MALT CARBOXYPEPTIDASE CATALYZED AMINOLYSlS REACTIONS

Transcription

1 Carlsberg Res. Cmmun. Vl. 49, p , 1984 MALT CARBOXYPEPTDASE CATALYED AMNOLYSlS REACTONS by KLAUS BREDDAM and MARTN OTTESEN Department f Chemistry, Carlsberg Labratry, Gamle Carlsberg Vej 1, DK-25 Cpenhagen Valby Keywrds: Phenylguanidine, carbxypeptidase, barley, malt, enzymatic synthesis, peptide synthesis. Malt carbxypeptidase catalyzes the frmatin f peptide bnds using N-benzyl-arginine esters as acyl cmpnents and amin acid amides r amin acid methyl esters as nuclephiles. With seven different nuclephiles yields f 5-95% were btained while n aminlysis was bserved with H-Pr-NH2 and H-Gu-a-NH2. The enzyme is easily saturated with nuclephile indicating the frmatin f a cmplex between nuclephile and acyl-enzyme intermediate prir t deacylatin. The nature f the side-chain f the amin acid esters used as nuclephiles strngly influences the apparent dissciatin cnstant f this cmplex (KN~app~) while less dependence n the side-chain is bserved with amin acid amides. KN~app~ is drastically increased by the presence fphenylguanidine in the reactin medium suggesting a cmpetitin between this substance and the nuclephile fr the same binding site. This site has previusly been identified as the S~ binding site f malt carbxypeptidase (Carlsberg Res. Cmmun. 48, (1983)). The influence f the nuclephile H-Va-NH2 n the kinetic parameters fr the cleavage f N,-CB-Lys-p-nitrphenylesler indicates that the acylatin step is rate-limiting, and that H-Val-NH2 has tw different mdes f binding t the enzyme. 1. NTRODUCTON t is well knwn that serine and sulfhydryl prteases in additin t hydrlysis f peptide bnds als hydrlyse N-blcked amin acid esters and peptide esters. These reactins prceed via an acyl-enzyme intermediate, and when ther nuclephiles are added t the reactin mixture, e.g. amin cmpunds, the acyl- enzyme intermediate will react bth with water t give a hydrlysis prduct and with the amine t give an aminlysis prduct. BENDER et al. ( 1 ) and FASTRE and FERSHT (9) fund that when methanl, ethanl, glycine amide r alanine amide were added as nuclephiles t chymtrypsin it reacted with ester substrates accrding t the simple reactin mdel shwn in Scheme Abbreviatins: Bu = butyl; Bz = N-benzyl; CB = Carbbenzxy; EDTA = ethylene diamine tetraacetic acid, sdium salt; HPLC = high perfrmance liquid chrmatgraphy; Mes = 2-(N-mrphlin)ethane sulfnic acid; Np = p-nitrphenyl; PhGu = phenylguanidine hydrgencarbnate. Amin acids abbreviated in upper case refer t the L-enantiner and thse in lwer case refer t the D-enantimer. Other abbreviatins f amin acids, amin acid derivatives and peptides are accrding t the guidelines f the UPAC-UB Cmmisin n Bichemical Nmenclature. The binding site ntatins fr the enzyme is that f SCHECHTER AND BERGER (11). Accrdingly, the binding site fr the C-terminal amin acid residue f the substrate is dented S~ and thse fr the amin acid residues in the amin-terminal directin away frm the scissile bnd are dented S,, S_,... K. Springer-Verlag /84/49/473/$1.8

