Proc. Natl. Acad. Sci. USA Vol. 75 No. 1, pp. 172-174, January 1978
Biochemistry
Absence of evidence for an intermediate in the deacetylation of acetylchymotrypsin (enzyme kinetics/acyl-enzyme mechanism)
P. W. RIDDLES, J. DE JERSEY, AND B. ZERNER* Department of Biochemistry, University of Queensland, St. Lucia, Queensland, Australia 4067
Communicated by F. H. Westheimer, October 21, 1977
of Eq. 1, we have reinvestigated this work. Our results, which are totally consistent with the scheme of Eq. 1, negate the existence of the proposed intermediate and once again point up the need for care not only in the design of meaningful enzymatic experiments but equally in the interpretation of whatever results are obtained.
ABSTRACT A recent paper [Chibber, B. A. K., Tomich, J. M., Mertz, E. T. & Viswanatha, T. (1977) Proc. NatL Acad. Sci. USA 74, 510-514] presented evidence that was taken to support the existence of an intermediate in the deacetylation of acetylchymotrypsin. It was observed that deacylation, as measured by following the decrease in [14C]acetylchymotrypsin (decrease in acid-precipitable radioactivity), occurred at 'o the rate of reactivation, as measured by return of activity toward N-acetyl-L-tyrosine ethyl ester. Our experiments have shown that, at pH 6, the deacylation rate constant (measured by the loss of [14C]acetylchymotrypsin and by the formation of [14C~acetate) is identical (within experimental error) with the rate constant for reactivation (measured by determining the activity of aliquots of reactivating enzyme against N-acetyl-L-tryptophan ethyl ester) and with kat for the turnover of p-nitrophenyl acetate by a-chymotrypsin. Part of the 10-fold greater reactivation rate observed by Chibber et a. has been shown to be due to the presence of 10% (vol/vol)'isopropanol in their reactivation mixture, and it is argued that the balance of the effect is a manifestation of the "indole effect" produced by the simultaneous presence of 10 mM N-acetyl-L-tyrosine ethyl ester throughout the reactivation experiments. The results presented are entirely consistent with the three-step mechanism of catalysis by a-chymotrypsin and negate the existence of the proposed additional acetylenzyme intermediate. The hypothesis that all reactions catalyzed by a-chymotrypsin (EC 3.4.21.1) proceed through an intermediate acyl-enzyme (ES', Eq. 1) has not been entirely without merit in accounting for the mass of data available on this enzyme (1). ES
ki kl
vES
k2
io-ES' +
k3
oE +P2
MATERIALS AND METHODS [14C]Acetic anhydride (0.55 Ci/mol) was prepared by heating sodium [U-'14C]acetate (1 mCi; 57 Ci/mol; Radiochemical Centre) with cold acetic anhydride (170 mg) for 2 hr at 1100 in an evacuated sealed tube, followed by high-vacuum distillation of the resulting radioactive anhydride. p-Nitrophenyl [14C]acetate was prepared by reaction of [14C]acetic anhydride (164 mg; 1.6 mmol) with p-nitrophenol (446 mg; 3.2 mmol) and dicyclohexylcarbodiimide (330 mg; 1.6 mmol) in dichlorpmethane (7 ml) for 2 hr at 25°. p-Nitrophenyl acetate isolated from this reaction mixture (162 mg) was mixed with pure cold ester (325 mg) and recrystallized from chloroform/hexane to constant specific radioactivity (67 mCi/mol); melting point, 76.8°-77.70. The yield of p-nitrophenolate ion (f4w = 18.314) on complete hydrolysis of an aliquot of ester in 0.1 M NaOH was 99.7% of the calculated value for pure p-nitrophenyl acetate. a-Chymotrypsin (3 times crystallized, Worthington Biochemical Corp.) was further purified by chromatography on Amberlite CG-50 (8) and shown to be free of autolysis products (3). Purified enzyme was concentrated by ultrafiltration (Diaflo PM-10 membrane), dialyzed against 0.1 M KCI, pH 3.08 (with dilute HCU), and stored at 4°. Enzyme concentration was determined spectrophotometrically [A"~mat 280 nm = 20.0 (9)] and by titration of active sites with p-nitrophenyl acetate (10). In preliminary experiments, the procedure of Chibber et al. (7) was used for preparation of acetylchymotrypsin. However, adjustment of the pH of the acetyl-enzyme solution from 3.0 to 6.0 repeatedly caused partial precipitation of the protein. Consequently, it was decided to carry out the entire procedure at pH 