Intramolecular Catalysis of Acylation and ... - ACS Publications

(8) J. W. Bunting, J. Murphy, C. D. Myers, and G. G. Cross, Can. J. Chem., 52,. (9) E. P. Kang, C. B. Storm, and F. W. Carson, J. Am. Chem. SOC., 97, ...
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2780 (4) P. L. Hall, B. L. Kaiser, and E. T. Kaiser, J. Am. Chern. SOC., 91,485 (1969). (5) D. S. Auld and 8. L. Vallee, Biochemistry, 9,4352 (1970). (6) D. S. Auld and B. L. Vallee. Biochemistry, IO, 2892 (1971). (7) D. S. Auld and B. Holmquist, Fed. Proc., Fed. Am. SOC.Exp. Bioi., 31,435 Abs (1972). (8) J. W. Bunting, J. Murphy, C. D. Myers, and G. G. Cross, Can. J. Chem., 52, 2648 (1974). (9) E. P. Kang, C. B. Storm, and F. W. Carson, J. Am. Chem. SOC., 97,6723 (1975). (IO) J. Suh and E. 1.Kaiser, J. Am. Chem. SOC., 98, 1940 (1976). (1 1) J. W. Bunting and S. S.-T. Chu, Biochemistry, 15,3237 (1976). (12) E. T. Kaiser. T.-W. Chan, and J. Suh in "Protein-Metal Interactions". M. Friedman, Ed., Plenum Press, New York, N.Y., 1974, pp 49-80. (13) For a full discussion of this problem see ref 8. The most recent reasonable assignments of pK. values are given in ref 10. (14) M. L. Bender and F. J. Kezdy, Annu. Rev. Biochem.. 34,49 (1965). (15) J. F. Riordan, Biochemistry, 12,3915 (1973). (16) D. S. Auld and B. Holmquist, Biochemistry, 13,4355 (1974). (17) L. G. Lange, D. S. Auld, and J. F. Riordan, Biochemistry, 13, 1983 (1974). (18) J. W. Bunting and C. D. Myers, Can. J. Chem., 53, 1993 (1975). (19) Glycolic, L-lactic. D- and DL-mandelic, and L-phenyllactic acids were obtained commercially. DL-3-Phenyllactic and ~~-2-hydroxy-4-phenylbutanoic acids were prepared by acidic hydrolysis of the cyanohydrins of phenylacetaldehyde and 3-phenylpropionaldehyde,respectively. Other 2hydroxy acids were prepared from the commercially available 2-bromo acids.20

(20) J. Murphy and J. W. Bunting, Can. J. Chem., 53,283 (1975). (21) Our previous report' that the sodium salt of 2 (R = CeH5)is insoluble in water at these concentrations is in error. The acetone procedure described herein was not used previously. Our earlier work was complicated by the very low aqueous solubility of the free acid of this ester. (22) J. W. Bunting and J. Murphy, Can. J. Chem., 52,2640 (1974). (23) G. N. Wilkinson, Biochem. J., 80, 324 (1961). (24) J. W. Bunting and C. D. Myers, Can. J. Chem., 51,2639 (1973). (25) J. W. Bunting and C. D.Myers, Can. J. Chem.. 52,2053 (1974). (26) J. W. Bunting and C. D. Myers, Can. J. Chem., 53, 1984 (1975). (27) S. S.-T. Chu, M.Sc. Thesis, University of Toronto, 1974. (28) D. S. Auld and B. L. Vallee. Biochemistry, 9,602 (1970). (29) L. D. Byers and R. Wolfenden, Biochemistry, 12,2070 (1973). (30) C. Hansch. Acc. Chem. Res., 2, 232 (1969). (31) A. Leo, C. Hansch, and D. Elkins, Chem. Rev., 71,525 (1971). (32) C. Tanford, J. Am. Chem. SOC.,84, 4240 (1962). (33) P. Mukerjee, Adv. Colloidlnterface Sci., 1, 241 (1967). (34) J. Murphy and J. W. Bunting, Can. J. Chem., 52,3829 (1974). (35) W. N. Lipscomb, G. N. Reeke, Jr., J. A. Hartsuck, F. A. Quiocho, P. H. Bethge. M. L. Ludwig, T. A. Steitz, H. Muirhead, and J. C. Coppola, Brookhaven Symp. Biol., 21, 24 (1968). (36) W. N. Lipscomb, G. N. Reeke. Jr., J. A. Hartsuck, F. A. Quiocho, and P, H. Bethge, Phiios. Trans. R. SOC.London, Ser. 6, 257, 177 (1970). (37) The current status of the acyl-enzyme intermediate hypothesis for carboxypeptidase A is summarized by Suh and Kaiserlo and Breslow and Werni~k.~* (38) R . Breslow and D.Wernick. J. Am. Cheni. SOC., 98,259 (1976).

