1976 589Synthesis and Catalytic Properties of Peptides containing Amino-acidsinvolved in the Active Centres of Hydrolytic EnzymesBy lphigenia Photaki * and Maria Sakarellou-Daitsiotou, laboratory of Organic Chemistry, the UniversitySeven oligopeptides of the type indicated in the title have been synthesised and found to catalyse the hydrolysis ofp-nitrophenyl acetate with a catalytic activity of ca. 30--50% of that of imidazole.of Athens, Athens 144, GreeceBOTH the amino-acid sequence in the region of the ' activeserine ' residue and the sequence near the histidine residueof the ' active site ' are similar in such hydrolytic enzymesas chymotrypsin, trypsin, and e1astase.l This similarityin primary structure suggests that the tertiary structurescould also be similar, in which case the amino-acidresidues comprising the active centres of these enzymeswould be in similar conformations.2 The histidinesequence, which is far removed from the active serinesequence in the primary structures of these enzymes,contains a second histidine residue close to the first one,and a mechanism of action of a-chymotrypsin has beenproposed 3 in which both histidine residues participate inthe catalytic process.Many relatively small peptides containing the amino-acid residues corresponding to the active centres ofenzymes have been synthesised and studied as ' hydrolasemodels ' in comparison with a-chymotrypsin andimidaz~le.~.~ In most cases the observed catalyticactivity is significant but low, probably owing to theinability of the molecule to realise a conformationsimilar to that of the active centre of a real enzyme.Histidine and serine residues are usually contained insuch enzyme models.In the present investigation thepeptides (D)-(G), each containing one serine and twohistidine residues, and the smaller peptides (A)-(C) havebeen prepared and used as hydrolase models in compari-son with chymotrypsin and imidazole. Molecular modelsindicate that a molecule containing one serine and twohistidine residues could show considerable flexibility ifthere are two amino-acid residues between the two histi-dines and also between the serine and the first histidineunit. To separate the serine and histidine in this wayglycine was chosen, in order to avoid complicationsresulting from side-chain interactions or steric hindrance.Gly-His-GIy (A)Phe-Gly-His-Gly (B)Gly-His-Gly-Gly-His-Gly (C)Ser-Gly-Gly-His-Gly-Gly-His-Gly (D)Asp-Ser-Gly-Gly-His-Gly-Gly-His-Gly-OEt (E)Ser-Gly-Gly-His-Gly-Gly-His-Gly-Asp (F)Gly-Asp-Ser-Gly-GIy-His-Gly-Gly-His-Gly-OEt (G)In addition to serine, histidine, and glycine, aspartic acidis included in the nonapeptides (E) and (F), and thedecapeptide (G) contains the sequence around the activeserine residue of some proteolytic enzymes, i.e.Gly-Asp-M. L. Bender and F. J. Kezdy, Ann. Rev. Biochem., 1965,34,A. Williams, Quart. Rev., 1969, 23, 1.B. S. Hartley in ' Structure and Activity of Enzymes,' ed.T. W. Goodwin, J. I. Harris, and B. S. Hartley, Academic Press,1964, p.47.49.Ser-Gly-Gly. A glycine residue was included at the endof the peptide chain to give the molecule still moreflexibility.' The tetrapeptide (B) was prepared in orderto test the influence of the phenylalanine * residue on