2 K. BREDDAM & M. OTTESEN." Aminlysis with malt carbxypeptidase E+S. Ks ~ES dp_,/dt = k~[w][es'] dp~/dt = ~[N][ES'] P, k2 > ES' + Pi ~[w] Scheme 1. Cmpetitin between water (W) and ther nuclephile (N) in the serine prtease catalyzed hydrlysis f ester substrates as prpsed by BENDER et al. (1). The enzyme (E) and substrate (S) initially frms a cmplex (ES) with the dissciatin cnstant K,. k2 is the rate cnstant fr the essentially irreversible cnversin f ES cmplex t the acyl-enzyme intermedidate (ES'). k~ and k4 are the rate-cnstants fr the deacylatin reactins with W and N as nuclephiles, respectively. P, is the alchl leaving grup f the ester substrate. P~ and P3 are the hydrlysis and aminlysis prducts, respectively. This mdel assumes that neither water nr added nuclephile bind t the enzyme prir t the reactin with the acyl-enzyme intermediate. 1, in which it is assumed that neither water nr the added nuclephile bind t the enzyme prir t the reactin with the acyl-enzyme intermediate. Hence, the rati f the tw reactin prducts being frmed, [P3]/[P2], is a linear functin f the rati [N]/[W] between the cncentratins f added nuclephile and water. FNK and BENDER (1) shwed that the sulfhydryl prtease papain catalyzed hydrlysis f esters with methanl and ethanl as added nuclephiles accrding t Scheme 1. Hwever, when higher alchls were added as nuclephiles deviatins were bserved and t explain this they had t intrduce a mre cmplex reactin scheme which tk int accunt specific binding f the nuclephiles t the enzyme and the enzyme-substrate cmplex (1). The serine expeptidase, carbxypeptidase Y frm baker's yeast, functins similar t the serine and sulfhydryl endpeptidases: it hydrlyzes bth peptide and ester bnds, and in the presence f nuclephiles it catalyzes bth aminlysis and alchlysis reactins, a prperty which has been demnstrated t be useful in enzymatic peptide synthesis (2-6, 13-15). Anther serine carbxypeptidase frm malt has recently been islated in high purity in this labratry and characterized with respect t hydrlysis f peptide and ester substrates (7, 8). Phenylguanidine has a specific functin as a mdifier f this enzyme by binding t the S'L binding site, i.e. the binding site f the leaving grup f the substrate. n the present paper this malt carbxypeptidase is characterized with respect t aminlysis reactins and the effect f additin f phenylguanidine is used t demnstrate that amin acid amides and amin acid methyl esters bind t the S; binding site prir t their attack n the acyl-enzyme intermediate. 2. MATERALS AND METHODS 2.1. Materials Malt carbxypeptidase and Bz-Arg-OBu were prepared as previusly described (7, 8). Mes, Bz-Arg-OMe and H-Ser-NH2 HC were btained frm Sigma, USA. Phenylguanidine hydrgencarbnate was btained frm EGA Chemie, W. Germany. All ther derivatives f amin acids were purchased frm Bachem, Switzerland. Other reagents and slvents were f analytical purity and btained frm Merck, W. Germany Methds The reactins were perfrmed in a ph stat (12). The reactin vlumes were 2 ml and Bz-Arg-OMe and Bz-Arg-OBu were used as substrates in the presence f 5 mm-edta. Mst experiments were perfrmed at ph 7.5 because the enzyme is unstable at higher ph values. Unless specifically stated, the reactins were allwed t prceed until 8-95% f the substrate had been cnsumed. At this stage aliquts were remved fr determinatin f the reactant cmpsitin by HPLC, using equipment frm Waters Assciates, USA, and a C-18 reverse phase clumn. The fllwing eluant system was used: 5 mm-triethyl ammnium phsphate, ph 3. (A-buffer) and 5 mm-triethyl ammnium phsphate, ph 3. in 5% CH3CN (Bbuffer) emplying varius linear and cncave 474 Carlsberg Res. Cmmun. Vl. 49, p , 1984

