triad [ 5,9]. Inaddition, TPP II has a long C-terminal extension [5,9].The widespread distribution and conserved amino-acidsequence would suggest that TPP II plays a role in generalcytosolic protein turnover, probably in association with theproteasome [7]. When TPP II w as induced in proteasome -deficient cells, it appeared to compensate for the partial lossof the proteasome activity [10,11], and over-expression ofTPP II protected the cells from the effect of proteasomeinhibitors [12]. In addition to this general role, more sp ecificfunctions have also been suggested, e.g. an involvement of amembrane-bound form of TPP I I in t he inactivation of theneuropeptide cholecystokinin [6], and a role upstream ofcaspase-1 in Shigella-induced apoptosis [13]. It is thereforenot surprising that when an efficient proteolytic system hasevolved, it will be used for specific degradation of certaintargets as well as functioning in less specific processes. Thisappears to be the case not only for th e proteasome but alsofor TPP II, which shows that also e xopeptidases areimportant in protein degradation [7].An important question is how the enzymatic activity ofTPP II is regulated, because, in contrast to most o thersubtilases, TPP II does not appear to be synthesized as apro-protein [9], a nd specific p hysiological inhibitors of theenzyme have not been identified as yet. The substratespecificity of TPP II is fairly broad, i.e. a variety of differenttripeptides can be released, even though the enzymeapparently cannot attack peptide bonds before or after aproline residue [1,2]. TPP II is highly dependent on a freeN-terminus and t he re cently reported endopeptidase activityof the enzyme [11] is very low compared to the exopeptidaseactivity. All substrates that have been identified so far areoligopeptides of 4–41 amino acids [1,2,6,11] and thecleavage of native proteins by TPP II has not beendescribed. The substrate specificity and oligomeric structureof TPP II could indicate that it is a self-compartmentalizingpeptidase, similar to the proteasome [14]. The self-compart-mentalization would thus protect the cell from uncontrolledproteolysis. This agrees with the observation that theenzyme is only fully active in the oligomeric complex.Native TPP I I has been shown to dissociate spontaneously,resulting in a loss of 90% of the original specific activity.The dissociated enzyme can reassociate and the activity isconcomitantly restored. This reactivation is enhanced bysubstrates and different competitive inhibitors [15], thussuggesting the involvement of the catalytic domain. There-fore, as suggested previously [8,15], association/dissociationCorrespondence to B. Tomkinson, Department of Biochemistry,Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala,Sweden. Fax: + 46 18 55 84 31, Tel.: + 46 18 4714659,E-mail: B irgitta.Tomkinson@biokem.uu.seAbbreviations: pNA, para-nitroanilide; TPP II, tripeptidyl-peptidaseII; DMEM, Dulbecco’s modified Eagle’s medium.(Received 31 December 2001, accepted 14 January 2002 )Eur. J. Biochem. 269, 1438–1443 (2002) Ó FEBS 2002of the oligomeric complex could be a way of regulating theenzymatic activity.In order to study the structural basis for complexformation, a previously developed expressio n system forTPP II has been used [16]. It was found that recombinanthuman TPP II and murine TPP II displayed differentassociation/dissociation characteristics when overexpressedin human 293-cells. The main objective of the prese nt workwas to find an explanation for this phenomenon. It isdemonstrated that the fo rmation of the active complex isprofoundly influenced by a single amino acid difference, i.e.G252R, in a region within the catalytic domain. This is thefirst evidence that this region is involved in the formation oftheactivecomplex.MATERIALS AND METHODSConstruction of expression clonesA3.9-kbKpnI fragment, corresponding to the completecoding sequence of human TPP II and 23 and 145 bp of theuntranslated 5¢ and 3 ¢ ends, respectively [17], was clonedinto the pcDNA 3 expression vector (Invitrogen, Groenin-gen, the Netherlands) by conventional cloning techniques[18]. C lones with the insert in the sense direction wereselected and purified. Chimeras were constructed