Intramolecular Catalysis of Acylation and Deacylation in Peptides Containing Cysteine and Histidine Michael J. Heller, Joseph A. Walder, and Irving NI. Klotz"

Contribution f r o m lhe Department of Biocheniistry tind Molecular Biology, rind the> Depurtnient of Chemistry, North Msrstern Unirersity, Eranston. Illinois 60201, Receioed August 27. 1976

Abstract: A series o f peptides having various arrangements o f cysteine and histidine was synthesized to determine the potential o f thiol-imidazole intramolecular systems for catalysis o f h y d r o l y t i c reactions. W i t h substituted phenyl acetates as substrates. n o cooperative effect o f histidine was observed for the acylation o f the cysteine residue. In the deacylation o f the S-acylated peptides, again the intramolecular histidine h a d l i t t l e or n o effect. However, i n the presence of the t h i o l specific reagents. 5.5'dithiobis(2-nitrobenzoic acid) (Ellman's reagent) a n d N-ethylnialeimide, the rates o f appearance o f free SH f r o m S-acyluted peptides were m u c h greater than the observed deacylation rates in the absence o f these reagents. For the peptide N - A c - G l y Arg-Phe-Cys(Ac)-Phe-His-Gly-COOH,the rate constant for the appearance o f free SH iri the presence o f Ellman's reagent was 3.1 X IO-* inin-' under conditions for w h i c h the observed deacylation rate was 3.2 X m i n - I (pH 8.1, 25 "C). This trapping effect was not observed for cysteine peptides l a c k i n g histidine o r for mixtures of N,,T-diacetylcysteirie and imidazole free i n solution. These observations indicate the existence of a rapid, reversible intramolecular transfer o f the acetyl group between the cysteine and histidine residues which greatly favors cysteine. A mechanism i n h i b i t i n g back-attack b y cysteine o f the acyl histidine would allow efficient deacylation t o occur through intramolecular nucleophilic catalysis b y the iinida7ole group. T h e cysteine proteases m a y have evolved such a mechanism.

There exists i n nature a homologous series of proteolytic enzymes having cysteine and histidine a t their active sites. In these enzymes, such a s papain, the thiol group of cysteine serves as the primary nucleophile, cleaving the acyl linkage of the substrate with the resultant formation of a n acyl-thiol intermediate. The imidazole moiety of histidine is thought to assist in this process as well as in the deacylation of the acylenzyme required to regenerate the catalyst.' A number of model thiol-imidazole intramolecular systems have been studied previously in efforts t o demonstrate the catalytic ability of this combination for hydrolytic reactions. With cysteine-histidine peptides,2-h little or no assistance by imidazole has been found for the nucleophilic attack of the SH group on p-nitrophenyl acetate. With the same substrate, only a sixfold increase in rate was observed6 for the reaction with 4-(2-mercaptoethyl)imidazoleover that with glutathione. For the deacylation step the largest acceleration reported

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was 30-fold, found for S-Ac-c)iclo-(Cys-His) compared with ethyl thiolacetate.h However, as shown below, when comparisons are made to more appropriate models lacking imidazole, little if any o f this effect can be attributed to the imidazole group. Thus, there has yet to be demonstrated any significant cooperative effect in the catalysis of hydrolytic processes by thiol-imidazole intramolecular systems as is thought to occur in the cysteine proteases. Since in certain of these model systems the imidazole and S H groups are in extremely close proximity, interactions with other functional groups must be required for the imidazole moiety to exert its catalytic effects. With this in mind we studied a series of cysteine--histidine peptides having other functional groups, looking not only for overt catalytic efficacy, but also for potential catalytic effects with thiol-specific trapping reagents. O u r results show the nature and magnitude o f the intramolecular effects that d o

April 13, 1977

278 1 occur within the peptides and explain why no significant cooperative effects were observed in the systems studied by other workers.

