Thermolysin-Catalyzed Peptide Bond Synthesis (Enzyme Kdnetics/Peptides/High-Performance Liquid Chromatography) SUSAN I

Proc. NatL Acad. Sci. USA Vol. 80, pp. 3241-3244, June 1983 Biochemistry

Thermolysin-catalyzed peptide bond synthesis (enzyme kdnetics/peptides/high-performance liquid chromatography) SUSAN I. WAYNE AND JOSEPH S. FRUTON* Kline Biology Tower, Yale University, New Haven, Connecticut 06520 Contributed byJoseph S. Fruton, February 28, 1983

ABSTRACT The rates of the thermolysin-catalyzed synthesis catalyzed synthesis of oligopeptides of the type A-X-Leu-NHPh of peptides have been determined by means of HPLC. In the con- by the condensation of A-X-OH (where A is an amino-blocking densation of various N-substituted amino acids and peptides with group and X is an amino acid or peptide residue) with H-Leu- L-leucinanilide, the enzyme exhibits preference for a hydrophobic NHPh. These data have given information about the primary L-amino acid as the donorof the carbonyl group of the newlyformed and secondary specificities of the enzyme with respect to the bond. The presence of another hydrophobic amino acid residue RCOOH component, and a comparison may be made with the adjacent to the carbonyl-group donor markedly enhances the rate available kinetic data on the hydrolytic action of thermolysin on of synthesis. In general, the effect of structural changes in both the carboxyl and amine components of the condensation reaction comparable oligopeptide substrates. Similar experiments were is in accord with the available data on the primary and secondary performed on the initial rates of the thermolysin-catalyzed syn- specificities of the thermolysin-catalyzed hydrolysis of oligopep- thesis of Z-Phe-Y-B (where Y is an amino acid or peptide res- tide substrates. A kinetic study of the condensation of benzyl- idue and B is a carboxyl-blocking group) by the condensation oxycarbonyl-L-phenylalanine with various amine components has of Z-Phe-OH with H-Y-B. Second, a study has been made of given data on the apparent kept and Km values for the entry of the the effect of changes in the nature of the H-Y-B component on acidic component into the condensation reaction. The results are the apparent K. and kay values associated with a single RCOOH consistent with the behavior of rapid-equilibrium random bi- component-namely, Z-Phe-OH. This work was undertaken to reactant systems leading to ternary enzyme-substrate complexes, examine the possibility that the interaction of the two com- with a synergistic effect in the binding of the two reactants at the ponents in the condensation reaction with the active site of active site. Because the changes in the apparent kct for the entry thermolysin is synergistic, so that changes in the structure of of the same acidic component into reaction with different amine one of them may influence the manner in which the other com- components are greater than those in the apparent K., it is sug- ponent is bound productively for reaction. gested that this synergism is largely expressed at the level of the Thermolysin has been shown to consist of two rounded do- transition-state complex. mains with a deep cleft between them that contains an extended active site (4). Its catalytic action involves the partici- In a previous report from this laboratory (1), some features of pation of a zinc atom (5) and its thermostability depends in part the specificity of swine pepsin as a catalyst of condensation re- on the presence of 4 atoms of calcium per molecule (6). The actions leading to peptide bond synthesis were described. In available data indicate that the role of the zinc atom in the hy- reactions of the type RCOOH + NH2R' = RCO-NHR' + H20, drolytic mechanism of thermolysin action is to serve as a Lewis the unfavorable equilibrium for synthesis in aqueous solution acid in attacking the carbonyl oxygen of the sensitive bond and may be counteracted by the removal of the peptide product that the carbonyl carbon is attacked by a water molecule, with through insolubility and