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Enzyme peptide synthesis and semisynthesis: Kinetic and thermodynamic aspects
J. theor. Biol. (1982) 98,419-425
Enzyme Peptide Synthesis and Semisynthesis: Kinetic and Thermodynamic
Aspects
D. D. PETKOV Institute of Organic Chemistry and Centre of Phytochemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. (Received
11 August
1981, and in revised form 22 March
1982)
The hydrolysis/synthesis equilibrium of the peptide bond is governed by the relative magnitudes of the corresponding Gibbs’ energies of hydrolysis to non-ionized products and of their ionization. The positive energy change in peptide hydrolysis to non-ionized products is the thermodynamic basis for the acyl and leaving group specificity of proteinases. With a proteinase of suitable specificity, some peptide bonds can be synthesized by a thermodynamically controlled enzyme aminolysis of specific acylamino or peptide acids; any peptide bond can be formed by a kinetically controlled enzyme aminolysis of the corresponding acylamino or peptide esters.
Introduction
Enzyme peptide synthesis is undergoing a burst of development after having been neglected for a long time. This revival of interest has been stimulated by the advantages offered by enzyme catalysis in the synthesis and semisynthesis of biologically active peptides and proteins. After the enzymecatalyzed semisynthesis of proteinase protein inhibitors (Sealock & Laskowski, Jr., 1969; Jering & Tschesche, 1976) and human insulin (Inouye et al., 1979; Morihara, Oka & Tsuzuki, 1979), as well as the preparation of angiotensin II derivative by enzyme fragment condensation (Isowa et al., 1977a), the total enzyme synthesis of enkephalins (Kullmann, 1980) has been reported. On the other hand, recent achievement in the elucidation of molecular conformation and the mechanism of action of proteinases (Fersht, 1977), as well as the new experimental techniques (e.g. highperformance liquid chromatography) have provided the basis for a new approach to the problems of enzyme-catalyzed peptide synthesis. In the present paper, some kinetic and thermodynamic aspects of enzyme peptide synthesis and semisynthesis are discussed, which have either been neglected or are subject to controversial discussion in the literature. 419
0022-5193/82/190419+07$03.00/0
@ 1982 Academic
Press Inc. (London)
Ltd.
420
D.
IX
PETKOV
Thermodynamic
Considerations
Due to the lack of reactivity of carboxyl anions and alkylammonium cations, the hydrolysis or synthesis of a peptide bond is a two-step process: K
RCONHR’
+ Hz0 2
RCOOH
K, 15
+ NHZR’ e
RCOO-
+NH:R’
(1)
The first step, peptide hydrolysis to non-ionized products, is an endergonic process (AG& >O); the second step, ionization of the carboxy group and neutralization of the amino group, is an exergonic process (AGi,” < 0) (Carpenter, 1960). If IAG&~I I < IAGionI
(2)
peptide hydrolysis is favoured; when IAGtycz~I > jAGion/ the equilibria
(3)
(1) are shifted towards peptide bond synthesis and if IAGOhydI z IAGionI
(4)
the equilibria (1) can be controlled by mass action. Since, for a typical peptide bond, the Gibbs’ energy change in the hydrolysis step (AG&) has approximately the same value (Carpenter, 1960), peptide hydrolysis or synthesis depends on the Gibbs’ energy change in the ionization steps: AGi,, = -RT
ln (I+ l()pH-pKl + 10pK~-pH+ 10pK2-pKl)
(5)
Consequently, the degree of conversion in a peptide synthesis at a certain pH- value will depend on the acid ionization constants of the carboxy (pK1) and amino (pKZ) components. In aqueous solutions pK1 values of amino acids increase by 1 pK unit upon acylation of the amino group and the pK2 values decrease by more than 1 pK unit upon blocking of the carboxy group (Perrin, 1965). This suggests that a suitable combination of blocking groups could bring about AGi,,,,-values that satisfy the requirements for peptide synthesis of the relations (3) and (4). In aqueous-organic buffer solutions there is a considerable effect of the organic cosolvent on the equilibria (1) apart from the reduction of water concentration (Martinek, Simeonov & Berezin, 1981). As has been shown by Homandberg, Mattis & Laskowski, Jr. (1978), it is the ionization steps which are mainly affected: the organic cosolvent reduces the Gibbs’ energy change of proton transfer from the ammonium cation to the carboxy anion during the synthesis.