6.0, as follows: (i) a solution of a-chymotrypsin in 0.1 M KCl at pH 3.08 (2 ml, 21.9 mg/ml) and 0.05 M phosphate buffer at pH 6.02 (2 ml) was dialyzed at 4° against the same phosphate buffer; (ii) p-nitrophenyl [14C]acetate in acetonitrile (150 ,1, 0.793 mM) was added to dialyzed a-chymotrypsin (3 ml, -10 mg/ml) at 250, and the reaction was followed in a Cary 17 spectrophotometer at 360 nm until a steady state was reached; (Mii) the reaction mixture was cooled to 40 and loaded onto a precalibrated column of Sephadex G-25 (2.2 X 22 cm; flow rate, 170 ml/hr) equilibrated with the same phosphate buffer, at 40; (iv) a fraction containing the peak 70% of the acetylchymotrypsin (7.1 ml) was immediately warmed to 250
II]
P1 Nonetheless, there have been more than a few attempts to show that the mechanism implied in Eq. 1 does not apply to all substrates or that it is a simplification of a more elaborate scheme containing more obligatory intermediates. Such attempts, however, have been found to provide a totally unsatisfactory account of the available experimental data when they were amenable to simple interpretation (2, 3); even when the experiments were confused by dye-binding studies or supposed "single turnover" reactions (4), the data were amenable to simple alternative explanations totally consistent with Eq. 1 (5,
6).
The most recent addition (7) to the a-chymotrypsin literature arguing for a reappraisal of Eq. 1 purports to provide evidence for an active intermediate in the deacylation step when the substrate is p-nitrophenyl acetate. Because the existence of such an intermediate would constitute an important amplification The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
*
172
To whom reprint requests should be addressed.
Biochemistry: Riddles et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
.
173
arnd maintained at that temperature. Subsequent- atialysis. showed a protein concentration of 3.19 mg/ml, with 0.946 acetyl group per molecule of enzyme. Aliquots were removed at suitable time intervals to permit concurrent determination of the rate of deacylation and the rate of recovery of activity.
Deacylation. Aliquots (0.5 ml) were taken at 8-min intervals, and the deacylation reaction was quenched by addition of 0.5 ml of 0.014 M HCO to give a final pH of 3.00. Each sample was then loaded onto a column of Sephadex G-25 (1.1 X 21 cm) that was precalibrated to allow collection of three fractions, the first containing [14C]acetylchymotrypsin (and free a-chymotrypsin), the second with no radioactivity, and the third containing [14C]acetate. Fractions were collected in weighed tubes, and 1-ml portions were added to scintillation fluid [15 ml; toluene containing Omnifluor (4 g/liter), and Triton X-100; 1:0.7 (vol/vol)] for assay of radioactivity in a Beckman LS250 scintillation system. Hence, the decrease in the concentration of acetylchymotrypsin and the increase in the concentration of acetate ion were measured concurrently and independently. Reactivation. The activity of aliquots of the reaction mixture against N-acetyl-L-tryptophan ethyl estert was determined spectrophotometrically at 25° by following the increase in absorbance (AE = 240) at 300 nm (11). Aliquots (50 Al) were taken at 8-min intervals and added to a spectrophotometer cell containing 3 ml of 0.05 M phosphate buffer (pH 6.02) and 100 Al of 32.4 mM N-acetyl-L-tryptophan ethyl ester [Mann Research Laboratories; melting point, 109.40-109.70; literature (11, 12) melting point, 110°-111.5°, 1090-109.5o] in acetonitrile. Under these conditions, with a mixing time of 10 sec, initial rates were linear, and an accurate estimate of the activity could be obtained within 30 sec. The a-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate was studied at 250 in 0.05 M phosphate buffer (pH 6.02) with [S]o > [E]o. Reaction mixtures contained p-nitrophenyl acetate in water (2 ml), 0.15 M phosphate buffer (1 ml), and a-chymotrypsin in 0.1 M KCI, pH 3.0 (100AId), giving a final pH of 6.02. In each run, the "burst" of p-nitrophenol corresponding to acylation and the turnover rate were both measured at 347.5 nm. Details of the turnover experiments in the presence of isopropanol are included in Table 1.