the use of organic co-solvents to shift glutamate-143 serving as a general base (7). Extensive studies the pK' of the RCOOH component (2). At suitable concentra- on the hydrolytic specificity of the enzyme (8-12) have shown tions of the enzyme, of the appropriate reaction components, that it preferentially cleaves peptide bonds in which the imino and of the co-solvent, the condensation reaction may be driven group is donated by a hydrophobic amino acid residue (leucine, to 95-100% completion within a reasonable time period. Anal- phenylalanine, etc.) and that the rate of cleavage is enhanced ysis by means of HPLC of the incubation mixture during the by the presence of a hydrophobic amino acid residue as the do- initial stages of the reaction permitted determination of the rel- nor of the carbonyl group of the sensitive bond. A widely used ative rates of peptide bond synthesis by pepsin, when the na- substrate is N-3(2-furyl)acryloylglycyl-L-leucinamide (Fagla); its ture of RCOOH and of NH2R' was varied, and thus to deter- hydrolysis may be followed spectrophotometrically at 345 nm mine the specificity of the enzyme as a catalyst in the con- (13). Thermolysin has also been shown to hydrolyze the ester densation reaction (1). It was found that the primary specificity analogues of suitable peptide substrates-for example, Bz-Phe- of pepsin with respect to the nature of the amino acid residues Pla-Ala-OH (14). In specificity, therefore, thermolysin resem- joined in the reaction, as well as the secondary specificity with bles pepsin and related aspartyl proteinases (3). respect to the nature of amino acids further removed from the As part of the recent renewed interest in the possibility of site of peptide bond synthesis, was similar to the specificities using proteinases as catalysts in preparative peptide synthesis observed previously in the study of the kinetics of the hydro- (2, 15, 16), studies on the catalysis of peptide bond synthesis lysis of oligopeptide substrates by swine pepsin (3). by thermolysin have shown that high yields of dipeptide de- This approach has been applied to the proteinase thermo- rivatives can be obtained by the condensation of Z-Phe-OH with lysin. In what follows, two aspects of the specificity of this en- H-Leu-NH2 or H-Leu-NHPh (17, 18). Also, the reaction has zyme as a catalyst of peptide bond formation are considered. First, data are presented on the initial rates of the thermolysin- Abbreviations: Z, benzyloxycarbonyl; Fagla, N-3(2-furyl)acryloylglycyl- L-leucinamide; Pla, -phenyl-L-lactyl; Phe(NO2), p-nitro-L-phenyl- The publication costs ofthis article were defrayed in part bypage charge alanyl; Bzl, benzyl; Boc, t-butyloxycarbonyl. The abbreviated desig- payment. This article must therefore be hereby marked "advertise- nation ofamino acid residues denotes the L form, unless otherwise stated. ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 3241 Downloaded by guest on October 2, 2021 3242 Biochemistry: Wayne and Fruton Proc. Natl. Acad. Sci. USA 80 (1983) been shown to be stereospecific with respect to both amino acid Phe-OH (n = 0, 1, 2) with H-Leu-NHPh is most rapid with Z- residues forming the peptide bond. A kinetic study of the ther- Phe-OH and progressively slower upon the insertion of one or molysin-catalyzed synthesis of Z-Asp-Phe-OMe from Z-Asp-OH two glycyl residues into the acidic component. This result is the and H-Phe-OMe has been reported (19). reverse of that obtained with swine pepsin (1) and is in qual- itative agreement with the relative rates of thermolysin-cata- MATERIALS AND METHODS lyzed hydrolysis of Z-(Gly)n-Phe(NO2)-Leu-Ala-OH at the Phe(NO2)-Leu bond (11). Replacement of the glycyl residue of Crystalline thermolysin (Calbiochem) was handled in the man- Z-Gly-Phe-OH by an alanyl residue markedly enhanced the rate ner described by Latt et al. (5), and the spectrophotometric as- of synthesis. The slow rate of the condensation of Z-Gly-Gly- say of its activity toward Fagla (13) gave a kt/Km value of 0.001 Phe-OH with H-Leu-NHPh is