in pUC19by seq uential su bcloning [18] using different clones isolatedpreviously [5,19,20]. Full-length constructs were excisedwith KpnIorEcoRI and inserted into the pcDNA3 vector.Clones with the insert in the sense direction were se lectedand purified.The rat EcoRV–SacI fragment was amplified from ratliver RNA by use of two specific primers: 5¢-GGTCACGACTGATGGGAAAC-3¢ and 5¢-CCATGAGCTCCTCCACTGGT-3¢ and the RT-PCR kit (PerkinElme r, Boston,MA, U SA), except that Advantage polymerase (Clontech,Palo Alto, CA, USA) was u sed. The amplified fragment wasdigested with EcoRV and SacI and cloned into thepBluescript SK+ vector (MBI Fermenta, Vilnius, Lithu-ania) and the sequence was determined by sequencing in a nABI Prism 310 automatic sequencer. The Eco RV–SacIfragment was cloned into a chimeric construct and the full-length chimera transferred to the pcDNA3 vector.The Dhum clone, containing the human sequenceresulting in a R252G substitution, was constructed byreplacing the EcoRV–SacI f ragment in clone Bhum with theEcoRV–SacI fragment from the human F5 clone describedpreviously [19,20].Cells and transfectionThe human embryonic kidney cell line 293 (ATCC CRL1573) was maintained in Dulbecco’s modified Eagle’smedium (DMEM) (Gibco -BRL, Paisley, Scotland, UK)with 10% (v/v) heat-inactivated fetal bovine serum,100 U ÆmL)1penicillin and 100 lgÆmL)1streptomycin, at37 °C i n a humidified 5% CO2atmosphere. F or thepreparation of stable t ransformants, the constructs wereintroduced into 293-cells by the calcium phosphate preci-pitation method, and stable clones were selected bygrowing cells in 400 lgÆmL)1geneticin (Duchefa, Haarlem,the Netherlands), as described previously [16]. Clonesexpressing murine TPP II were i solated [16]. C ellstransfected with the p cDNA3 vector alone were u sed ascontrols. The expression efficiency of the constructs wasdetermined by Western blot a nalysis, and the two mostefficient clones of each construct were selected for furthercharacterization.Preparation of cell extractsCells from stable transformants expressing recombinantTPP I I [16 ] were harvested and lysed with 50 mMTris buf-fer, pH 7.5, containing 1% (w/v) Triton X-100 (10 lLper106cells). The lysate was centrifuged for 30 min at 4 °Cand14 500 g. The supernatant was collected and diluted 10-foldwith 100 mMpotassium phosphate buffer, pH 7.5, contain-ing 30% (w/v) g lycerol and 1 mMdithiothreitol. Dilutedsupernatants were used for activity assays, Western blotsand gel filtration, as indicated.Enzyme assayEnzyme aliquots were incubated with 0.2 mMAla-Ala-Phe-pNA (Bachem, Bubendorf, Switzerland) in 0.1Mpotassiumphosphate buffer, pH 7.5, containing 15% (w/v) glyceroland 2.5 mMdithiothreitol at 37 °C, in a total volume of200 lL. The rate of change in absorbance at 405 nm wasmeasured in a Multiscan PLUS ELISA plate reader(Labsystems, Helsinki, Finland) [21]. A molar a bsorbanceof 9600M)1Æcm)1for pNA was used [22]. The activity wasrelated to the total amount of protein in the sample,determined with a modified Bradford method [23,24], usingBSA as the standard.Gel filtrationCell extracts were prepared as desc ribed above. The d ilutedsupernatant (1.8 mL, corresponding to 1–2 · 107cells) wasloaded onto a Sepharose CL-4B (AP Biotech, Uppsala,Sweden) column ( 1 · 90 cm, several columns being usedfor the experiments). The column was e quilibrated andeluted with 0.1Mpotassium phosphate buffer, pH 7.5,containing 30% (w/v) glycerol and 1 mMdithiothreitol, at aflow rate of 6 mL Æh)1. Fractions of 1 mL were collected.The void-volume (Vo) and total volume (Vt)ofthecolumnwere determined from the elution positions of Blue dextran(AP B iotech, Uppsala, Sweden) and dinitrophenol-b-Ala(Sigma), respectively. Kavvalues for different elutionvolumes (Ve) were calculated f rom Kav¼ Ve) Vo/Vt) Vo.Individual fractions were investigated through activitymeasurements and Western blot analysis.Western blot analysisAliquots from fractions of the chromatography were mixedwith SDS/PAGE sample buffer to give final concentrationsof 2.3% (w/v) SDS, 5% (v/v) 2-mercaptoethanol and 10%(w/v) glycerol. The samples were h eated for five minutes at95 °C before they were loaded onto an 8% polyacrylamidegel. The S DS/PAGE and Western blot analysis w ereperformed as described previously usin g a ffinity purifi edpolyclonal chicken anti-(human TPP II) Ig [25]. Theimmunoreactivity was quantitated from scanned X-rayfilms by use of theMOLECULAR ANALYSTsoftware (Bio-Rad,Hercules, CA, USA).