I 0.3

I

'

I

I

I

I

I

-

Experimental Section Materials. N-t- Boc-Ninf-Tos-L-His,N-t-Boc-S-p-MeOBzl-~-Cys, N-t-Boc-Ng-NOz-L-Arg as well as all other Boc-L-amino acids were obtained from Beckman Instruments, Inc. Bio-Beads S-X-I chloromethylated resin was purchased from Bio-Rad Laboratories. NAcetylcysteine, imidazole, p-nitrophenyl acetate, and N-ethylmaleimide were the products of Sigma Chemical Co. Ellman's reagent [ 5,5'-dithiobis(2-nitrobenzoic acid)] was obtained from Pierce Chemical Co., and phenyl acetate from Aldrich Chemical CO. p-Chlorophenyl acetate was prepared from the reaction of 1 equiv ofp-chlorophenol, 1 equiv of acetyl chloride, and 1 equiv of pyridine for 1 h at 25 "C. Cold ethyl ether was added and the precipitated pyridinium chloride was filtered off. The ether was evaporated under reduced pressure. The product, p-chlorophenyl acetate, revealed only one component in chromatographic analysis on silica gel plates with the solvent mixture, benzene-dioxane-acetic acid (1 8:2: I ) . Spectrophotometric analysis at 280 nm, before and after complete hydrolysis, indicated the product was >95% p-chlorophenyl acetate. Peptide Synthesis. The peptides, N-Ac-Gly-Arg-Phe-Cys-PheHis-Gly-COOH ( I ) . N-Ac-Gly-His-Phe-Cys-Phe-His-Gly-COOH ( I I ) , N-Ac-His-Pro-Cys-Pro-His-Gly-COOH (111), N-Ac-CysGly-His-Gly-COOH (IV), and N-Ac-Gly-Cys-Gly-C00H (V), were synthesized by the Merrifield solid-phase method.'" Boc-glycine, the carboxyl terminal amino acid, was esterified to S-X- 1 -chloromethylated resin ( I . 3 mequiv of Cl/g of resin) by refluxing in ethanol with triethylamine for 8 h. The amount of Boc-glycine esterified was 0.1 mequiv of amino acid/g of resin, tert-Butoxycarbonyl (t-Boc) groups were removed by treatment with 30% (v/v) of trifluoroacetic acid in chloroform for 30 min. Couplings of successive amino acids were done in methylene chloride using a six- to eightfold excess of Boc-amino acid and of dicyclohexylcarbodiimide above the amount of the first amino acid coupled to the resin. Each coupling step was checked for completeness by mixing a small amount of peptide-resin with 2,4.6-trinitrobenzenesulfonicacid in buffer solution (Bistris, 0.02 M, pH 8.0). I f no orange color was detectable within 2 min, coupling was assumed complete. The N-terminal group of all completed peptides was acetylated by dicyclohexylcarbodiimide coupling with acetic acid. The completed N-acetylated peptides were cleaved from the resin with CoF3-dried hydrogen fluoride. About 15 mL of H F and 2 g of peptide/resin were mixed for 30 niin at 0 "C. Anisole was added to protect sensitive amino acids during the H F cleavage. The cleaved peptide/resin mixture was thoroughly dried by vacuum to remove H F and anisole. The peptide/resin was then washed several times with ethyl acetate to remove organic impurities and again dried in vacuum. The peptides were extracted from the resin with a small amount of 1 % acetic acid-water solution. The resin was then removed by filtration and the resulting solution lyophilized. The peptides were analyzed by thin-layer chromatography on silica gel plates in butanol-acetic acid-water (8:2:2) and in 2-butanoneacetic acid-water (1:3:2.5). The chromatograms were sprayed with Pauly reagent (for histidine) to visualize the peptides. The peptides were pure by chromatographic analysis. Chromatograms of peptides were also sprayed with ninhydrin, and the results established that N-terminal acetylation was complete. Histidine content of the peptides was determined spectrophotometrically, with a Beckman DU spectrophotometer set at 500 nm, by means of the Pauly reaction.7bCysteine assays used Ellman's reagent* and the spectrophotometer set at 41 2 nm. Arginine was determined qualitatively by the Sakaguchi test.7b Amino acid analyses, with a Durrum D-500 analyzer, were carried out on acid (HCI) hydrolysates of peptides I-IV. The observed values were in good agreement with those expected from the sequence shown in Table I. Cysteine was not determined in the amino acid analyzer. Kinetics. For kinetic determinations all buffers were made fresh and degassed. All samples and reaction mixtures were kept under argon to minimize oxidation of the thiol group. The pK, of the cysteine S H group within the peptides was determined from the difference spectra at 230 nm as a function of pH, according to the method of D o n ~ v a n . ~ The catalyzed hydrolysis of substituted phenyl acetates was studied