ENZYME
PEPTIDE
421
SYNTHESIS
For preparative purposes, the states of equilibria (1) must be reached in a finite period of time. This may be achieved by highly efficient catalysts such as proteinases. As catalysts, the proteinases accelerate the forward and the reverse reaction without altering the position of equilibrium. That is why the enzyme peptide synthesis and semisynthesis can be realized only if equations (3) and (4) hold. Actually, more than 40 years ago, Bergmann & Fruton (1938) synthesized benzoyl-L-Tyr-Gly-NH2 by means of chymotrypsin-catalyzed coupling of benzoyl-L-Tyr-OH and H-Gly-NH*. Later Kozlov, Ginodman, Orekhovich & Valueva (1966) calculated the Gibbs’ energy change in the hydrolysis of this peptide to ionized products, (AG& + AGi,,), to be 0.49 kcal/mol. Therefore, the success of Bergmann and Fruton can be attributed to IAG&I = lAGi,” and to the fact that this dipeptide is insoluble in aqueous solutions. Due to its insolubility, the product can be accumulated in preparative quantities according to the law of mass action. For a long time, it has been incorrectly assumed that insolubility of the peptide is a necessary condition for enzymatic peptide synthesis. Even in recent studies (Luisi et al., 1977; Saltmann, Vlach & Luisi, 1977; Pellegrini & Luisi, 1978; Oka & Morihara, 1978, 1980; Isowa et al., 19776; Isowa, Ichikawa & Ohmori, 1978; Isowa & Ichikawa, 1979) it was implied that the enzyme coupling method can be used successfully only in the case of water-insoluble peptides. Quite recently using trypsin as catalyst, Tsuzuki, Oka & Morhara (1980) successfully synthesized the water-soluble derivatives of benzyloxycarbonyl-L-Arg-L-Leu-OH simply by carrying out the reaction with a large excess of amine. These authors, however, have incorrectly attributed their results to some features of the mechanism of proteinase action. Enzyme Kinetic Considerations The acyl enzyme pathway is the major hydrolysis (Fastrez & Fersht, 1973): RCONHR’
+E -
k+zfK,
RCOE -
kmZ[NH2R’] K,fK,
+E _
RCOO-
route for enzyme peptide k :JH201
RCOOH
k-d&
+ NHfR’
(6)
Application of the Haldane relationship (Haldane, 1930; Fastrez & Fersht, 1973) to this reaction scheme gives the following expression for the equilibrium constant of enzymatic peptide hydrolysis to non-ionized products:
422
D.
K
W
D.
