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 ex- tended 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.