thecatalytic activity of the tripeptide (A).The synthetic route to the peptides (A)-(G) is shownin Schemes 1 and 2. The starting material N-benzyl-oxycarbonylglycyl-L-histidylglycine ethyl ester (I) wasprepared by a slight modification of a literature m e t h ~ d . ~Saponification of the protected hexapeptide (V) andoctapeptide (VII) proved difficult, so the catalytichydrogenolysis was performed first .lo Compounds (X)and (E) in the crystalline state proved insoluble even indimethylformamide ; for coupling purposes, these com-pounds were therefore used in the oily state, i.e.beforecrystallization.Peptides (A)-(G) showed a catalytic activity for thehydrolysis of 9-nitrophenyl acetate (p-NPA) (expressedby the catalytic coefficient A,") of ca. 30-50% of thatof imidazole (see Table). First-order kinetics wereHydrolysis of P-nitrophenyl acetate in 0.2M-phosphatebuffer (pH 7.7) containing 2.7% dioxan (v/v) at 27-28 "C; [p-nitrophenyl acetate] = 32 pmol 1-l[His] x lo4/ K, x lo4/ k,/l mol-1Catalyst n1011-1 min-1 min-1None 42Imidazole 4.5 186 324.4 104 144.1 84 104.4 183 161.5 76 112.25 93 114.5 136 104.4 145 124.7 124 91.48 75 114.5 134 10~ ~ ~ _ _ _ _Cf. e . g . (a) E. Katchalski, G. D. Fasman, E. Simon, F. R.Blout, F.R. N. Gurd, and W. L. Koltun, Arch. Biochem. Biophys.,1960, 88, 361; K. D. Kopple, D. E. Nitechi, J . Amer. Chem. Soc..1961,83,4103; C. G. Overberger, T. St. Pierre, N. Vorschheimer,and S. Yaroslavsky, J . Amer. Chem. SOC., 1963, 85, 3513; F.Schneider. 2. physiol. Chem., 1967, 348, 1034; G. Losse and H.Weddige, Annalen, 1964, 678, 148; J. Sheehan, G. Bennett, andJ. Schneider, J . Amer. Chem. SOC., 1966, 88, 3455; W. H. Carr,Diss. Abs., 1968, B28 (7). 2711; K. Nakajima and K. Okawa,Bull. Chem. SOC. Japan, 1973, 46, 1811; M. Fridkin and H. J.Goren, European J . Biochem., 1974, 41, 273; (b) I. Photaki, V.Bardakos, A. W. Lake, and J. Lowe, J . Chem. SOC. (C), 1968,1860;(c) I. Photaki and S. Moschopedis, Expevientia, 1969, 25, 903.W.P. Jencks and J. Carriulo, J . B i d . Chem., 1959, 234,1272, 1280.B. Zerner and M. L. Bender, J . Amer. Chem. SOC., 1964, 86,3669; M. L. Bender and B. W. Turnquest, ibid., 1967, 79, 1662. ' F. B. Abramson, D. F. Elliott, D. G. Lindsay, and R. Wade,J . Chem. SOC. (C), 1970, 1042. * A. Kapoor, Progr. Peptide Res., 1971, 2, 44.R. F. Fischer and R. R. Whetstone, J . Amer. Chem. SOC.,1954, 56, 5078.lo R. Schwyzer, B. Iselin, H. Kappeler, B. Riniker, W. Rittel,and H. Zuber, Helv. Chim. Acta, 1968, 41, 1287J.C.S. Perkin Iconcentrations of the peptides (A)-(G) were calculatedon the basis of histidine residues. The catalytic coeffici-ent k, of peptides (D)-(G) would be doubled, but stilllower than that of imidazole itself, if the concentrationsof the peptides were calculated in terms of a mechanisminvolving both histidine residues.2A change in optical density at 245 nm, indicating theprobable formation and decomposition of an Nh-acetylpeptide inter~nediate,~ was observed in the case of thetripeptide (A) when used at a concentration of 5 xmol 1-1 in the presence of p-NPA (1.2 x 104 mol 1-l).The peptides (D)-(G) containing two histidine residuescould not be compared since solutions in this range ofconcentration could not be prepared.Finally, the possibility