3 z 1~ L).- 6 '~ 4On- 2" L B Xm ~ 7 m C x D,i x--'x././. :, ~. - 1.O xmx~x~x x co.8 ~.6 u.4- ~ < rr" LL.2 2 OmM 5 1 1ram 5 to 3raM mM MNUTES 3 mm [VALNE AMDE] Figure 1. Malt carbxypeptidase catalyzed aminlysis f Bz-Arg-OBu using H-Va-NH, as nuclephile at different cncentratins. Reactin cnditins: 2 mm-bz-arg-obu, M-H-Val-NH_~, 5 mm-edta,.3 pm-enzyme, ph 7.5. Reactins were quenched at the indicated times and analyzed by HPLC (see sectin 2.2). -O-Q-, Bz-Arg-OBu; -e-o-, Bz-Arg-OH; --- Bz-Arg-Val-NH~; -x-x-, fractin faminlysis, i.e. Bz-Arg-Va-NH=/ (Bz-Arg-OH + Bz-Arg-Va-NH_,). gradients. All separatins were carried ut at rm temperature and mnitred at 254 nm. The per cent cmpsitin f the reactin mixtures culd be calculated directly frm the integrated peak areas since all cmpnents had the benzyl grup as a cmmn chrmphre. Aminlysis and hydrlysis prducts were initially cllected and identified by amin acid analysis. The fractin f aminlysis was expressed as the rati between the frmed aminlysis prduct and the sum f all prducts being frmed, i.e. uncnsumed substrate was disregarded in the calculatins. 3. RESULTS 3.1. nfluence f added nuclephiles Previusly it was bserved that malt carbxypeptidase had high activity twards ester substrates with a psitively charged side-chain (8), and fr this reasn, Bz-Arg-OBu and Bz-Arg- OMe were selected as substrates in the aminlysis reactins. The actin f malt carbxypeptidase n Bz-Arg-OBu with the amin acid amide H-Va-NH2 as added nuclephile resulted in the frmatin f bth Bz-Arg-OH and Bz-Arg-Val- NH2 due t the partitining f the acyl-enzyme intermediate between the hydrlysis reactin and the aminlysis reactin. The fractin underging aminlysis reactin increased with increasing cncentratins f H-Va-NH2 but it did nt exceed apprximately.9 (Figure l). t is seen that the fractin faminlysis was cnstant at each cncentratin f nuclephile and thus essentially independent f the cncentratin f substrate remaining in the reactin mixture. Hwever, the rate f substrate disappearance decreased significantly with increasing cncentratins f H-Va-NH2 indicating an inhibitr functin f this nuclephile. The crrelatin between fractin f aminlysis and cncentratin f H-Va-NH2 shwn in Figure 2 was unaffected by the additin f 2 M-NaC r by replacing Bz-Arg-OBu with Bz-Arg-OMe as substrate. Using Bz-Arg-OBu as substrate and the amin acid ester H-Va-OMe as nuclephile resulted in frmatin f the aminlysis prduct Bz-Arg-Val-OMe. Als in this case the fractin faminlysis reached a cnstant value but it was at a lwer level than was bserved with H-Val- NH2 (Figure 2). The bservatin that malt carbxypeptidase becmes saturated with nuclephiles such that the fractin f aminlysis des nt exceed.9 Carlsberg Res. Cmmun. Vl. 49, p ,