not a consequence of the es- mM-'min-'. Protein concentrations were determined spectro- tablishment of an equilibrium below 95% synthesis owing to photometrically at 280 nm, with the assumption that thermo- the solubility of the product because at a higher enzyme con- lysin has a molar absorptivity of 66,400 and a molecular weight centration (0.01 mM), this reaction attained 98% completion of 34,600. The amino acid and peptide derivatives used as within 2 hr. Replicate estimates from the initial rate data of ket/ RCOOH and H2NR' components were drawn from our collec- Km for the condensation of Z-Phe-OH and H-Leu-NHPh by tion or purchased from Sigma or from Research Organics means of the integrated Michaelis-Menten equation gave a value (Cleveland, OH). Their identity and purity were checked by of approximately 170 mM-1min-1. It may be added that the melting point determinations and HPLC; when necessary, they addition of 1.5 M (NH4)2SO4 did not alter the rate of reaction were recrystallized before use. significantly, in agreement with earlier experiments (1) show- The rates of the enzyme-catalyzed condensation reactions ing that the precipitation of the product is not rate-limiting in were determined at 370C, and each point of the rate curve was the overall process. provided by a separate incubation mixture (100 1.l) containing Table 1 summarizes the relative kcat/Km values for the con- the RCOOH and H2NR' components, thermolysin, and di- densation of various acidic components with H-Leu-NHPh un- methyl sulfoxide at the concentrations indicated in the tables. der the conditions stated in Fig. 1. The relative primary spec- The incubation mixture also contained 0.05 M calcium acetate ificity of thermolysin, with a preference for hydrophobic amino and 0.5 M Tris buffer; the apparent pH (glass electrode) of the acids as donors of the carboxyl group, is in accord with the mixture was 7.5. The reaction was stopped by dilution with 1 available data on the hydrolytic action of this enzyme (8). The ml or more of the HPLC eluant mixture, and the HPLC anal- slow rate observed with Z-Val-OH indicates that, as in the case ysis was performed with the apparatus and in the manner de- of swine pepsin, p-substituted amino acid residues are not suit- scribed by Bozler et aL (1). able acyl donors in the enzyme-catalyzed synthesis despite their For all the products whose elemental analysis (C, H, N) was hydrophobic character. determined, the results accorded satisfactorily with theory. To As noted in Fig. 1, the replacement of a glycyl residue by conserve space, the descriptions (melting point, elemental an alanyl residue in the so-called P2 position of the acid com- analysis, HPLC retention time) of the many compounds pre- ponent markedly enhanced the rate of synthesis. Table 1 shows pared by enzymatic synthesis are not included. that this effect is especially marked and is stereospecific. Whereas Z-Gly-Gly-OH was a relatively ineffective component, Z-Ala- RESULTS AND DISCUSSION Gly-OH was an excellent reactant and Z-D-Ala-Gly-OH did not In Fig. 1, representative rate curves are shown for the ther- give a product under the conditions of our studies. In this con- molysin-catalyzed condensation of Z-A-Phe-OH with H-Leu- nection it should be noted that, for the hydrolysis by ther- NHPh. The data are plotted in terms of the amount of product molysin of Z-Gly-Gly-Leu-Gly-OH and Z-Ala-Gly-Leu-Gly-OH formed, expressed as a percentage of the theoretical maximum at the Gly-Leu bond, the Km values were found (20) to be nearly (25 tumol/ml of incubation mixture). It is evident that under the same, but the kat value for the Ala-containing substrate was the conditions of these experiments the condensation of Z-(Gly)n- approximately 14 times greater. The presence of the hydrophobic side chain of methionine or phenylalanine in the P2 po- Table 1. Relative initial rates of thermolysin-catalyzed peptide c bond synthesis C) 80 Carboxyl Rate, Carboxyl Rate, 00., component component S rnCl) 60 Z-Phe-OH 100 Z-Gly-OH (0) w Z-Gly-Phe-OH 