RESULTS AND DISCUSSION Fig. 1 shows first-order plots for the deacetylation of acetylchymotrypsin (measured both by the loss of enzyme-bound acetyl groups and by the formation of acetate ion) and for the reactivation (measured against N-acetyl-L-tryptophan ethyl ester as substrate). The first-order rate constants obtained from the data of Fig. 1 are listed in Table 1, which also includes the values of kob (i.e., kcat) determined in the steady-state (turnover) experiments. The following conclusions may be drawn from these results. (i) The rate constant obtained for the deacetylation of acetylchymotrypsin at pH 6.0 is in good agreement with the value (3.33 + 1.33 X 10-4 sect at 25) determined by Chibber et al. (7). (ii) The rate constant for return of activity toward N-acet
This ester was chosen because of its desirable spectral characteristics, of its availability in essentially pure form, of the difficulty experienced on many occasions in purifying N-acetyl-L-tyrosine ethyl ester from various commercial sources, and, finally, the evidence for existence of the proposed intermediate must be independent of the substrate used to assess reactivation.
&5.s
-a5
T~~~~~~~~~
8. ao1
-4.0
4
7.5 0
-4.5 40 60 80 TIME (min) FIG. 1. Deacetylation of [14C]acetylchymotrypsin as measured by loss of ['4C]acetylchymotrypsin (@), by formation of [14C]acetate (u), and by reactivation (A); solid lines were calculated by the method of least squares. Ct, cpm at time t; Rt, rate of hydrolysis of N-acetylL-tryptophan ethyl ester at time t. 20
1_
1
tyl-L-tryptophan ethyl ester is the same as the rate constant for deacetylation, indicating that a simple one-step process is sufficient to account for the data. (Mi) Similarly, kat (determined by measuring the zero-order rate immediately following the stoichiometric acylation reaction) agrees well with the deacetylation and reactivation rate constants. Given the mechanism proposed by Chibber et al. (7), the release of p-nitrophenol would be triphasic, with a rapid "burst" of p-nitrophenol corresponding to acylation ([P1]burst -. [E10), a slower release of p-nitrophenol corresponding to the proposed acetyl transfer reaction, and a still slower zero-order release of p-nitr6phenol. Thus, the behavior observed is consistent with a simple one-step deacylation and argues strongly against an intraenzyme acetyl transfer as proposed by Chibber et al. (7). We submit that the results of Chibber et al. (7) and earlier Table 1. Rate constants associated with the a-chymotrypsincatalyzed hydrolysis of p-nitrophenyl acetate* Reaction observed
Deacetylation: Loss of acetylchymotrypsin Formation of acetate
kobsq
104 X sec-1 3.44 3.27 3.59
Reactivation Turnover: Zero isopropanolt 3.75 10% (vol/vol) isopropanolt 11.3 10% (vol/vol) isopropanol§ 14.7 * First-order rate constants at 250 in 0.05 M phosphate buffer, pH 6.02. t [Slo varied from 63 to 135 AsM; [Elo = 3.77 AtM. At higher [S]o, marked substrate activation was observed as previously noted (13, 14), and this activation may explain the slightly higher kobs (kcat = 3.54 X 10-4 sec-l at pH 6.02, from the data of ref. 13). [Slo = 101 uM; pH 6.024. The pH was adjusted after addition of 1P% (vol/vol) redistilled reagent grade isopropanol. § [SIo = 99.4 uM; pH 6.21. The pH was not adjusted after addition of 10% (vol/vol) redistilled reagent grade isopropanol.