PETKOV
= [RCOOHl[NHzR’l=
bk+dK)~:dI-Wl
[RCONHR’]
b-JKp)k-z
Since for peptides
AGEyd >O (Carpenter,
In Khyd, Khyd<< 1. Therefore,
(k+JK,)kL3
(7)
1960) and as hGf,d = -RT (k-3/K,)k-2. This could be
valid if (k+zlKs) CC(k-,/K,)
(8)
and k:s K km2
(9)
The inequalities (8) and (9) imply that a proteinase should possess a specificity towards both the acyl and the leaving group in order to be an effective catalyst in the hydrolysis or synthesis of a peptide bond. Actually, Fersht, Blow & Fastiez (1973) have shown that H-L-Ala-NH2 as a nucleophile is 2400-fold better than water in the nucleophilic attack of the specific acylenzyme, acetyl-L-Phe-chymotrypsin, and although H-L-AlaNH2 and H-Gly-NH2 have similar pK,-values, H-L-Ala-NH2 reacts 4 times faster. Therefore, the inequalities (8) and (9) express the thermodynamic basis for the acyl and leaving group specificity of proteinases. Since for enzyme peptide synthesis the inequalities (3) and (4) hold, (bG&d + AGi,,) 5: 0. Therefore, the acyl and leaving group specificity of proteinases should affect the peptide synthetic yield, as has been observed (Luisi et al., 1977; Oka & Morihara, 1978; Isowa & Ichikawa, 1979). Kinetic and Thermodynamic
Controlled
Aminolysis
A congruent reaction of enzyme peptide hydrolysis to non-ionized products (scheme (1)) is the enzyme alcoholysis of specific esters: RCONHR’
+E-
k+zfKs
k_2[NH2R’l
k+,[R”OHl
RCOE V
RCOOR”
(10)
k-d/K:
Since the Gibbs’ energy change for this reaction AGZ,, = AGiYd > 0 (Kezdy, Clement & Bender, 1964), (k+2/Ks) <<(km4/Ki), k+4 < k-2, the equilibrium is shifted towards aminolysis of the ester RCOOR”. Therefore, in non-aqueous solutions, a specific ester RCOOR” could be quantitatively converted into a peptide RCONHR’. The natural reaction medium for enzyme action, however, is water and it is not only solvent but a reactive nucleophile too, its concentration being 55.5 M. The presence of water molecules in the reaction medium, causing deacylation of the acyl enzyme RCOE, leads to the hydrolysis of both ester and peptide:
ENZYME
RCONHR’
+E 1
PEPTIDE
k&K,
423
SYNTHESIS k+,[R”OHJ
RCOE -
ke2[NH2R’l
RCOOR”
+E
k-.,/K;
k-,/K,
RCOOH
?J k:d%Ol KI/Kz
+E_
(11)
RCOO-+NH:R’
Therefore, in aqueous and aqueous-organic buffer solutions there are four equilbiria: aminolysis of the specific ester, hydrolysis of the peptide to non-ionized products, hydrolysis of the specific ester to non-ionized products and aminolysis of specific acids. Since all these reactions are reversible, the products can be both thermodynamically and kinetically controlled. Under thermodynamic control the products with the lowest free energy will accumulate. If inequality (2) holds, the ionized products RCOO- and NHiR’ are thermodynamically more stable. After a period of time sufficiently long for attaining all equilibria, r,, the hydrolysis products will accumulate regardless of the starting point of reaction (11). This has been observed: after T., starting from equimolar amounts of acetyl-L-Phe-OMe and H-Gly-L-Leu-OH, Morihara & Oka (1977) isolated only 3% acetyl-lPhe-Gly-L-Leu-OH in chymotrypsin-catalyzed coupling. On the other hand, if equations (3) and (4) hold, after T,, accumulation or mass action controlled accumulation of the peptide should be observed. Under kinetic control the products that appear with the highest rate and disappear with the lowest rate would accumulate. Provided the reaction is stopped well before equilibrium is reached, the more rapidly formed products will dominate regardless of the relative magnitues of AG&, and AGi,,. If the reaction is permitted to approach equilibrium, however, the products will be thermodynamically controlled. Therefore, the concentration of the products formed under kinetic control will go through a maximum. Deacylation is the rate-limiting step in enzyme hydrolysis of specific ester and acylation controls the rate in enzyme amide hydrolysis (Bender & Kezdy, 1965): (k-JK:)[RCOOR”]
>>k:JH20]
This means that under kinetic accumulate provided k-JNHZR’] i.e. at a concentration
the peptide
>>(k :3[HzO] + k+JR”OH])
RCONHR’
(12)
should (13)
of the amine component
[NHzR’] Therefore,
control
>>(k+2/K,)[RCONHR’]
>>(k:JH20]+
the enzyme aminolysis
k+&R”OH])/kv2
(14)
of specific esters under kinetic control
424
D.
D.