27 Z-Gly-Gly-OH (0.01) Z-Gly-Gly-Phe-OH 4 Z-Gly-Gly-Gly-OH (0) z 40 u) w Z-Ala-Phe-OH 129 Z-Ala-Gly-OH 32 a Z-Val-Phe-OH 56 Z-D-Ala-Gly-OH (0.01) 20 Boc-Phe-OH 85 Z-Ala-Gly-Gly-OH (0) wa. a. Z-Trp-OH 126 Z-Met-Gly-OH 36 Z-Tyr(Z)-OH 25 Z-Phe-Gly-OH 37 20 40 60 80 Boc-Tyr(Bzl)-OH 51 TIME (min) Z-Lys(Z)-OH 18 Z-Ala-OH (0.03) Z-Val-OH (0.03) Z-Ala-Ala-OH 55 FCIG. 1. Thermolysin-catalyzed peptide synthesis at pH 7.5 and 370C. In all experiments, the incubation mixture initially contained 50 mM Experimental conditions as specified in legend for Fig. 1. Rate is per- H-Leu-NHPh, the indicated RCOOH component at 25 mM, 1 AM ther- centage ofrate with Z-Phe-OH. Numbers in parentheses denotethe ab- molysin, 20% (vol/vol) dimethyl sulfoxide, and 0.5 M Tris/0.05 M cal- sence of observed synthesis during the first 2 hr or a rate too slow to cium acetate buffer. be estimated with satisfactory precision. Downloaded by guest on October 2, 2021 Biochemistry: Wayne and Fruton Proc. NadtAcad. Sci. USA 80 (1983) 3243

sition also markedly promoted the synthesis of the Gly-Leu bond of oligopeptide substrates as the explanation of the kinetic data (Table 1). Clearly, the secondary specificity of thermolysin in- but apparently overlooked the fact that k,,t/Km values are rel- cludes a stereospecific preference for a hydrophobic P2 residue, atively independent of such binding (26). Moreover, James et and the effectiveness of the Z-amino acids (especially Z-Phe- at (27) have demonstrated that with the aspartyl proteinase OH and Z-Trp-OH) as acyl donors is related to the ability of the penicillopepsin there is clear evidence of a sizable conforma- Z group to mimic the behavior of such residues. It is note- tional change upon the binding of a pepstatin analogue. worthy that Z-Ala-Gly-Gly-OH is no more effective as a car- Thus far, experimental efforts in our laboratory to determine boxyl component than Z-Gly-Gly.-Gly-OH, suggesting the ab- apparent kcat and Km values for the swine pepsin-catalyzed pep- sence, in the extended active site of thermolysin, of a-comple- tide bond synthesis in the systems described earlier (1) have not mentary region for the side chain of an L-amino acid in the so- been successful because of the limited solubility of most acyl- called P3 position. amino acids at acidic pH values (unpublished data). With ther- As was to be expected from the known primary specificity molysin, however, which acts optimally at pH values near 7.5, of thermolysin, the replacement of H-Leu-NHPh by H-Phe- such data could be obtained and are presented in Table 2. NHPh as the amine component in the condensation with Z-Phe- It is evident that the kinetics of the entry of Z-Phe-OH into OH gave the expected product at a rapid rate. Under the con- its thermolysin-catalyzed condensation with various amine ditions of the experiments for which data are given in Fig. 1 and components to form a Phe-Phe or Phe-Leu bond is character- Table 1, the estimated kct/Km was 82% of that found with H- ized by different apparent kat and Km values. Moreover, changes Leu-NHPh as the amine component. Under the same condi- in the structure of the amine component lead to much larger tions, H-Phe-OEt reacted with Z-Phe-OH at about 10% of the differences in the k, values than in-the Km values. Thus, there rate found for H-Phe-NHPh, indicating the contribution of the appears to be a synergistic effect that is expressed largely at the hydrophobic anilino group to the rate enhancement. This rel- level of the transition-state complex. We believe that the most atively slow peptide bond synthesis was not a consequence of plausible explanation is in terms of changes in the conformation the establishment of an equilibrium below 95-100% because of of the active site as a consequence of the binding of one of the the solubility of the product (Z-Phe-Phe-OEt) because an in- two reactants in the synthetic process, so that the structure of crease of the enzyme concentration from 1 AM to 0.01 mM gave the active-site locus for binding Z-Phe-OH in the