174
Biochemistry: Riddles et al.
observations along the same lines (15) are in part manifestations of the "indole effect" (16-18). Foster (16) showed that the presence of 1 mM indole increased kcat for the a-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate at pH 7.9 by a factor of 1.6. Bender et al. (19) observed that the rate of reactivation of acetylchymotrypsin is increased 5-fold at 250 and pH 8.6 by the presence of 2.33 mM N-acetyl-L-tyrosine ethyl ester and interpreted this result in terms of the indole effect. Analogous observations have been made with trypsin: the trypsin-catalyzed hydrolysis of N-acetylglycine ethyl ester is accelerated by alkylamines and alkylguanidines (20). Similarly, benzene (8.44 mM) causes a 2-fold increase in kcat for the pig liver carboxylesterase-catalyzed hydrolysis of phenyl butyrate (21). The conditions used by Chibber et al. (7) to study the reactivation of acetylchymotrypsin differ in two important aspects [the presence of 10 mM N-acetyl-L-tyrosine ethyl ester and simultaneously 10% (vol/vol) isopropanol throughout the reactivation] from the conditions used to study the deacetylation. As discussed above, the presence of 10 mM N-acetyl-Ltyrosipe ethyl ester would be expected to increase the rate of reactivation. Moreover, the results in Table 1 show that 10% isopropanol also causes a marked increase in the rate of deacylation of acetylchymotrypsin. Faller and Sturtervant (14) have'previously reported that, in the Tris-dependent deacylation of acetylchymotrypsin (0.4 M Tris-HCl/O. 1 M NaCl, pH 7, 25'), the presence of 10% (vol/vol) isopropanol produces a 2.78-fold increase in the'deacylation rate constant. The remaining observation of Chibber et al. (7)-that immediately after reactivation of acetyl-chymotrypsin, kcat for the hydrolysis of N.acetyl-L-tyrosine ethyl ester is 27% higher than that for the native enzyme-is not directly commented on in the present work. We note, however, that attempted repetition of their procedure for the preparation of acetylchymotrypsin repeatedly caused partial precipitation of the protein when the pH was adjusted from 3.0 to 6.0 and that the anomalous' results observed by them at short time intervals may be due to this precipitation. The fact that, according to their work, "deacetylation [decrease in acid-precipitable radioactivity] is slow during the first 5 min of exposure of the protein to pH 6.0 but occurs exponentially after that time interval" goes without further comment. Yet this is the critical time period on which their complex analysis is based. Furthermore, there is a possible difference in the purity of the enzymes (i.e., reactivated versus native) used in their two experiments (7), owing to the removal of contaminating peptides present in commercial a-chymotrypsin by the Sephadex G-25 chromatography used
Proc. Natl. Acad. Sci. USA 75 (1978)
to prepare acetylchymotrypsin (3). However, although this proposition is of importance because protein concentration was measured by absorbance at 280 nm, it could not account per se for the decrease in kat reported for N-acetyl-L-tyrosine ethyl ester. In conclusion, our experiments demonstrate that, although the observations by Chibber et al. (7) may be as reported, their explanation of them is untenable. Furthermore, the mechanism implied in Eq. 1 still provides an adequate account of a-chymotrypsin-catalyzed reactions. This work was supported in part by the Australian Research Grants Committee. 1. Zerner, B. & Bender, M. L. (1964) J. Am. Chem. Soc. 86, 3669-3674. 2. de Jersey, J. & Zerner, B. (1969) Biochemistry 8, 1975-1983. 3. de Jersey, J., Keough, D. T., Stoops, J. K. & Zerner, B. (1974) Eur. J. Biochem. 42, 237-243. 4. Gutfreund, H. (1971) Annu. Rev. Biochem. 40,315-341. 5. Himoe, A. & Hess, G. P. (1967) Biochem. Biophys. Res. Commun.