Ó FEBS 2002 Formation of the tripeptidyl-peptidase II complex (Eur. J. Biochem. 269) 1439RESULTS AND DISCUSSIONComplex-forming characteristics of recombinanthuman and murine TPP IIExpression of recombinant human TPP II, encoded by full-length cDNA, in 293-cells indicated that only part of theexpressed protein was active. Although there was 8 - to10-fold more immunoreactive material in the high-expres-sing clones than in t he control, according to d ensitometerscanning of a Western b lot of cell lysates, t he enzymeactivity increased only threefold (data not shown). Investi-gation of the cell lysate by gel filtration demonstrated that asubstantial part of the immunoreactive protein from theextract o f an individual clone with a high expression ofhuman TPP II eluted with a Kavof 0.55 and was virtuallyinactive (Fig. 1A). The Mrof this protein was 2–3 · 105asdetermined through chromatography on a calibratedSepharose CL-6B column (cf. [15]; data not shown). Theexperiment was repeated with t wo other high-expres singhuman clones with the same result. Evidently, only afraction of the expressed p rotein had formed t he large,active oligomers, which eluted a t a Kavof 0.26. This was incontrast to stable transformants expressing the murineenzyme, where activity increased about eightfold, comparedto the control cells. T he majority of t he protein was in theoligomeric form and coeluted with t he activity upon gelfiltration (Fig. 1B; [16]). The 293-cells used for the experi-ments have an endogenous expression of TPP II [16], andthe activity in control cells, untransfected or transfected withvector alone, were used as a comparison (Fig. 1). In thecontrol cells, t he immunoreactivity followed the activity(data not shown).The two forms of the enzyme, eluting at a Kavof 0.26and a Kavof 0.55, respectively, will be referred to asÔassociatedÕ and ÔnonassociatedÕ throughout this work. It isnot possible, however, to know whether the human enzymenever associates or whether it transiently associates andthen dissociates. In general, the total amount of immuno -reactive protein obtained from the human clone was lowerthan from the murine clone (Fig. 1). This may be due tothe fact that nonassociated enzyme is more sensitive t oproteolytic digestion than enzyme associated into thecomplex, as has been seen previously for purified humanTPP II [2 6].Identification of the region causing differentassociation characteristicsThe difference in association characteristics of the enzymefrom the two sources was surprising because the sequenceis extremely well conserved between the two species, i.e.96% of the amino acids are identical and a number o f theamino-acid differences are conservative [5]. A comparisonshows that there is a cluster of amino-acid differences inthe C-terminal part o f the enzyme (Fig. 2A) where 13 o f 44amino acids are different. Therefore, chimeric enzymeswith the N-terminal part from the human and theC-terminal part from the murine enzyme a nd vice versawere co nstructed by u se of an XmnI site. When stabletransformants expressing these chimeric constructs werestudied, it was evident that the sequence differenceresponsible f or the lack o f a ssociation of the humanenzyme resi ded in t he N-terminal part of the humanenzyme (Figs 2B,C), not in the hypervariable C-te rminalpart. As 23 amino acids differ between the N -terminal partof the human and mouse enzyme, new chimeras wereconstructed by use o f the EcoRV and SacI sites in thecDNAandwerethenusedtotransform293-cells.Theregion responsible for the different degree of association ofthe human and murine enzyme could be defined as beingwithin the EcoRV–SacIfragmentoftheenzyme(Figs 2 B,C). This 591-bp fragment corresponds to 197amino acids located between the Asp and His of thecatalytic triad. M ost other subtilases h ave about 20 aminoacids in this region and the large in sertion is a specialfeature of TPP II and pyrolysin [9,21]. There are, in total,12 amino-acid differences between the human and