transition state a rate comparable to that found with H-Phe-NHPh at the lower is somewhat different when the locus for the amine component concentration of thermolysin, and 95% synthesis was attained is occupied by say H-Phe-OMe than when it is occupied by H- within 2 hr. Phe-Gly-OMe. A similar substrate synergism has been ob- It is evident from the data in Table 1 that thermolysin is a served for other bireactant systems involving ternary enzyme- useful catalyst for the preparation of blocked dipeptide and tri- substrate complexes, most notably with yeast hexokinase (28) peptide derivatives, and it has already been used for this pur- which has been shown to undergo significant conformational pose by earlier workers (17-19). changes upon the binding of glucose (29). The main objective of our study of proteinase-catalyzed pep- Further kinetic studies are needed on thermolysin-catalyzed tide bond synthesis has been to contribute to the understanding peptide synthesis because the data in Table 2 only give the ap- of the mechanism of the action of these enzymes. Because the parent kc., and Km values for the entry of Z-Phe-OH at a single catalytic process must pass through the same transition state in excess concentration of the various amine components. De- both the hydrolysis and synthesis of a peptide bond by a given termination of the initial rates of the reaction of Z-Phe-OH as enzyme, the study of the kinetics of peptide bond synthesis is the variable component (5-25 mM) with H-Phe-OMe at dif- relevant to the mechanism of the hydrolytic reaction. In the ferent initial excess concentrations (50-200 mM) gave data in case of the aspartyl proteinases, the formation of covalent in- accord with the kinetics of rapid-equilibrium random bireac- termediates of both the acyl-enzyme and imino-enzyme type tant systems that obey Michaelis-Menten kinetics. From a has been postulated on the basis of apparent transpeptidation graphical analysis (30) of the data for this condensation reaction, reactions (3), but later evidence (21-24) has thrown doubt on k,, was estimated to be approximately 125 min-, Km for Z-Phe- the formation of such covalent intermediates. Rather, the avail- OH to be approximately 20 mM, and Km for H-Phe-OMe to be able data are more consistent with a general acid-base catalytic approximately 0.3 M, with a synergistic factor (a) of about 0.125. mechanism of the kind proposed for thermolysin (7). It had previously been suggested (3) on the basis of kinetic data on the Table 2. Apparent kinetic parameters for the entry of Z-Phe-OH action of swine pepsin on oligopeptide substrates that covalent into thermolysin-catalyzed condensation reactions intermediates are not formed but that the interaction of the hy- Amine component Km, mM kcat, min' k' drolytic products with the extended active site of an aspartyl tIK proteinase may be coupled so that the nature of one product H-Phe-OEt 8.2 ± 0.4 87 ± 9 10.6 influences the rate of departure of the other product through H-Phe-OMe 9.7 ± 0.3 104 ± 8 10.7 an influence on the conformational state of the active site. H-Phe-NH2 22.4 ± 0.6 1,540 ± 105 69 A study of the effect of variation of substrate systematic H-Phe-Gly-OMe 62.8 ± 1.2 11,500 ± 350 188 structure on the kinetic parameters of swine pepsin action showed differences in and small in H-Phe-Ala-OMe 35.1 ± 0.5 9,800 ± 400 222 large kat only differences Km (3). H-Phe-Ala-Ala-OMe 40.2 ± 0.7 5,650 ± 530 141 This may be taken to indicate that the effect on the conformation of the active site of structural changes in pepsin sub- H-Leu-NH2 41.7 ± 1.0 105 ± 10 24 strates is exerted at the level of the transition state of the re- H-Leu-NHPh 7.8 + 0.2 2,040 ± 125 262 action rather than on the ground state of the Michaelis complex. A recent paper by Bott et at (25) on a x-ray crystallographic study Initial concentration ofZ-Phe-OH variedfrom 5 to 25 mM (five runs); of the of chinensis the that of the amine component was held at 100 mM. Depending on the aspartyl proteinase Rahizopus questioned initial rate, the