4 9 1. <,5- - < {E ix. S f [NUCLEOPHLE], M Figure 2. The influence f the cncentratin fnuclephile n the fractin faminlysis using Bz-Arg-OBu as substrate and H-Va-NH2 (-O-9 r H-Va-OMe (-O-O-) as nuclephile. The reactin cnditins were the fllwing: 2 mm-bz-arg-obu, 5 mm-edta, MM-malt carbxypeptidase, ph 7.5. The cncentratin f nuclephile is indicated. Reactin time: 2-2 rain. 8-95% f the substrate was cnsumed in the reactin. with H-Val-NH_, and.5 with H-Va-OMe is incmpatible with a prprtinality with the nuclephile cncentratin as predicted frm Scheme 1. These results indicate that the nuclephiles bind t the acyl-enzyme cmplex prir t the deacylatin reactin and the capability f the enzyme t bind nuclephiles may cnveniently be estimated frm the cncentratin f nuclephile required t prduce a fractin f aminlysis which is half the maximal aminlysis fractin. This cncentratin is designated KN(,p) since it is related t the dissciatin cnstant f the cmplex between acyl-enzyme and nuclephile. Table lists the KN(,pp) values fr a series f amin acid amides and amin acid methyl esters and presents in additin the fractins faminlysis bserved at saturatin with the nuclephiles. t is seen that KN(app) fr the amin acid esters varies much mre with the nature f the amin acid side-chain than Km(,pp) fr the crrespnding amin acid amides. Furthermre, the fractin faminlysis with amin acid amides is cnsistently higher than with amin acid methyl esters. The amin acid amides H-Pr-NH2 and H-Gu-tt-NH:, the D-enantimers H-va-NH2 and H-ala-OMe, and glycine N-methyl amide gave n aminlysis prducts. The influence f ph n malt carbxypeptidase catalyzed aminlysis reactins was studied with Bz-Arg-OBu as substrate and H-Val-NH2 as nuclephile (Figure 3). The enzyme reached saturatin with H-Val-NH2 at all ph values Table. Malt carbxypeptidase catalyzed synthesis f peptide bnds using Bz-Arg-OBu as substrate and amin acid amides and esters as nuclephiles Substrate Nuclephile Aminlysis prduct Fractin f KN(app) KN(app) aminlysis at with PhGu saturatin Bz-Arg-OMe H-Gy-NH_, Bz-Arg-Gy-NH_, Bz-Arg-OMe H-Ser-NH_, Bz-Arg-Ser-NH_~.9.3 Bz-Arg-OMe H-Leu-NH: Bz-Arg-Leu-NH, Bz-Arg-OBu H-Va-NH, Bz-Arg-Va-NH,.9.15 ~ >.3 Bz-Arg-OBu H-Gly-OMe Bz-Arg-Gly-OMe.65 > 3. ~ > 3. ~' Bz-Arg-OBu H-Aa-OMe Bz-Arg-Ala-OMe Bz-Arg-OBu H-Val-OMe Bz-Arg-Val-OMe KN,apm was determined frm plts f yield f aminlysis versus cncentratin f amine cmpnent (see Figure 2) in the absence and in the presence f 1 mm-phgu. Reactin cnditins: 2 mm-substrate, 5 mm-edta,.3-3 /am-malt carbxypeptidase,.2-4. M-amine cmpnent, ph 7.5, 25 ~ Reactin time: 2-2 minutes. Cnditins were selected such that 8-95% f the acyl cmpnent was cnsumed in the reactins, a) the same value f KN(~p) was btained with Bz-Ala-OMe as substrate, b) saturatin was nt btained with H-Gy-OMe as amine cmpnent and PhGu was fund nt t influence the yield f aminlysis at cncentratins f H-Gy-OMe up t 4. M. 476 Carlsberg Res. Cmmun. Vl. 49, p , 1984

5 ,~ t l ~9 m t~ z [a. z V- t,).8".6",4"" B t [.VALNE AMDE], M Figure 3. nfluence f the cncentratin f H-Va-NH_, n the fractin faminlysis at different ph values. Reactin cnditins: 2 mm-bz-arg-obu, 5 mm-edta,.3-3 pm-malt carbxypeptidase. Reactin time 2-2 minutes. ph 8., -Q-C)-; ph 7.5, -Q-O-; ph 6.5, -B-B-; ph 6., -x-x-; ph 5.5, -7-7-; ph 5., -A-A-. except ph 5. and the maximal fractin f aminlysis varied nly slightly with ph. Hwever, the cncentratin fnuclephile needed t saturate the enzyme increased drastically with decreasing ph with KN~app~ reflecting a grup with a pk, belw 5.5 (Figure 4). The cleavage fpeptide bnds by malt carbxypeptidase prceed like the cleavage f ester bnds via an acyl-enzyme intermediate. Cnsequently, it is t be expected that peptides als can functin as substrates fr aminlysis reactins. T