PETKOV
depends on the enzyme leaving specificity, expressed by the deacylation rate constants ke2, k13 and k+.+ The higher the enzyme specificity for the amino component NHzR’, the lower the minimal concentration [NHzR’],i,, necessary for peptide accumulation. Actually, as the kpz-values for H-LAla-NH* and H-Gly-NH2 are 6340 and 1660 set’ (Fersht et al., 1973), at the same concentration (0.1 M) 30 set after the beginning of chymotrypsin ammonolysis of acetyl-L-Phe-OMe, Fastrez & Fersht (1973) have obtained 81.8% of acetyl-L-Phe-L-Ala-NH2 and 51.8% of acetyl-L-Phe-Gly-NH*, respectively. Furthermore, Morihara & Oka (1977) have shown that not only the Pi-amino acid but the Pi-amino acid as well, affects the peptide yield. This effect could be attributed to the significant increase of the leaving group kpz-values caused by $-Pi interaction (Bauer, 1976; Christova, Petkov & Stoineva, in preparation). In order to carry out enzyme aminolysis of specific acids (scheme (6)) under kinetic control, inequalities analogous to (12) and (13) should hold: (k-JK,)[RCOOH]
>>k:JH20] k-JNHZR’]
>>(k+JK,)[RCONHR’]
(15)
>>k:JH20]
(16)
At any pH- value, at least one of these inequalities is not valid, since either [RCOOH] or [NHTR’] or both are very low. Therefore, preparative enzyme synthesis from ionized products can be realised under thermodynamic control only, i.e. when equations (3) or (4) hold. In conclusion, any peptide bond could be formed by enzyme-catalyzed synthesis provided a proteinase is available with a specificity for the corresponding carboxy and amine components. If the synthesis can not be carried out under thermodynamic control, formation of the peptide bond can be achieved by kinetically controlled enzyme aminolysis of the corresponding ester of the carboxy component. The author wishesto thank Dr Iv. Pojarlieff and Mrs V. Yomtova for assistance in preparation of the English version of the manuscript,criticism and stimulating discussions. REFERENCES BAUER, C. A. (1976).Biochim. biophys. Acta 438, 495. BENDER, M. L. & KEZDY, F. J. (1965).Ann. Rev. Biochem. 34,49. BERGMANN,M. & FRUTON,J. S. (1938).J.biol. Chem. 124,321. CARPENTER, F. H. (1960).J. &em. Sot. 82,llll. CHRISTOVA,E.,PETKOV,D.D.& STOINEVA, I.Arch.Biochem.Biophys. FASTREZ,J.& FERSHT,A.(~~~~). Biochemistry 12,2025. FERSHT, A., BLOW,D.M.& FASTREZ,J. (1973). Biochemistry 12,2035.
(In press).
ENZYME
PEPTIDE
SYNTHESIS
425
FERSHT, A. (1977). In: Enzyme Structure and Mechanism, p. 1. San Francisco: W. H. Freeman and Company. HALDANE, J. B. C. (1930). In: Enzymes p. 123, London: Longmans. HOMANDBERG, G. H., MARS, J. A. & LASKOWSKI, JR., M. (1978). Biochemistry 17,522O. INOUYE, K., WATANABE, K., MORIHARA, K., TOCHINO, Y., KANAYA, T., EMIJRA, J. & SAKAKIBARA, S. (1979). J. Am. Chem. Sot. 101,702. ISOWA, Y., OHMORI, M., ICHIKAWA, T., SATOH, H. & MORI, K. (1977a). Bull. them. Sot. Japan 50, 2762. ISOWA, Y., OHMORI, M., SATOH, H. & MORI, K. (19776). Bull. them. Sot. Japan 50,2766. ISOWA, Y., ICHIKAWA, T. & OHMORI, M. (1978). Bull. them. Sot. Japan 51,271. ISOWA, Y. & ICHIKAWA, T. (1979). Buff. them. Sac. Japan 52,796. JERING, H. & TSCHESCHE, H. (1976). Eur. J. Biochem. 