1

o.2

-

0.1

.

4

I

260

220

I

300

Wavelength, nm

Figure I . ( I ) Ultraviolet difference spectrum with peptide I at about 1 X I O W "I.I in both sample and reference cells ( I cm. 3 mL volume); 0.07 M borate-0.1 M NaCl buffer, pH 9.6, at 25 "C; (2) difference spectrum recorded about 30 s after injection of 4 p L of 10% acetic anhydride ( i n

acetonitrile) into the sample cell; ( 3 ) same solution as in ( 2 ) recorded 30 min after acetylation; the absorbance near 260 nm was completely gone after about 10 min; (4) 80 min after acetylation; (5) 155 min after acetylation; (6) 215 min after acetylation. Table I. Amino Acid Composition of Peptides

I

I1

IV

111

Obsd Theor Obsd Theor Obsd Theor Obsd Theor Gly His Phe Arg Pro

2.0 1.0

1.9 1.2

2.0 1.0 2.0

2.0

2.2 2.1

2.0 2.0 2.0

2.1

1.0 2.0

2.2

2.0

1.0

2.0 1.1

2.0 1.0

1.0

with imidazole, with N-acetylcysteine and with each of the peptides I-IV. The substrates employed were p-nitrophenyl acetate, m-nitrophenyl acetate, p-chlorophenyl acetate, and phenyl acetate. Their cleavage reactions were followed with a Beckman DU spectrophotometer measuring the release of the corresponding phenol at 400,400, 280, and 280 nm, respectively. Second-order rate constants for the acylation process were determined from the initial rates according to initial rate - spontaneous rate k, = (1) [catalyst] [substrate] The nitrophenyl esters were examined in the pH range 7.5-8.5 (in 0.01 M Bistris buffer with 0.1 M NaCI), p-chlorophenyl acetate and phenyl acetate in the pH range 9.0-9.5 (in 0.01 M Tris buffer with 0.1 M NaCI). The concentration of the ester was 6-8 X M and the concentration of potential catalyst 3-5 X M. All experiments were performed at 25 "C. The deacylation studies were carried out in 0.02-0.07 M borate buffer containing 0.1 M NaCl and in 0.02 M Tris buffer with 0.1 M NaCI, buffers covering the pH range of 8 to IO. The peptides, at a concentration of approximately 5 X M, were acetylated with a 10- to 15-fold excess of acetic anhydride (at the pH at which deacylation was to be followed), injected as a 10% solution in acetonitrile. The volume of acetonitrile in the final solution was -

kgen. base

coo-

+

phenol or acetate

pathway

cHFcOO-

or

0

CI$-C'o'C-CH3 I?