demnstrate this, Bz-Lys-Ala-OH was used as substrate and H-Va-NH2 as nuclephile, and the reactin was studied in the ph range 3.5 t 7.. t was bserved that the fractin faminlysis was maximal at ph 4.25 where it reached apprximately.4 (Figure 5) while at ph 7. nly a small amunt f Bz-Lys-Va-NH2 was frmed. Hwever, due t the relatively lw yield f this reactin n further studies were perfrmed..4- =E.3 6. Q..2.1 i ph Figure 4. The influence fph n KN~.pp~ derived frm the curves shwn in Figure 3. t is assumed that the maximal yield at ph 5. is 85%. Carlsberg Res. Cmmun. Vl. 49, p ,

6 K. BREDDAM & M, OTTESEN: Aminlysis with malt carbxypeptidase r) Or) (~).4 <1:.3 L_.2 F- < nr".1 LL ph Figure 5. The influence f ph n the fractin f aminlysis in malt carbxypeptidase catalyzed transpeptidatin reactin. Reactin cnditins: 5 mm-bz- Lys-Ala-OH, 1.5 M-H-Val-NH2, 5 mm-edta,.3 /am-malt carbxypeptidase. Reactin time: 5-15 rain The influence f phenylguanidine (PhGu) n aminlysis reactins The results described in sectin 3. l. demnstrate that the acyl-enzyme cmplex has a binding site fr the added nuclephiles and it is likely that this binding site is identical with the S] binding site fr the leaving grup in the acylatin reactin. Hence, it wuld be expected that additin f PhGu, which previusly has been shwn t bind t the S~ binding site in malt carbxypeptidase (8), wuld have an effect n the aminlysis reactins. This was investigated by additin f PhGu t the malt carbxypeptidase catalyzed aminlysis f Bz-Arg-OBu using H-Val-NH2 and H-Va-OMe as nuclephiles. Althugh the limited slubility f the nuclephiles did nt allw sufficiently high cncentratins t be used t btain cmplete saturatin it appeared (Figure 6) that the the same maximal fractins f aminlysis was being apprached as was bserved withut PhGu (see Figure 2). Hwever, as seen frm a cmparisn between Figure 6 and Figure 2 much higher cncentratins f nuclephile were needed t saturate the enzyme in the presence f PhGu than in the absence f this substance, i.e, PhGu caused an increase in tt~.s - z.6 c~,,. O.4 O ~.zt.)! tr a_ [NUCLEOPHLE], M Figure 6. nfluence f PhOu n malt carbxypeptidase catalyzed aminlysis reactins using Bz-Arg-OBu as acyl cmpnent and H-Va-NH, (-O-O-) r H-Val- OMe (-e-o-) as amine cmpnents. The reactin cnditns were the fllwing: 2 mm-bz-arg-obu, 5 mm-edta, 1 mm-phgu,.3 /am -.6 lam-malt carbxypeptidase, ph 7.5. Reactin time: 2-2 minutes. The cncentratin f nuclephile is indicated. 8-95% f the awl cmpnent was cnsumed in the reactins. KN~~ As seen frm Table similar effects f PhGu were bserved with ther nuclephiles, with the exceptin f H-Gly-OMe, suggesting that PhGu cmpetes with these nuclephiles fr the S', binding site Kinetic studies f malt carbxypeptidase catalyzed aminlysis reactins The influence f H-Val-NH2 n the cleavage f Bz-Arg-OBu by malt carbxypeptidase (sectin 3.1.) was studied at 2 mm-substrate cncentratin which was apprximately 1 fld higher than Km (8). Hence, the rate f substrate disappearance reflected k~a, which was seen t decrease by the additin f H-Val-NH: (Figure 1). Hwever, nitrphenylesters are mre cnvenient substrates fr kinetic studies f this type since it is pssible spectrphtmetrically t fllw the rate f release f the leaving grup, i.e. nitrphenl. N,-CB-Lys-ONp is an excellent substrate f malt carbxypeptidase and it was used as substrate with H-Va-NH2 as nuclephile. When the fractin faminlysis was studied by HPLC 478 Carlsberg Res. Cmmun. Vl. 49, p , 1984