69,453. KEZDY, F. J., CLEMENT, C. E. & BENDER, M. L. (1964). J. Am. Chem. Sot. 86,3690, KOZLOV, L. V., GINODMAN, L. M., OREKHOVICH, V. N. & VALUEVA, T. A. (1966). Biokhimiya 31, 3 15. KULLMANN, W. (1980). J. biol. Chem. 2X$8234. LUISI, P. L., SALTMAN, R., VLACH, D. & GUARNACCIA, R. (1977). J. mol. Catalysis 2,133. MARTINEK, K., SIMEONOV, A. N. & BEREZIN, I. V. (1981). Biochim. Biophys. Acta 658, 5220. MORIHARA, K. & OKA, T. (1977). Biochem. J. 163,531. MORIHARA, K., OKA, T. & TSUZUKI, H. (1979). Nature 280,572l. OKA, T. & MORIHARA, K. (1978). J. Biochem. 84,1277. OKA, T. & MORIHARA, K. (1980). J. Biochem. 88,807. PELLEGRINI, A. & LUISI, P. L. (1978). Bfopolymers 17,2573. PERRIN, D. D. (1965). In: Dissociation Constants of Organic Bases in Aqueous Solutions, p. 353, London: Butterworths. SALTMAN, R., VLACH, D. & LUISI, P. L. (1977). Biopolymers 16,631. SEALOCK, R. W. & LASKOWSKI, JR., M. (1969). Biochemistry 8,3703. TSUZUKI, H., OKA, T. & MORIHARA, K. (1980). J. Biochem. 88,669.
Enzyme Peptide Synthesis and Semisynthesis: Kinetic and Thermodynamic
Aspects
D. D. PETKOV Institute of Organic Chemistry and Centre of Phytochemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. (Received
11 August
1981, and in revised form 22 March
1982)
The hydrolysis/synthesis equilibrium of the peptide bond is governed by the relative magnitudes of the corresponding Gibbs’ energies of hydrolysis to non-ionized products and of their ionization. The positive energy change in peptide hydrolysis to non-ionized products is the thermodynamic basis for the acyl and leaving group specificity of proteinases. With a proteinase of suitable specificity, some peptide bonds can be synthesized by a thermodynamically controlled enzyme aminolysis of specific acylamino or peptide acids; any peptide bond can be formed by a kinetically controlled enzyme aminolysis of the corresponding acylamino or peptide esters.
Introduction
Enzyme peptide synthesis is undergoing a burst of development after having been neglected for a long time. This revival of interest has been stimulated by the advantages offered by enzyme catalysis in the synthesis and semisynthesis of biologically active peptides and proteins. After the enzymecatalyzed semisynthesis of proteinase protein inhibitors (Sealock & Laskowski, Jr., 1969; Jering & Tschesche, 1976) and human insulin (Inouye et al., 1979; Morihara, Oka & Tsuzuki, 1979), as well as the preparation of angiotensin II derivative by enzyme fragment condensation (Isowa et al., 1977a), the total enzyme synthesis of enkephalins (Kullmann, 1980) has been reported. On the other hand, recent achievement in the elucidation of molecular conformation and the mechanism of action of proteinases (Fersht, 1977), as well as the new experimental techniques (e.g. highperformance liquid chromatography) have provided the basis for a new approach to the problems of enzyme-catalyzed peptide synthesis. In the present paper, some kinetic and thermodynamic aspects of enzyme peptide synthesis and semisynthesis are discussed, which have either been neglected or are subject to controversial discussion in the literature. 419
0022-5193/82/190419+07$03.00/0
@ 1982 Academic
Press Inc. (London)
Ltd.
420
D.