rea gent

-0oc

coo-

+ thioni trobenzoate anion

In any event, the interactions necessary for imidazole to assist in the acylation of a thiol have yet to be demonstrated in model systems. In the deacylation process (Scheme I), we have found for the S-acetylated peptides accelerations as large a s approximately 120-fold above the deacylation rate reported for ethyl thiolacetate (Table 111). Schneider6 observed a n acceleration of about 30-fold for the deacylation of S-Ac-cyclo-(Cys-His) compared with the same reference compound. However, the amide bonds in cysteine peptides are electron withdrawing and in themselves should labilize the thiol ester. Consequently a reference compound more appropriate than ethyl thiolacetate is required. For such, we have chosen the peptide N-Ac-GlyCys(Ac)-Gly-COOH(V). T h e deacylation rate of peptide V was found to be 20 times greater than that of ethyl thiolacetate. With peptide V as the reference compound, tha largest possible acceleration attributable to the imidazole moiety in S-Accyclo-(Cys-His)6 is only lS-fold, and in even our best peptide, I, only 5.6-fold. Thus, large variations in peptide structures show only small differences in the deacylation rates. T h e imidazole group could, in principle, participate in the deacylation of the acyl-thiol through either a general base or nucleophilic mechanism (Scheme I). T h e deacylation rate of peptide I was found to be linear in (OH-) between pH 8.0 and 10 (Figure 2) and was zero within experimental error when extrapolated to zero (OH-) concentration. Since a general base pathway with imidazole should be independent of (OH-) in this p H region, its contribution to the observed rate must be kinetically insignificant. The contribution of the nucleophilic pathway to the observed deacylation rate can be best appreciated in the light of the trapping experiments with Ellman's reagent and N-ethylmaleimide. Both compounds a r e thiol specific reagents and have not been observed to react with acylated thiols. For every one of our S-acetylated thiol-imidazole peptides, however, the , the presence of either rate of appearance of free S H , k t r a pin of these agents was much greater than the observed deacylation rate in the absence of the thiol specific reagent, kobsd [approximately 100-fold greater a t p H 8 (Tables IV and V)]. This trapping effect thus demonstrates the existence of a rapid transfer of the acetyl group from cysteine, not reflected in the observed rate of deacylation in solutions without the sulfhydryl reagents. Since a corresponding effect was not observed for

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N-Ac-Gly-Cys(Ac)-Gly-COOH(V) or for N,S-diacetylcysteine, the transfer in peptides I-IV must represent the intramolecular nucleophilic attack by imidazole on the acetylated thiol. T h e rate ( V N )for the nucleophilic mechanism based on a steady-state assumption applied to the concentration of the imidazole-acetylated peptide (A) is given by VN =

+ k2 (S-acetylated peptide)

k2kl

k-1

V N = kN(S-acetylated peptide)

(3)

where kN is the effective first-order rate constant for the nucleophilic pathway and the other rate constants are defined in Scheme I. T h e term kl(S-acetylated peptide) gives the rate of formation of A, and k 2 / ( k - 1 k2) represents the probability that A will hydrolyze to products rather than transacetylate to regenerate the S-acetylated peptide. Once the acetyl group is transferred to the imidazole moiety, the trapping reagent can react rapidly with the free tliiol (Scheme 1). Since the trapping rate was independent ot the concentration of Ellman's reagent, ktrdPwith Ellman's redgent must equal k 1. Accordingly, ktrapwith Ellman's reagent was found to be independent of (OH-) from p H 8.0 to 10 (Figure 2).20 For the S-acetylated peptide I, kl = ktrdpN 3 X IO-? min-l. Similar trapping rates were obtained for the other S-acetylated thiol imidazole peptides (Table IV). From eq 2 and 3, one can show that

+

(4)

From eq 4 we can obtain a lower limit for k-1. The rate ot hydrolysis of the acetylimidazole moiety in thiol-imidazole min-I. Since the diacetylated peptide I a t p H 8.1 is 4.6 X thiol group in this peptide is also acetylated, this observed rate is not rigorously k2, but if it is a t all different it should be less than k2, as the free thiol might be able to serve as a general acid-base catalyst. T h e value for kN must be less than the observed deacylation rate constant, 3 X 1 0-4 min-' a t pH 8.1, for peptide I (Figure 2 ) . Substituting these values into eq 4, we obtain about 5.0 min-l as a lower limit for k-1 a t pH 8. I . From this result it is also possible to estimate the equilibrium I ~ the transfer of the acetyl group from constant K S H -for