7 _u).8 to.6.4 g.2 '- to n- la_ J i ///1.8- v '1~7~ 1 2 [VALNE AMDE], M Figure 7. nfluence f the cncentratin f H-Va-NH, n the fractin f aminlysis using N,,-CB-Lys-ONp as substrate. Reactin cnditins: 2 mm-n,-cb-lys- ONp,.1 M-Mes, 1 mm-edta,.7 p_m-malt carbxypeptidase, ph 5.5. The cncentratin f H-Va-NH, varied as described in the Figure. 8-95% f the substrate was cnverted in the reactin which lasted 5-15 minutes. determinatin f the amunts f N-CB-Lys- OH and N-CB-Lys-Va-NH2 being frmed the shape f the curve (Figure 7) again indicated a saturatin with nuclephile with a maximal fractin f aminlysis f apprximately.8 and a Ky~ap~ f apprximately.4 M. When the rate f disappearance f N~-CB-Lys-ONp was determined spectrphtmetrically as a functin f the cncentratin f H-Val-NH2 the kinetic parameters indicated that k,a, rapidly decreased t apprximately 5% and then remained cnstant, suggesting that H-Val-NH~ exhibited partial nn-cmpetitive inhibitin with an apprximate Kj f.3 M. The cnstant value f k~,, at increasing nuclephile cncentratins abve.2 M suggested that the rate-limiting step in malt carbxypeptidase catalyzed actin n ester substrates lies ahead f the deacylatin step, i.e. acylatin is presumably rate-limiting (12). n cntrast t this, Km increased linearly ver the entire cncentratin range f H-Val- NH2 ( M), indicating that the nuclephile als exhibited cmpetitive inhibitin. The Kz f this inhibitin is.5 M which is much larger than the K, f.3 M fr the nn-cmpetitive inhibitin and this suggests that H-Val-NH2 binds t the enzyme at tw different psitins..4-- ' ' ;/ [VALNE AMDE], M 1.5 O 5 ca Figure 8. nfluence f the cncentratin f H-Va-NH2 n the kinetic parameters fr the disappearence f N,-CB-Lys-ONp. The reactins were perfrmed in.5 M-Mes, 1 mm-edta, ph 5.5. The cncentratin f -Lys-ONp varied frm.1-1. mm, and 7 x 1 9 M-malt carbxypeptidase was used. The kinetic parameters were determined frm linear Lineveawer- Burk plts at different cncentratins at H-Val-NH2 included in the assay mixture. 4. DSCUSSON The results presented in sectin 3 fr the actin f malt carbxypeptidase n ester substrates in the presence f added amine nuclephiles shwed saturatin phenmena which deviated frm the simple mdel in Scheme 1 which predicted a prprtinal partitining f the acyl-enzyme cmplex between hydrlysis and aminlysis. FNK and BENDER (1) have previusly bserved similar binding effects when larger n-alkyl alchls were added t papain acting n p-nitrphenyl substrates. They fund that their results culd nly be explained by adpting the reactin scheme presented in Scheme 2 in which the added nuclephile binds t the enzyme at tw different psitins. ES is the nncvalent enzyme-substrate cmplex, ES' is the awl-enzyme, P, is the substrate leaving grup, Pz is the hydrlysis prduct and P3 is the aminlysis prduct frmed when the nuclephile N is present. KN is the dissciatin cnstant fr nuclephile bund t the same site as the leaving grup, i.e. the S; binding site f the enzyme. K, is the dissciatin cnstant fr nu- Carlsberg Res. Cmmun. Vl. 49, p ,