IX
PETKOV
Thermodynamic
Considerations
Due to the lack of reactivity of carboxyl anions and alkylammonium cations, the hydrolysis or synthesis of a peptide bond is a two-step process: K
RCONHR’
+ Hz0 2
RCOOH
K, 15
+ NHZR’ e
RCOO-
+NH:R’
(1)
The first step, peptide hydrolysis to non-ionized products, is an endergonic process (AG& >O); the second step, ionization of the carboxy group and neutralization of the amino group, is an exergonic process (AGi,” < 0) (Carpenter, 1960). If IAG&~I I < IAGionI
(2)
peptide hydrolysis is favoured; when IAGtycz~I > jAGion/ the equilibria
(3)
(1) are shifted towards peptide bond synthesis and if IAGOhydI z IAGionI
(4)
the equilibria (1) can be controlled by mass action. Since, for a typical peptide bond, the Gibbs’ energy change in the hydrolysis step (AG&) has approximately the same value (Carpenter, 1960), peptide hydrolysis or synthesis depends on the Gibbs’ energy change in the ionization steps: AGi,, = -RT
ln (I+ l()pH-pKl + 10pK~-pH+ 10pK2-pKl)
(5)
Consequently, the degree of conversion in a peptide synthesis at a certain pH- value will depend on the acid ionization constants of the carboxy (pK1) and amino (pKZ) components. In aqueous solutions pK1 values of amino acids increase by 1 pK unit upon acylation of the amino group and the pK2 values decrease by more than 1 pK unit upon blocking of the carboxy group (Perrin, 1965). This suggests that a suitable combination of blocking groups could bring about AGi,,,,-values that satisfy the requirements for peptide synthesis of the relations (3) and (4). In aqueous-organic buffer solutions there is a considerable effect of the organic cosolvent on the equilibria (1) apart from the reduction of water concentration (Martinek, Simeonov & Berezin, 1981). As has been shown by Homandberg, Mattis & Laskowski, Jr. (1978), it is the ionization steps which are mainly affected: the organic cosolvent reduces the Gibbs’ energy change of proton transfer from the ammonium cation to the carboxy anion during the synthesis.
ENZYME
PEPTIDE
421
SYNTHESIS
For preparative purposes, the states of equilibria (1) must be reached in a finite period of time. This may be achieved by highly efficient catalysts such as proteinases. As catalysts, the proteinases accelerate the forward and the reverse reaction without altering the position of equilibrium. That is why the enzyme peptide synthesis and semisynthesis can be realized only if equations (3) and (4) hold. Actually, more than 40 years ago, Bergmann & Fruton (1938) synthesized benzoyl-L-Tyr-Gly-NH2 by means of chymotrypsin-catalyzed coupling of benzoyl-L-Tyr-OH and H-Gly-NH*. Later Kozlov, Ginodman, Orekhovich & Valueva (1966) calculated the Gibbs’ energy change in the hydrolysis of this peptide to ionized products, (AG& + AGi,,), to be 0.49 kcal/mol. Therefore, the success of Bergmann and Fruton can be attributed to IAG&I = lAGi,” and to the fact that this dipeptide is insoluble in aqueous solutions. Due to its insolubility, the product can be accumulated in preparative quantities according to the law of mass action. For a long time, it has been incorrectly assumed that insolubility of the peptide is a necessary condition for enzymatic peptide synthesis. Even in recent studies (Luisi et al., 1977; Saltmann, Vlach & Luisi, 1977; Pellegrini & Luisi, 1978; Oka & Morihara, 1978, 1980; Isowa et al., 19776; Isowa, Ichikawa & Ohmori, 1978; Isowa & Ichikawa, 1979) it was implied that the enzyme coupling method can be used successfully only in the case of water-insoluble peptides. Quite recently using trypsin as catalyst, Tsuzuki, Oka & Morhara (1980) successfully synthesized the water-soluble derivatives of benzyloxycarbonyl-L-Arg-L-Leu-OH simply by carrying out the reaction with a large excess of amine. These authors, however, have incorrectly attributed their results to some features of the mechanism of proteinase action. Enzyme Kinetic Considerations The acyl enzyme pathway is the major hydrolysis (Fastrez & Fersht, 1973): RCONHR’
+E -
k+zfK,
RCOE -
kmZ[NH2R’] K,fK,
+E _
RCOO-
route for enzyme peptide k :JH201
RCOOH
k-d&
+ NHfR’
(6)
Application of the Haldane relationship (Haldane, 1930; Fastrez & Fersht, 1973) to this reaction scheme gives the following expression for the equilibrium constant of enzymatic peptide hydrolysis to non-ionized products:
422
D.