/ April 13, 1977

2785 cysteine to histidine:

K s H . =~ k~l 1~k - l

< 4.4 X

IO-'

(5)

This value is consistent with that of about 2.7 X calculated on the basis of the free energies of hydrolysis of ethyl thiolacetate and acetylimidazole.21Thus, the preequilibrium for acylimidazole formation is highly unfavorable. With increasing p H , K S H . ~ increases "~ attendant to the ionization of the S H group. Accordingly if k&sd were equal to kr\i the titration of the thiol group (pK, 8.7) would be manifested in the pH-rate profile for peptide I (Figure 2). Since it is not, k N cannot contribute appreciably to kobsd. I n large measure, then, kobad appears to simply represent the hydroxide ion attack, koli-, on the acyl-thiol, with subtle structural and electronic factors contributing to the slightly higher deacylation rates that are observed for the cysteine-.histidine peptides over peptide V lacking histidine. The trapping studies clearly demonstrate that if the backattack of the thiol group were inhibited in cysteine-histidine peptides, intramolecular imidazole would be able to function effcctively as a nucleophilic catalyst for the deacylation process. This conclusion is strongly supported by results from Bruice'sZ2" study of the model compound N-propyl-y-(4'it1iidazolyl)thiolbiityrate (structure 1 in Scheme 11). T h e rate

-

Scheme I1

Prevailing theory holds that imidazole functions as a general base catalyst for the deacylation of the cysteine proteases.'*25 However, significant general base catalysis by imidazole in the deacylation of a n acyl-thiol has never been demonstrated in a n intramolecular model system. On the other hand, it is clear from Bruice's studies22aof structure 1 (Scheme 11) that in a well-approximated system in which back-attack by the thiol is inhibited, in this case because of the leaving group effect, nucleophilic catalysis by imidazole would result in a deacylation rate of approximately the same order of magnitude as occurs in the cysteine proteases. T h e main evidence which suggests general base catalysis in the proteases is a deuterium isotope effect of approximately 3 for the deacylation proC ~ S S . ~ ~However, .~' this is also compatible with the nucleophilic mechanism: the nucleophilic attack by imidazole or the deacylation of the acylimidazole, or both, may be subject to general acid-base catalysis. Although one must proceed cautiously in extrapolating from model systems to an enzyme, the existing evidence would certainly favor nucleophilic catalysis over general base catalysis for the role of imidazole in the deacylation of the cysteine proteases.

Acknowledgment. This investigation was supported in part by grants from the National Science Foundation ( N o . BMS72-02204) and from the National Institute of General Medical Sciences, I J S . Public Health Service ( N o . G M 09280). References and Notes

1

2

of hydrolysis of 1 a t pH 7.0 is -IOs tinics faster than that of methyl thiolacetate and is limited by the hydrolysis of the intermediate lactam. I n compound 1, the thiol moiety is the leaving group and cannot reattack the acylimidazole that is formed.22bFor the comparable intramolecular system in which back-attack is possible, the S-acetyl derivative of 4-(2-mercaptocthy1)iniidazole (structure 2 in Scheme I I ) , Schneiderh observed little if any increase in the rate of deacylation over simple thiol esters. To inhibit the back attack of the thiol in an intramolecular catalytic system such as the cysteine -histidine peptides, one needs other functional groups, properly positioned to stabilize the thiol in a n unreactive conformation while the imidazole is deacylated and to enhance the rate of hydrolysis of the acylimidazole. Although this type of mechanism might be difficult to attain synthetically, the cysteine proteases, such as papain, may have evolved a mechanism that embodies this effect. T h a t such reaction mechanisms can be achieved has been demonstrated by Fersht and Kirby23 with the hydrolysis of aspirin-like compounds. They have pointed outZ4that in aspirin itself back-attack by the phenolate group on the intermediate anhydride prevents the ortho carboxyl group of aspirin from functioning effectively as a nucleophilic catalyst for aspirin hydrolysis. On the other hand, incorporation of a second carboxyl group to that of aspirin, giving 3 - a ~ e t o x y p h t h a l a t e , ~ ~ allowed the nucleophilic pathway to function efficiently.