8 k3[wl ~,E+P z KN[N] K S k z ] k4 EN -"..- E + S ""..-ES ES ~ -. ES~N ~'E+P 3 + N E N.,'----T. N E + S ~-----~ N E S ) NESm~..~7 NESN k4"~, N E + p3 KNEN:] K s P, /3 k3rw] ~NE+P 2 Scheme 2. Mdel prpsed by UNK and BENDER (1) to accunt fr the binding f nuclephiles t papain. The added nuclephile binds at tw different psitins f the enzyme with the dissciatin cnstants KN and K~, respectively. Water is assumed nt t bind t the enzyme prir t its participatin in the deacylatin reactin, ct and fl are factrs which accunt fr the pssible influence f bund nuclephile n the magnitudes f k~ and k3, respectively. Other abbreviatins used are defined in Scheme 1. clephile bund at a different site. a and 13 are factrs which accunt fr the pssible influence f nuclephile bund at this psitin n the magnitudes f k2 and k3, respectively. n ur studies, the influence f H-Val-NH2 n the steady-state parameters k,, and Km fr the actin f malt carbxypeptidase n N,-CB-Lys-ONp similarly demnstrated the existence f tw binding mdes fr the added nuclephile, ne causing cmpetitive inhibitin with a K, f.5 M, the ther causing nn-cmpetitive inhibitin with an apprximate K~ f.3 M. These results are cmpatible with the mechanism presented in Scheme 2 when it is assumed that KN =.5 M and K, =.3 M fr H-Va-NH2 at ph 5.5. The fact that KN(,p,~ was determined t apprximately.4 M frm the crrelatin f fractin faminlysis, P3/(P2 + P3), versus nuclephile cncentratin, [N], and that this is in agreement with the kinetically determined K~ value f.5 M SUpprts that Scheme 2 is a gd describtin f the actin f malt carbxypeptidase n ester substrates and that KN(app~ is a reasnably gd measure fr KN. PhGu has previusly been demnstrated t bind t the S', binding site f malt carbxypepti- dase since it weakened the binding f substrates with bulky leaving grups (8). The present results demnstrated that additin f PhGu t malt carbxypeptidase catalyzed aminlysis reactins drastically increased KN(ap~ fr all the tested nuclephiles (except H-Gly-OMe), but it had n effect n the maximal level faminlysis. This is cnsistent with a cmpetitin between PhGu and the binding f nuclephiles at the S~ psitin. The fact that PhGu had n effect n the aminlysis reactins with H-Gly-OMe but strngly affected KN(ap~ fr H-Ala-OMe and H-Val-OMe might suggest that it is the sidechains f amin acid methyl esters which are bund t the S; binding site in such a manner that they ccupy r verlap with the binding site fr PhGu. N such differences were bserved with amin acid amides since PhGu affected KN(app~ fr all amin acid amides tested including H-Gly-NH2. This suggests that these tw types f nuclephiles exhibit slightly different binding mdes within the S; binding site f the enzyme. These different mdes f binding may explain why the fractin faminlysis at saturatin with nuclephile is much higher with amin acid amides than with amin acid methyl esters. 48 Carlsberg Res. Cmmun. Vl. 49, p , 1984

9 Hwever, the binding f bth types f nuclephiles appear t be sterespecific since the D- enantimers H-va-NH2 and H-ala-OMe gave n aminlysis prducts. The nuclephiles used in the present study exist in slutin bth in an unprtnated (amine) frm and a prtnated (ammnium) frm. Hwever, nly the amine frm participates as nuclephile in the deacylatin reactin and cnsequently, KNap1 refer t the dissciatin cnstant f the cmplex between the amine frm f the nuclephile and the acyl-enzyme intermediate. Thus, in the