mousesequences in this region, and a number of them areconservative changes (e.g. Val fi Ile) (Fig. 3).Fig. 1. Gel filtration of extracts of cells expre ssing recombinant humanor murine TPP II. Cell lysates (corresponding to 1–2 · 107cells) fromstable transformants or control cells were loaded onto a SepharoseCL-4B colum n and chromato graphy was perfo rmed as describe d inMaterials a nd methods. Enzyme activity was a nalysed by the standardassay and the i mmunoreactivity was detected by Western blot analysisand quantitated as described i n Materials and methods. Open andfilled circles indicate the activity, and open and filled bars the immu-noreactivity (PD, pixel density) fo r human and murine TPP II,respectively. The enzyme activity in control cells is indicated ( ·).(A) Human TPP II and control ce lls (V0¼ 27.5 mL; Vt¼ 76.7 mL).(B) Murine TPP II and control cells (V0¼ 26.5 mL; Vt¼ 74.7 mL).1440 B. Tomkinson et al. (Eur. J. Biochem. 269) Ó FEBS 2002As seen in Fig. 3, the corresponding rat sequence [6] ismore or less a m ix between the human and the murinesequence. Therefore, the Ec oRV–SacI fragment was ampli-fied from rat RNA by use of PCR, as described in Materialsand methods. This fragment was used to create a human–murine–rat chimera, as outlined in Fig. 2; the chimera wasused fo r t ransfecting 293 cells. This c himera behaved like themurine enzyme (Fig. 2B), demonstrating that seven amino-acid substitutions of potential importance for the differentassociation remained (Fig. 3).It is important to note that there is a single nucleotidedifference between the sequences of two human clonesreported, one encoding a Gly at position 252 [19], andanother an A rg [20]. The Arg252-encoding cDNA clonewas employed for construction of the human full-lengthcDNA-clone used for expression [17]. Currently availablesequence information indicates that the Arg252 variant isatypical, as all hitherto sequenced variants of TPP II ( i.e. rat,mouse, fruit fly, Arabidopsis thaliana, Caenorhabdit is elegansand Schizosaccharo myces pombe), and at least three humanEST-clones covering this area (GenBank a ccession numbersAU118610, AW452455, BF511874) encode a Gly in thisposition. In order to test the consequence of this singleamino-acid difference, a construct containing the humanN-terminal part with an R252G substitution was made.This construct associated and had a high activity (Fig. 2B,Dhum), which was in contrast to the construct Bhum. Theonly difference between these two clones is the amino acid inposition 252. Evidently, changing Gly252 to an Arg wascritical for the association properties of the enzyme.The nonassociated form is inactiveFor purified human TPP I I and recombinant murineTPP II, it has been shown that the smallest active form ofTPP I I appears to be dimers, which have a bout one tenth ofthe specific activity of the oligomeric complex [15]. For therecombinant human enzyme the nonassociated form alsoappeared to be dimers of the 138 kDa subunit, since theirMrwas determined to be 2–3 · 105.However,noactivitypeak eluting at a Kavof 0.55 could be detected, indicatingthat they were inactive (Fig. 1). This nonassociated form ofthe recombinant human enzyme has been isolated after g elfiltration an d a variety of experiments have been performedFig. 2. Comparison of human and murine TPP II and propertie s of chimeric cons tructs. (A) Black vertical lines indicate amino-acid differencesbetween human and murine TPP II. D, H , and S denote the catal ytic triad (Asp44, His264 and S er449, respectively). The restriction sites used forcreation of the chim eras are shown. (B) Mur ine and human fragm ents in the co nstructs are indicated by filled and open bars, respectively. Thefragment originating from the rat gene is indicated by a hatched bar. The activity in cell extracts of stable transformants was measured as describedin Materials and methods. The values represent mea ns of two to fi ve measurements each of two ind ividual clones with the highest express ion of eachof the chimeras. The activity in control cells transformed with vector alone is 4 nmolÆmin)1Æmg)1. Association was investigated by gel filtration ofcell extracts on a Sepharo se CL-4B column, as de scribed in Materials and methods. A t least two individual c lones of each chimera were i nvestigated(except Bhum), and both clones displayed the same result. +, the immunoreactivity at Kav¼ 0.26>the immunoreactivity at Kav¼ 0.55; –, theimmunoreactivity at Kav¼ 0.55>the immunoreactivity at Kav¼ 0.26 (cf. Figure 2C). *, indicates a clone with a relatively low expression rate.