27,494-499. 6. Miller, C. G. & Bender, M. L. (1968) J. Am. Chem. Soc. 90,
6850-6852. 7. Chibber, B. A. K., Tomich, J. M., Mertz, E. T. & Viswanatha, T. (1977) Proc. Natl. Acad. Sci. USA 74,510-514. 8. Hirs, C. H. W. (1955) J. Am. Chem. Soc. 77,5743-5744. 9. Edelhoch, H. (1967) Biochemistry 6, 1948-1954. 10. Bender, M. L., Begue-Cant6n, M. L., Blakeley, R. L., Brubacher, L. J., Feder, J., Gunter, C. R., Kezdy, F. J., Killheffer, J. V., Marshall, T. H., Miller, C. G., Roeske, R. W. & Stoops, J. K. (1966) J. Am. Chem. Soc. 88,5880-5889. 11. Zerner, B., Bond, R. P. M. & Bender, M. L. (1964) J. Am. Chem. Soc. 86,3674-3679. 12. Spies, J. R. (1948) J. Am. Chem. Soc. 70,3717-3719. 13. Kezdy, F. J. & Bender, M. L. (1962) Biochemistry 1, 10971106. 14. Faller, L. & Sturtevant, J. M. (1966) J. Biol. Chem. 241, 48254834. 15. Viswanatha, T. & Lawson, W. B. (1961) Arch. Biochem. Biophys. 93, 128-134. 16. Foster, R. J. (1961) J. Biol. Chem. 236,2461-2466. 17. Cane, W. P. & Wetlaufer, D. (1966) Abstr. Annu. Meet. Amer. Chem. Soc. 152d, 110C. 18. Moore, L. & de Jersey, J. (1970) Aust. J. Biol. Sci. 23, 607615. 19. Bender, M. L., Kezdy, F. J. & Gunter, C. R. (1964) J. Am. Chem. Soc. 86,3714-3721. 20. Inagami, T. & York, S. S. (1968) Biochemistry 7,4045-4052. 21. Stoops, J. K., Horgan, D. J., Runnegar, M. T. C., de Jersey, J., Webb, E. C. & Zerner, B. (1969) Biochemistry 8,2026-2033.
Biochemistry
Absence of evidence for an intermediate in the deacetylation of acetylchymotrypsin (enzyme kinetics/acyl-enzyme mechanism)
P. W. RIDDLES, J. DE JERSEY, AND B. ZERNER* Department of Biochemistry, University of Queensland, St. Lucia, Queensland, Australia 4067
Communicated by F. H. Westheimer, October 21, 1977
of Eq. 1, we have reinvestigated this work. Our results, which are totally consistent with the scheme of Eq. 1, negate the existence of the proposed intermediate and once again point up the need for care not only in the design of meaningful enzymatic experiments but equally in the interpretation of whatever results are obtained.
ABSTRACT A recent paper [Chibber, B. A. K., Tomich, J. M., Mertz, E. T. & Viswanatha, T. (1977) Proc. NatL Acad. Sci. USA 74, 510-514] presented evidence that was taken to support the existence of an intermediate in the deacetylation of acetylchymotrypsin. It was observed that deacylation, as measured by following the decrease in [14C]acetylchymotrypsin (decrease in acid-precipitable radioactivity), occurred at 'o the rate of reactivation, as measured by return of activity toward N-acetyl-L-tyrosine ethyl ester. Our experiments have shown that, at pH 6, the deacylation rate constant (measured by the loss of [14C]acetylchymotrypsin and by the formation of [14C~acetate) is identical (within experimental error) with the rate constant for reactivation (measured by determining the activity of aliquots of reactivating enzyme against N-acetyl-L-tryptophan ethyl ester) and with kat for the turnover of p-nitrophenyl acetate by a-chymotrypsin. Part of the 10-fold greater reactivation rate observed by Chibber et a. has been shown to be due to the presence of 10% (vol/vol)'isopropanol in their reactivation mixture, and it is argued that the balance of the effect is a manifestation of the "indole effect" produced by the simultaneous presence of 10 mM N-acetyl-L-tyrosine ethyl ester throughout the reactivation experiments. The results presented are entirely consistent with the three-step mechanism of catalysis by a-chymotrypsin and negate the existence of the proposed additional acetylenzyme intermediate. The hypothesis that all reactions catalyzed by a-chymotrypsin (EC 3.4.21.1) proceed through an intermediate acyl-enzyme (ES', Eq. 1) has not been entirely without merit in accounting for the mass of data available on this enzyme (1). ES
ki kl
vES
k2
io-ES' +
k3
oE +P2