case f H-Val- NH2 it wuld be expected that KN~app) wuld reflect the deprtnatin f a grup with a pka f 8. which is the pka f H-Val-NH2. Hwever, the aminlysis was dependent n a grup with a pka belw 5.5 which suggested that H-Val-NH2 when bund t the enzyme exhibited a pka signifcantly different frm that bserved in slutin. The deacylatin reactin with H-Val- NH2 as nuclephile represents the reverse f an alkylatin reactin with H-Val-NH2 as leaving grup. Thus, after cleavage f a peptide bnd it is cnceivable that the leaving grup remains unprtnated while still bund t this particular binding site even at ph values far belw its pka in slutin, and the dissciatin f this grup frm the enzyme will be dependent n the dissciatin cnstant. t remains t be established whether this feature is functinally significant but ne might imagine that the prtnatin and subsequent ejectin f the leaving grup culd be linked t a cnfrmatinal change f the active site regin f the enzyme. REFERENCES 1. BENDER, M.L., G.E. CLEMENT, C.R. GUNTER & F.J. KEDY: The kinetics f et-chymtrypsin reactins in the presence f added nuclephiles. J. Am. Chem. Sc. 86, (1964) 2. BREDDAM, K., F. WDMER & J.T. JOHANSEN: Carbxypeptidase Y catalyzed transpeptidatins and enzymatic peptide synthesis. Carlsberg Res. Cmmun. 45, (198) 3. BREDDAM, K.. F. WDMER & J.T. JOHANSEN: nfluence f the substrate structure n carbxypeptidase Y catalyzed peptide bnd frmatin. Cadsberg Res. Cmmun. 45, (198) 4. BREDDAM, K., F. WDMER & J.T. JOHANSEN: Carbxypeptidase Y catalyzed C-terminal mdificatins f peptides. Carlsberg Res. Cmmun. 46, (1981) 5. BREDDAM, K., F. WDMER & J.T. JOHANSEN: Carbxypeptidase Y catalyzed C-terminal mdificatin in the B-chain f prcine insulin. Carlsberg Res. Cmmun (1981) 6. BREDDAM, K., F. WDMER & J.T. JOHANSEN: Amin acid methyl esters as amine cmpnents in CPD-Y catalyzed peptide synthesis: Cntrl f side reactins. Carlsberg Res. Cmmun. 48, (1983) 7. BREDDAM, K., S. SORENSEN & M. OTTESEN: slatin fa carbxypeptidase frm malted barley by affinity chrmatgraphy. Carlsberg Res. Cmmun. 48, (1983) 8. BREDDAM, K. & M. OTTESEN: nfluence f guanidine derivatives n the specificity f malt carbxypeptidase. Carlsberg Res. Cmmun. 48, (1983) 9. FASTRE, J. & A.R. FERSH'r: Demnstratin f the awl-enzyme mechanism fr the hydrlysis f peptides and anilides by chymtrypsin. Bichemistry 12, (1973) 1. FNK. A.L. & M.L. BENDER: Binding sites fr substrate leaving grups and added nuclephiles in papain-catalyzed hydrlyses. Bichemistry 8, (1969) 11. SCHECHTER,. & B. BERGER: On the size f the active site f prteases.. Papain. Bichem. Biphys. Res. Cmmun. 27, (1967) 12. SEYDOUX, F. & J, YON: Cmpetitin nuclephite dans les reactins d'hydrlyse enzymatique. Analyse cinetique et applicatin a l'hydrlyse trypsique de quelques esters. Eur. J. Bichem. 3, (1967) 13. WDMER, F. & J.T. JOHANSEN: Enzymatic peptide synthesis. Carbxypeptidase Y catalyzed frmatin f peptide bnds. Carlsberg Res. Cmmun. 44, (1979) 14. WDMER, F., K. BREDDAM & J.T. JOHANSEN: Carbxypeptidase Y catalyzed peptide synthesis using amin acid alkyl esters as amine cmpnents. Carlsberg Res. Cmmun. 45, (198) 15. WDMER. F.. K. BREDDAM & J.T. JOHANSEN: nfluence f the structure f amine cmpnents in carbxypeptidase Y catalyzed amide bnd frmatin. Carlsberg Res. Cmmun. 46, (1981) Carlsberg Res. Cmmun. Vl. 49, p ,