(c) Western blot analysis of fractions from gel chroma tography (com pare to Figure 1) was perform ed as describ ed i n M aterials and m ethods. F oreach construct, one of the clones with the highest expression was selected. Two fractions eluting at a Kavof about 0.26, and two fractions eluting at aKavof about 0.55 are shown.Ó FEBS 2002 Formation of the tripeptidyl-peptidase II complex (Eur. J. Biochem. 269) 1441to activate the material, as previously described [15].However, all attempts so far to associat e this material havefailed. Thus, it appears that the isolated Arg252-containingdimers cannot form the active oligomers.Formation of active heterocomplexesEven if the r ecombinant human enzyme appe ared to forminactive dimers, the total activity in cells overexpressingrecombinant human TPP II or different chimeras was atleast t wice as high as the endogenous TPP II-activity incontrol cells (Fig. 2B). The active enzyme e luted at a Kavofabout 0.26 (Fig. 1), which shows that the expressed subunitscan, in fact, be part of an active complex. It appears thatcomplex formation involves molec ular interactions on atleast t wo le vels, dimerization and oligomerization, where t heoligomeric complexes have a 10-fold higher s pecific activitythan the dimers [15]. Even though inactive dimer s areformed when over-expressing the Arg252-variant, thesedimers may contribute to the formation of active oligomersin the p resence of the endogenously expressed G ly252-containing subunits. T he exact c omposition of the hetero-complexes could not be established, i.e. if heterodimers wereformed by endogenous and recombinant monomers or ifthe a ctive complexes were assembled from the two types ofhomodimers.The insert within the catalytic domain is of importancefor complex formationNo functional significance has previously been ascribed tothe insert between Asp and His of the catalytic domain ofTPP II. We can now report that the region surroundingArg252 is of importance for the formation of the oligomericenzyme complex, which is a prerequisite for obtaining afully active enzyme [8,15]. Upon removal of this entireregion (amino acids 68–255 ), no protein of the expected sizecould be detected, although mRNA was expressed i ntransformed cells (data not shown). One interpretation ofthis finding is that the protein did not oligomerize properly,with the consequence that the subunits were prone todegradation by p roteases. With such a large deletion, it isalso possible that the enzyme was not folded correctly andtherefore more easily subjected to proteolysis.Part of the subtilisin-like catalytic N-terminal partof TPP II has been modeled on the structure of subtilisinBPN¢ (http://biospace.stanford.edu) [27]. I n this model(Nr 03816 78/1), residues 211–507 of human TPP II werealigned with residues 18–273 of subtilisin BPN¢.Thecatalytic His264 and Ser449 residues were aligned correctly,whereas the catalytic Asp44 of TPP II was not aligned tothe active Asp36 of subtilisin, probably due to the l argeinsertion between the catalytic Asp and His in TPP II. Thisregion would, of course, be difficult to model, but as Arg252is so close to H is264, where the structure is conserved, themodel is still expected to be useful. In this model, Arg252 ispredicted to be on the surface of the enzyme where it couldbe directly in volved in a s ubunit–subunit interaction. Bysubstituting Gly252 with Arg, this interact ion c ould b edisturbed by electrostatic or steric interference. Moreover,the re lative short distance to the active site may explain theeffect of complex formation on activity [8,15]. Furtherstudies with a number of different Gly252 mutants andother amino-acid changes in this region will be required tofully elucidate the role of this interaction for oligomerizationand cata lytic a ctivity.Although the data presented here suggests that the regionsurrounding residue 