(1) G. Lowe, Tetrahedron, 32, 291 (1976). (2) K. Nakajima and K. Okawa, Bull. Chem. SOC.Jpn., 46, 1881 (1973). (3) I. Photaki, V. Bardokos, A. W. Lake, and G. L.owe, J. Chem. Soc C, 1860 (1968) (4) I. Photaki and S. Moschopedis, Experientia, 25, 903 (1969). (5) F. Schneider, E. Schaich, and H. Wenck, Hoppe-Seyler's 2. physiol. Chem., 349, 1525 (1968). (6) F. Schneider, Hoppe-Seyler's 2. Pbysiol. Chem, 348, 1034 (1967). (7) (a) J. M. Steward and J. D. Young, "Solid Phase Peptide Synthesis", W. H. Freeman, San Francisco, Calif., 1969; (b) ;bid., p 56. (8)G. L. Ellman, Arch. Biochem. Biophys., 82, 70 (1959). (9) J. W. Donovan, Biochemistry, 3, 67 (1964). (10) E. Roberts and G. Rouser, Anal. Cbem., 30, 1291 (1958). (11) J. F. Kirsch and W. P. Jencks, J. Am. Cbem. SOC.,86, 837 (1964). (12) M. Friedman, J. F. Cavins, and J. S . Wall, J. Am. Cbem SOC..87. 3672 (1965). P. N. Rylander and D. S. Tarbeil, J. Am. Cbem. SOC.,72, 3021 (1950). J. Drenth, J. N. Jansonius, R. Koekoek, and B. G. Wolthers, "The Enzymes", Vol. 111, P. D. Boyer, Ed., Academic Press, New York, N.Y.. 1971, pp 485-499. S. S. Husain and G. Lowe, Biochem. J., 108, 855 (1968). S. S. Husain and G. Lowe. Biochem. J., 108, 861 (1968). S. S. Husain and G. Lowe, Biochem. J., 117, 333 (1970). S. S. Husain and G. Lowe. Biochem. .1., 117, 341 (1970). D. M. Blow, Acc. Chem. Res., 9, 145 (1976). Although papain, like chymotrypsin, does have a carboxyl group (aspartate-158)in close proximity to the active-site histidine, the imidazole moiety of this histidine appears to be more closely approximated to an asparagine residue (see ref 14). kbapwith Nethylmaleimide. pH 7.7, was approximately one-half the value for kaap with Ellman's reagent, pH 8.0-10 (Tables IV and V). Although this difference may be due to differences in reaction conditions, saturation of the trapping rate was not demonstrated with Kethylmaleimide, whereas it was with Ellman's reagent, and hence ktrapwith Kethylmaleimide may be less than k , . Attack by Ellman's reagent of the acetylated thiol does not occur and hence cannot account for this difference. J. Gerstein and W. P. Jencks, J. Am. Chem. SOC., 86, 4655 (1964). The equilibrium constant KsH.,, was calculated using a value of 8.7 for the pKa of the thiol group, as occurs in the peptides. (a) T. C. Bruice, J. Am. Chem. SOC.,81, 5444 (1959). (b) Bruice showed in this study, that if thiol at very high concentration is added to the reaction, the hydrolysis of compound 1 could be completely inhibited. A. R. Fersht and A. J. Kirby, J. Am. Chem. SOC.,90, 5833 (1968). A. J. Kirbyand A. R . Fersht, Prog. Bioorg. Chem., 1, 11 (1971). A. N. Glazer and E. L. Smith, "The Enzymes", Vol. Ill, P. D. Boyer, Ed., Academic Press, New York, N.Y., 1971, pp 533-535. J. R. Whitaker and M. L. Bender, J. Am. Chem. SOC.,87, 2728 (1965). L. J. Brubacher and M. L. Bender, J. Am. Chem. SOC., 88,5871 (1966).

Heller, Walder, Klotz

/ Peptides Containing Cysteine and Histidine