enzyme concentration was 0.2-10 uM. All runs were occurrence of a conformational change during catalysis by en- conducted at 370C in the presence of 10% (vol/vol) dimethyl sulfoxide zymes of this group because no evidence was found for such and 0.5 M Tris/0.05 M calcium acetate buffer, pH 7.5. The Km and kct change upon the binding of the inhibitor pepstatin to the en- values were estimated from Lineweaver-Burk plots and are the mean zyme they studied. Bott et aL suggested nonproductive binding of two or three sets of runs for each pair of reactants. Downloaded by guest on October 2, 2021 3244 Biochemistry: Wayne and Fruton Proc. Natl. Acad. Sci. USA 80 (1983) A similar set of experiments with H-Phe-Gly-OMe as the amine 10. Holmquist, B., Blumberg, S. & Vallee, B. L. (1976) Biochemistry component in place of H-Phe-OMe gave k = 12,500 min1, 15, 4675-468. Km(Z-Phe-OH) = 65 mM, Km(H-Phe-Gly-OMe) = 0.5 M, and 11. Morgan, G. & Fruton, J. S. (1978) Biochemistry 17, 3562-3568. = 12. Pank, M., Kirret, O., Paberit, N. & Aaviksaar, A. (1982) FEBS a 0.125. The magnitude of the estimated Km value for H- Lett. 142, 297-300. Phe-OMe explains why Oyama et al (19) were unable to de- 13. Feder, J. & Schuck, J. M. (1970) Biochemistry 9, 2784-2791. termine it under the conditions of their studies on the kinetics 14. Holmquist, B. & Vallee, B. L. (1976) Biochemistry 15, 101-107. of its thermolysin-catalyzed condensation with Z-Asp-OH. We 15. Jakubke, H. D. & Kuhl, P. (1982) Pharmazie 37, 89-106. have repeated their experiments and have obtained data in fair 16. Chaiken, I. M., Komoriya, A., Ohno, M. & Widmer, F. (1982) agreement with their reported knit value of 2.65 M's-1 and AppL Biochem. BiotechnoL 7, 385-399. 17. Isowa, Y. & Ichikawa, T. (1979) Bull Soc. Chem. Jpn. 52, 796400. K., value for Z-Asp-OH of 0.01 M (19). The data presented above 18. Oka, T. & Morihara, K. (1980)J. Biochem. 88, 807-813. are not consistent with the formation, in thermolysin-catalyzed 19. Oyama, K., Kihara, K. & Nonaka, Y. (1981)J. Chem. Soc. Perkin reactions, of covalent intermediates of the acyl-enzyme or im- Trans. 2, 356-360. ino-enzyme type (31). 20. Morihara, K. & Tsuzuki, H. (1970) Eur. J. Biochem. 15, 374-380. 21. James, M. N. G., Hsu, I. N. & Delbaere, L. T. J. (1977) Nature This work was aided by Grant GM-18172 from the National Institutes (London) 267, 808-813. of Health. 22. Antonov, V. K., Ginodman, L. M., Kapitannikov, Y. V., Barshev- skaya, T. N. & Rumsh, L. D. (1978) FEBS Lett. 88, 87-90. 1. Bozler, H., Wayne, S. I. & Fruton, J. S. (1982) Int. 1. Peptide Pro- 23. Silver, M. S. & James, S. L. T. (1981) Biochemistry 20, 3177-3189. tein Res. 20, 102-109. 24. Dunn, B. M., Hofmann, T. & Fink, A. L. (1982) Fed. Proc. Fed. 2. Fruton, J. S. (1982) Adv. EnzymoL Relat. Areas Mol. Biol. 53, 239- Am. Soc. Exp. BioL 41, 762. 306. 25. Bott, R., Subramanian, E. & Davies, D. R. (1982) Biochemistry 3. Fruton, J. S. (1976) Adv. Enzymol Relat. Areas Mol BioL 44, 1- 21, 6956-6962. 36. 26. Bender, M. L. & Kezdy, F. J. (1965) Annu. Rev. Biochem. 34, 49- 4. Holmes, M. A. & Matthews, B. W. (1982)J. MoL BioL 160, 623- 76. 639. 27. James, M. N. G., Sielecki, A., Salituro, F., Rich, D. H. & Hof- 5. Latt, S. A., Holmquist, B. & Vallee, B. L. (1969) Biochem. Bio- mann, T. (1982) Proc. NatL Acad. Sci. USA 79, 6137-6141. phys. Res. Commun. 37, 333-339. 28. Viola, R. E., Rauschel, F. M., Rendina, A. R. & Cleland, W. W. 6. Dahlquist, F. W., Long, J. W. & Bigbee, W. L. (1976) Biochem- (1982) Biochemistry 21, 1295-1302. istry 15, 1103-1111. 29. Anderson, C. H., Stenkamp, R. E., McDonald, R. C. & Steitz, 7. Holmes, M. A. & Matthews, B. W. (1981) Biochemistry 20, 6912- T. A. (1978)J. MoL BioL 123, 207-219. 6920. 30. Segel, I. H. (1975) Enzyme Kinetics (Wiley, New York), pp. 273- 8. Morihara, K. (1974) Adv. EnzymoL Relat. Areas Mol BioL 41, 179- 281. 243. 31. Morihara, K., Tsuzuki, H. & Oka, T. (1978) Biochem. Biophys. Res. 9. Blumberg, S. & Vallee, B. L. (1975) Biochemistry 14, 2410-2419. Commun. 84, 95-101. Downloaded by guest on October 2, 2021