of hydrolysis of $-NPA by nucleo-philic attack by the a-NH, groupll was excluded byelectrophoresis of mixtures where the substrate concen-tration was greater (10 X) than that of the peptidecatalyst. After 2 4 days incubation only peptide withfree a(-NH, group was detected.A comparison of the k, values obtained for varioussynthetic peptides, including those reported here, doesnot produce any general conclusions which could help topredict the catalytic ability of a new peptide.Beforeany more new peptides are synthesised as esterase models,more information about their tertiary structure in rela-tion to that of the enzymes is needed.observed from ca. 10% to ca. 70% completion of thereaction. The liberation of P-nitrophenol was followediiZ-Gly-His-Gly-OH -B- Gly-His-Gly .). (11) (4ii(IV) iiiGly-Gly-(MeOH) I(1)___) Gly-His-Gly-OEtZ-Gl y-His-Gl y-OEt- Z-Glv-His-Glv-NH-NH(IV) Z-Gly-His-Gly-Gly-His-Gly-OEt Z-Gly-His-Gly-N,i(C)1" (v)iGly-His-Gly-Gly-His-Gly-OEt + Gly-His-Gly-Gly-His-Gly1z-:e21y-N3Z-Ser-Gly-Gly-His-Gly-Gly-His-Gly-OE t(VII)iii (Me,N*CHO)Z-Ser-Gly-Gly-His-Gl y-Gly-His-Gly-NHNH,I ( V W 1 ivt Z-Ser-Gly-Gl y-His-Gly-Gl y-His-Gly-N,Z-Ser-Gl y-Giy -His-GI y-Gly -His-Gl y- Asp-ONdNb(1x13.Ser-G1 y-G1 y-His- G1 y-G1 y-His- G1 y- Asp(F)SCHEME 1 Reagents: i, N-NaOH added to aqueous or alcoholicsolution; ii, Pd-H, in AcOH containing water or water andan alcohol; iii, NH,NH,; iv, H+-NaNO,; Nb = p-nitrobenzylEXPERIMENTALThe whole Experimental section is available as Supple-mentary Publication No. SUP 21 631 (17 pp., 1 microfiche).*Only key experiments are also described here.Anhydrous solvents were used for the coupling reactions.M.p.s were taken for samples in capillary tubes.Beforeanalysis, compounds were dried over P,O, at room temper-ature under high vacuum ; microanalyses were performed atthe Analytical Laboratory of F. Hoffman-La Roche and Co.,Basle, Switzerland, under the direction of Dr. A. Dirscherl,or by A. Bernhardt, Elbach, Germany.Amino-acid analyses were carried out a t the NuclearResearch Centre ' Demokritos ', Aghia Paraskevi Attikis,1(DlSer-Gly-Gly-His-Gly-Gly-His-Gly-OEt __e Ser-Gly-Gly-His-Gly-Gly-His-GlyiiZ-Asp-Ser-Gly-Gly-His-Gly-Gly-His-Gly-OEt + Asp-Ser-Gly-Gly-His-Gly-Gly-His-Gly-OEt(El(a) Z-Gly-ONp I (b) ii(XI)bBZ13. Gly-Xsp-Ser-Gly-Gl y-His-Gly-Gly-His-Gly-OEt(GISCHEME 2 Reagents: i, N-NaOH added to aqueous or alcoholic solution; ii, Pd-H, in AcOH containing waterby U.V.spectrometry at 400 nm.(G) were tested at various concentrations.Authors No. 7, J.C.S. Perkin I , 1975, Index issue.Peptides (D) andThe molar* For details of Supplementary Publications see Notice toGreece. RF Values refer to t.1.c. on Kieselgel G (Fluka)containing 13% Gibs in the following solvent systems:W. L. Koltun and F. R. N. Gurd, J. Amer. Chew. SOC., 1959,81, 3011976 591(proportions by volume) : ( 1) butan- 1-ol-acetic acid-water(6 : 2 : 2) ; (2) butan-l-ol-acetic acid-water (4 : 1 : 1) ; (3)butan-l-ol-acetic acid-water-pyridine (30 : 6 : 24 : 20) ; (4)phenol-water (3 : 1) ; (5) chloroform-methanol (4 : 1) ; (6)propan-l-ol-33yo ammonium hydroxide (67 : 33). Sub-stances with a free amino-group were detected with ninhy-drin, and N-protected derivatives with iodine.Histidine-containing