252 is directly involved in complexformation, it may instead have a more i ndirect function. Forexample, this region may f unction as an intramolecularchaperone. By promoting the folding of the protein itself, itwould have a similar role as that of pro-peptides in o therproteases [28,29]. Incorrect folding could also explain thereduced amount of immunoreactive protein observed for allenzyme forms with Arg252 (Fig. 2C), as this protein wouldbe more susceptible to proteolytic degradation. However,the enzyme activity in cells overexpressing all the Arg252variants still increases twofold to threefold (Fig. 2), indica-ting that these Arg-containing subunits may be part of anactive complex. This suggests that the subunits could stilladapt to the three-dimensional fold required f or interactionwith endogenously expressed subunits. Alternatively, theregion surrounding Arg252 may be of importance forinteraction with a chaperone or other factors i nfluencing theformation of t he active complex. For example, i t is possiblethat a p rotein in the 293-cells sequesters the Arg-contai ningsubunits, thereby preventing complex f ormation. This couldexplain why the nonassociated form, isolated by gelfiltration, cannot be made to associate [cf. 15]. Therecombinant protein incorporated into the active enzymecomplex together with endogenous TPP I I would then beprotected from sequestration. However, additional d ata isrequired to show whether the G252R substitution interfereswith activity and/or structure of the dimer or with theoligomerization, and whether this effect is direct or indirect.CONCLUSIONSWe have shown that a single amino-acid difference,G252R, is critical for formation of t he TPP II complex.Fig. 3. Alignment of the amino acid sequences be tween the catalyticAsp44 and His26 4 residues from human, murin e and rat TPP II. Adotindicates that the amino aci d is identical to that in the h uman sequence.The arrows indicate the part corresponding to the Eco RV–SacI frag-ment. The GenBank accession numbers for the sequence data areM73047, X81323 and U 50194. The catalytic Asp44 and His264 areindicated by asterisks.1442 B. Tomkinson et al. (Eur. J. Biochem. 269) Ó FEBS 2002This amino acid is located in the insert within the catalyticdomain, close to the catalytic His264, and the proximity tothe active site may explain the effect of oligomerization onenzyme activity. Even though the exact mechanism forcomplex formation and activation of the enzyme remainsto be determined, it can be concluded that the insertwithin the catalytic domain is of importance for oligome-rization.ACKNOWLEDGEMENTSThis work was supported by the Swedish Medical R esearch Council(project 09914). The critical reading of this manuscript by Pr o f. O¨rjanZetterqvist and Dr Helena Danielson are gratefully acknowledged.REFERENCES1. Ba˚lo¨w, R M., Ragnarsson, U. & Zetterqvist, O¨. (19 83) Tripepti-dylaminopeptidase in the extralysosomal fraction o f rat liver.J. Biol . Chem. 258, 11622–11628.2. Ba˚lo¨w, R M., Tomkinson, B., Ragnarsson, U. & Zetterqvist, O¨.(1986) Purification, substrate specific ity and classification oftripeptidyl peptidase II. J. Biol. 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In Proceedings of the Eighth InternationalConference on ISMB (Bourne, P., Gribskov, M., Altman, R.,Jensen, N., Hope, D., Lengauer, T., Mitchell, J., Sch eeff, E.,Smith, C., Strande, S. & Weissig, H., eds) pp. 395–4 06. AAAIPress, CA, USA.28.Shinde,U.P.,Liu,J.J.&Inouye,M.(1997)Proteinmemorythrough altered folding mediated by intramolecular chaperones.Nature 389, 520–522 .29. Yabuta, Y., Takagi, H., Inouye, M. & Shinde, U. (2001) Foldingpathway mediated by an intramo lecular chaperone. Propeptiderelease modulates activation precision of pr o-subtilisin. J. Bio l.Chem. 276 , 44427–44434.Ó FEBS 2002 Formation of the tripeptidyl-peptidase II complex (Eur. J. Biochem. 269) 1443 . The insert within the catalytic domain of tripeptidyl-peptidase II is important for the formation of the active complex Birgitta Tomkinson,. 2002This amino acid is located in the insert within the catalytic domain, close to the catalytic His264, and the proximity to the active site may explain the
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