K
W
D.
PETKOV
= [RCOOHl[NHzR’l=
bk+dK)~:dI-Wl
[RCONHR’]
b-JKp)k-z
Since for peptides
AGEyd >O (Carpenter,
In Khyd, Khyd<< 1. Therefore,
(k+JK,)kL3
(7)
1960) and as hGf,d = -RT (k-3/K,)k-2. This could be
valid if (k+zlKs) CC(k-,/K,)
(8)
and k:s K km2
(9)
The inequalities (8) and (9) imply that a proteinase should possess a specificity towards both the acyl and the leaving group in order to be an effective catalyst in the hydrolysis or synthesis of a peptide bond. Actually, Fersht, Blow & Fastiez (1973) have shown that H-L-Ala-NH2 as a nucleophile is 2400-fold better than water in the nucleophilic attack of the specific acylenzyme, acetyl-L-Phe-chymotrypsin, and although H-L-AlaNH2 and H-Gly-NH2 have similar pK,-values, H-L-Ala-NH2 reacts 4 times faster. Therefore, the inequalities (8) and (9) express the thermodynamic basis for the acyl and leaving group specificity of proteinases. Since for enzyme peptide synthesis the inequalities (3) and (4) hold, (bG&d + AGi,,) 5: 0. Therefore, the acyl and leaving group specificity of proteinases should affect the peptide synthetic yield, as has been observed (Luisi et al., 1977; Oka & Morihara, 1978; Isowa & Ichikawa, 1979). Kinetic and Thermodynamic
Controlled
Aminolysis
A congruent reaction of enzyme peptide hydrolysis to non-ionized products (scheme (1)) is the enzyme alcoholysis of specific esters: RCONHR’
+E-
k+zfKs
k_2[NH2R’l
k+,[R”OHl
RCOE V
RCOOR”
(10)
k-d/K:
Since the Gibbs’ energy change for this reaction AGZ,, = AGiYd > 0 (Kezdy, Clement & Bender, 1964), (k+2/Ks) <<(km4/Ki), k+4 < k-2, the equilibrium is shifted towards aminolysis of the ester RCOOR”. Therefore, in non-aqueous solutions, a specific ester RCOOR” could be quantitatively converted into a peptide RCONHR’. The natural reaction medium for enzyme action, however, is water and it is not only solvent but a reactive nucleophile too, its concentration being 55.5 M. The presence of water molecules in the reaction medium, causing deacylation of the acyl enzyme RCOE, leads to the hydrolysis of both ester and peptide:
ENZYME
RCONHR’
+E 1
PEPTIDE
k&K,
423
SYNTHESIS k+,[R”OHJ
RCOE -
ke2[NH2R’l
RCOOR”
+E
k-.,/K;
k-,/K,
RCOOH
?J k:d%Ol KI/Kz
+E_
(11)
RCOO-+NH:R’
Therefore, in aqueous and aqueous-organic buffer solutions there are four equilbiria: aminolysis of the specific ester, hydrolysis of the peptide to non-ionized products, hydrolysis of the specific ester to non-ionized products and aminolysis of specific acids. Since all these reactions are reversible, the products can be both thermodynamically and kinetically controlled. Under thermodynamic control the products with the lowest free energy will accumulate. If inequality (2) holds, the ionized products RCOO- and NHiR’ are thermodynamically more stable. After a period of time sufficiently long for attaining all equilibria, r,, the hydrolysis products will accumulate regardless of the starting point of reaction (11). This has been observed: after T., starting from equimolar amounts of acetyl-L-Phe-OMe and H-Gly-L-Leu-OH, Morihara & Oka (1977) isolated only 3% acetyl-lPhe-Gly-L-Leu-OH in chymotrypsin-catalyzed coupling. On the other hand, if equations (3) and (4) hold, after T,, accumulation or mass action controlled accumulation of the peptide should be observed. Under kinetic control the products that appear with the highest rate and disappear with the lowest rate would accumulate. Provided the reaction is stopped well before equilibrium is reached, the more rapidly formed products will dominate regardless of the relative magnitues of AG&, and AGi,,. If the reaction is permitted to approach equilibrium, however, the products will be thermodynamically controlled. Therefore, the concentration of the products formed under kinetic control will go through a maximum. Deacylation is the rate-limiting step in enzyme hydrolysis of specific ester and acylation controls the rate in enzyme amide hydrolysis (Bender & Kezdy, 1965): (k-JK:)[RCOOR”]
>>k:JH20]
This means that under kinetic accumulate provided k-JNHZR’] i.e. at a concentration
the peptide
>>(k :3[HzO] + k+JR”OH])
RCONHR’
(12)
should (13)
of the amine component
[NHzR’] Therefore,
control
>>(k+2/K,)[RCONHR’]
>>(k:JH20]+
the enzyme aminolysis
k+&R”OH])/kv2
(14)
of specific esters under kinetic control
424
D.