compounds were also detected by use of the Paulyreagent.12 For paper electrophoresis, an LKB 3 276 appar-atus was used, with Schleicher and Schiill2 043-B paper, or ahigh-voltage Pherograph-L Hormuth apparatus, withMX-Duren paper. The following solvent systems wereemployed : (a) acetic acid (5.7 m1)-pyridine (8 nil)-water(to 1 1) (pH 4.9) ; (b) acetic acid (18.25 m1)-pyridine (8 m1)-water (to 1 1) (pH 3.9); (c) 0.5~-acetic acid; (d) acetic acid-formic acid (pH 1.85) .' Unless otherwise stated compoundsiiioved as single bands toward the cathode. Neutralizationequivalents were found by titration with perchloric acid inacetic acid.l3Optical rotations were measured with a Perkin-Elmerautomatic polarimeter (1 dm cell).Kinetics were followedwith a Zciss RPQ 20A spectrophotometer.GZycyE-L-histidylglycine Ethyl Ester (IV) Acetate.-TheAT-protected tripeptide ester (I) (4 g, 0.0092 mol) in 90%acetic (130 ml) was hydrogenated a t room temperature andpressurc during 1 h over palladium-charcoal (10% ; 0.5 g).The mixture was then filtered and washed with water. Thefiltrate was evaporated to dryness in vacuo. The excess ofacid was removed by addition and evaporation of ethylalcohol several times, and the residue was extracted withethyl alcohol giving the ester acetate (2.3 g, 70%), m.p. 161"[ ( Z g, 60y0), m.p. 163-165" after trituration with hot aceto-nitrile], [ M ] , ~ ~ - 8.9" (c 2 in N-HCl), RF (3) 0.6, neutralizationequiv. 361 (calc.357) (Found: C, 46.8; H, 6.6; N, 19.4.C,,H,,N,O,,AcOH requires C, 46.9; H, 6.8; N, 19.5%).Glycyl-L-hzstidylglycine (A) Acetate.-A suspension ofcompound (11) (1.6 g, 0.0039 mol) in water (31 ml), methanol(62 ml), and acetic acid (3.5 ml) was hydrogenated as des-cribed for compound (IV). The residue was dissolved inmethanol and precipitated with ether to give the crudeacetate (1.1 g, 90%), m.p. 217" [218-219" after recrystal-lization from water (recovery SO%)], [aIDz4 -10.5' (c 1 inO.lx-KaOH), RF (4) 0.4; paper electrophoresis (350 V; 3h) in system (a) (Found: C, 41.95; H, 5.85; N, 22.1.C,H,,N,O,,AcOH requires C, 41.8; H, 5.7; N, 22.1%).N-BenzyZoxycarbonyZglycyZ-r2-histidyZglycyZgZycy 2-L-histidy Z-glycine Ethyl Ester (V).-(a) Compound (I) (1.3 g, 0.003mol) was dissolved in methyl alcohol (10 ml) by gentlywarming.The solution was allowed to cool to room temper-ature and hydrazine hydrate (1.2 ml) was added. Thecrystalline N-benzyloxycarbonylglycyl-L-histidylglycylhj-drazide (111) formed was isolated after 24 h ; m.p. 200-201' after boiling in methanol (yield 1 g, 95%), [0(ID34 - 15"(c 1 in N-HCl), RF (3) 0.8; (lit.,9 m.p. 199-201").(b) A solution of the hydrazide (111) (1.25 g, 0.003 mol)in N-hydrochloric acid ( 12 ml) and dimethylformamide (30nil) was treated at -5 "C with a cold aqueous M-sodiumnitrite (3.3 ml). After 5 min the ester acetate (IV) (1.071 g,0.003 mol) was added to the resulting solution of the azide,followed by triethylamine (2.3 ml; pH of the mixture ca.7).The niixture was stirred a t 0 "C for 2 h and left at 4 "C forl2 J; P.. Greenstein and M. Winitz, ' Chemistry of the hmino;Icicls, Wiley, New York, 1961, p. 1981.24 h. The precipitated product was filtered off, washed witha little dimethylformamide, water, O.S~-boric acid, andwater, and dried in vacuo (Pz05 and NaOH); m.p. 245",unchanged after recrystallization by addition of water to asolution in dimethylformamide ; yield 1.6 g (80%) , [0iJD2*-20" (c 1 in N-HCl), RF (3) 0.8 (Found: C, 52.6; H, 5.4; N,20.4. C,,H,,N,,O, requires C, 52.8; H, 5.6; N, 20.5%).GZycyl-L-histidylglycyZgZycy2-L-histidyZgZycine (C) Acetate.