D.
PETKOV
depends on the enzyme leaving specificity, expressed by the deacylation rate constants ke2, k13 and k+.+ The higher the enzyme specificity for the amino component NHzR’, the lower the minimal concentration [NHzR’],i,, necessary for peptide accumulation. Actually, as the kpz-values for H-LAla-NH* and H-Gly-NH2 are 6340 and 1660 set’ (Fersht et al., 1973), at the same concentration (0.1 M) 30 set after the beginning of chymotrypsin ammonolysis of acetyl-L-Phe-OMe, Fastrez & Fersht (1973) have obtained 81.8% of acetyl-L-Phe-L-Ala-NH2 and 51.8% of acetyl-L-Phe-Gly-NH*, respectively. Furthermore, Morihara & Oka (1977) have shown that not only the Pi-amino acid but the Pi-amino acid as well, affects the peptide yield. This effect could be attributed to the significant increase of the leaving group kpz-values caused by $-Pi interaction (Bauer, 1976; Christova, Petkov & Stoineva, in preparation). In order to carry out enzyme aminolysis of specific acids (scheme (6)) under kinetic control, inequalities analogous to (12) and (13) should hold: (k-JK,)[RCOOH]
>>k:JH20] k-JNHZR’]
>>(k+JK,)[RCONHR’]
(15)
>>k:JH20]
(16)
At any pH- value, at least one of these inequalities is not valid, since either [RCOOH] or [NHTR’] or both are very low. Therefore, preparative enzyme synthesis from ionized products can be realised under thermodynamic control only, i.e. when equations (3) or (4) hold. In conclusion, any peptide bond could be formed by enzyme-catalyzed synthesis provided a proteinase is available with a specificity for the corresponding carboxy and amine components. If the synthesis can not be carried out under thermodynamic control, formation of the peptide bond can be achieved by kinetically controlled enzyme aminolysis of the corresponding ester of the carboxy component. The author wishesto thank Dr Iv. Pojarlieff and Mrs V. Yomtova for assistance in preparation of the English version of the manuscript,criticism and stimulating discussions. REFERENCES BAUER, C. A. (1976).Biochim. biophys. Acta 438, 495. BENDER, M. L. & KEZDY, F. J. (1965).Ann. Rev. Biochem. 34,49. BERGMANN,M. & FRUTON,J. S. (1938).J.biol. Chem. 124,321. CARPENTER, F. H. (1960).J. &em. Sot. 82,llll. CHRISTOVA,E.,PETKOV,D.D.& STOINEVA, I.Arch.Biochem.Biophys. FASTREZ,J.& FERSHT,A.(~~~~). Biochemistry 12,2025. FERSHT, A., BLOW,D.M.& FASTREZ,J. (1973). Biochemistry 12,2035.
(In press).
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SYNTHESIS
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