-The ester (VI) acetate (0.6 g, 0.001 mol) was dissolved bygentle heating in 85% ethanol (13 ml).N-Sodium hydrox-ide (2.8 ml) was added and 3me solution was stirred for 2.5 hat room temperature 3men acidified with N-hydrochloric acid(2.8 ml). The precipitated crystalline p,roduct was recrystal-lized from water; yield 0.27 g (48%), m.p. 230", [aJD20- 11" (c 1 in N-HCl), RF (4) 0.5; paper electrophoresis insystem (a) or (b) (1 500 V; 2 h) and (d) (350 V; 3 h);neutralization equiv. 547 (calc. 582) (Found: C, 45.0;H, 5.5; N, 24.4. C,oH30N,o0,,AcOH requires C, 45.36;H, 5.9; N, 24.0%).L-Sery ZgZycy ZgZycyZ-L-his~~dy Zglycylglycy Z-L-~~ istidylgzycine(D) Acetate.-Compound (X) (0.74 g, 0.001 mol) was dis-solved in hot water (15 ml). h.-Sodium hydroxide (5.6 ml)was added a t room temperature ; after 3 h a small amount ofundissolved material was filtered off, and the solution wasacidified with N-hydrochloric acid (5.6 ml).The clearsolution was passed through a column of Dowex 50 W (H+),which was washed with water until chloride ions were com-pletely removed. The peptide was eluted with 3% ammon-ium hydroxide. The eluate and washings were concentratedin vacuo. Water was added and evaporated off repeatedlyand the peptide was precipitated with ethyl alcohol. It wascollected by centrifugation; yield 0.5 g (69y0), m.p. 233"-234" (from water; recovery 80y0), [a],2o -5.4" (c 1 inN-HCl), l i p (4) 0.3; paper electrophoresis (350 V; 2.6 h) insystem (d) and (1 500 V; 2 h) in system (a): amino acidanalysis: His, 2; Ser, 0.755; Gly, 5; neutralization equiv.727 (calc. 726) (Found: C, 44.5; H, 5.8; N, 23.15. C25Hae-N,,O,,,AcOH requires C, 44.7; H, 5.6; N, 23.2%).histidylglycine Ethyl Ester (E) .-The protected peptide ester(XI) (3 g, 0.003 rnol) was hydrogenated in 50% acetic acid(150 ml) for 3 h over palladium-charcoal (1.4 g). Themixture was filtered and evaporated (0.5-1 mmHg) and theoily residue was dissolved in ethyl alcohol and precipitatedwith ethyl acetate. The crystalline material obtained waspurified by dissolving in water and precipitating with ace-tone; yield 2.3 g (go%), m.p. 233--235", [aID2, - 13.9" (c 1 inN-HCl), -8.7" (c 1 in glacial AcOH); paper electro-phoresis (1 500 V; 2 h) in system (b) ; amino-acid analysis:His, 1.88; Ser, 0.93; Gly, 5; Asp, 1.11 (Found: C, 46.1;H, 5.7; N, 21.5. C31H46N13019rAcOH requires C, 45.7; H,L-A spartyZ-L-seryZgZycyZg1ycyZ-L-histidyZgZycyZgZycyZ-L-5.7; N, 21.0%).We thank the National Hellenic Research Foundation forfinancial support. We are grateful to Drs. R. Studer andA. Dirscherl, Hoffmann La Roche and Co., Basle, for ele-mental analyses, and to Dr. Chr. Zioudrou, Nuclear ResearchCentre ' Demokritos ', Aghia Paraskevi Attikis, Greece forthe amino-acid analyses.[6:6/1639 Received, 19th Azdgwst, 19761l3 A. Patchornik and S. Shaltiel, Bull. Res. Comm. Israel, 1962,11A, 224
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