MATERIALS AND METHODS [14C]Acetic anhydride (0.55 Ci/mol) was prepared by heating sodium [U-'14C]acetate (1 mCi; 57 Ci/mol; Radiochemical Centre) with cold acetic anhydride (170 mg) for 2 hr at 1100 in an evacuated sealed tube, followed by high-vacuum distillation of the resulting radioactive anhydride. p-Nitrophenyl [14C]acetate was prepared by reaction of [14C]acetic anhydride (164 mg; 1.6 mmol) with p-nitrophenol (446 mg; 3.2 mmol) and dicyclohexylcarbodiimide (330 mg; 1.6 mmol) in dichlorpmethane (7 ml) for 2 hr at 25°. p-Nitrophenyl acetate isolated from this reaction mixture (162 mg) was mixed with pure cold ester (325 mg) and recrystallized from chloroform/hexane to constant specific radioactivity (67 mCi/mol); melting point, 76.8°-77.70. The yield of p-nitrophenolate ion (f4w = 18.314) on complete hydrolysis of an aliquot of ester in 0.1 M NaOH was 99.7% of the calculated value for pure p-nitrophenyl acetate. a-Chymotrypsin (3 times crystallized, Worthington Biochemical Corp.) was further purified by chromatography on Amberlite CG-50 (8) and shown to be free of autolysis products (3). Purified enzyme was concentrated by ultrafiltration (Diaflo PM-10 membrane), dialyzed against 0.1 M KCI, pH 3.08 (with dilute HCU), and stored at 4°. Enzyme concentration was determined spectrophotometrically [A"~mat 280 nm = 20.0 (9)] and by titration of active sites with p-nitrophenyl acetate (10). In preliminary experiments, the procedure of Chibber et al. (7) was used for preparation of acetylchymotrypsin. However, adjustment of the pH of the acetyl-enzyme solution from 3.0 to 6.0 repeatedly caused partial precipitation of the protein. Consequently, it was decided to carry out the entire procedure at pH 6.0, as follows: (i) a solution of a-chymotrypsin in 0.1 M KCl at pH 3.08 (2 ml, 21.9 mg/ml) and 0.05 M phosphate buffer at pH 6.02 (2 ml) was dialyzed at 4° against the same phosphate buffer; (ii) p-nitrophenyl [14C]acetate in acetonitrile (150 ,1, 0.793 mM) was added to dialyzed a-chymotrypsin (3 ml, -10 mg/ml) at 250, and the reaction was followed in a Cary 17 spectrophotometer at 360 nm until a steady state was reached; (Mii) the reaction mixture was cooled to 40 and loaded onto a precalibrated column of Sephadex G-25 (2.2 X 22 cm; flow rate, 170 ml/hr) equilibrated with the same phosphate buffer, at 40; (iv) a fraction containing the peak 70% of the acetylchymotrypsin (7.1 ml) was immediately warmed to 250
II]
P1 Nonetheless, there have been more than a few attempts to show that the mechanism implied in Eq. 1 does not apply to all substrates or that it is a simplification of a more elaborate scheme containing more obligatory intermediates. Such attempts, however, have been found to provide a totally unsatisfactory account of the available experimental data when they were amenable to simple interpretation (2, 3); even when the experiments were confused by dye-binding studies or supposed "single turnover" reactions (4), the data were amenable to simple alternative explanations totally consistent with Eq. 1 (5,
6).
The most recent addition (7) to the a-chymotrypsin literature arguing for a reappraisal of Eq. 1 purports to provide evidence for an active intermediate in the deacylation step when the substrate is p-nitrophenyl acetate. Because the existence of such an intermediate would constitute an important amplification The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
*
172
To whom reprint requests should be addressed.
Biochemistry: Riddles et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
.
173
arnd maintained at that temperature. Subsequent- atialysis. showed a protein concentration of 3.19 mg/ml, with 0.946 acetyl group per molecule of enzyme. Aliquots were removed at suitable time intervals to permit concurrent determination of the rate of deacylation and the rate of recovery of activity.
Deacylation. Aliquots (0.5 ml) were taken at 8-min intervals, and the deacylation reaction was quenched by addition of 0.5 ml of 0.014 M HCO to give a final pH of 3.00. Each sample was then loaded onto a column of Sephadex G-25 (1.1 X 21 cm) that was precalibrated to allow collection of three fractions, the first containing [14C]acetylchymotrypsin (and free a-chymotrypsin), the second with no radioactivity, and the third containing [14C]acetate. Fractions were collected in weighed tubes, and 1-ml portions were added to scintillation fluid [15 ml; toluene containing Omnifluor (4 g/liter), and Triton X-100; 1:0.7 (vol/vol)] for assay of radioactivity in a Beckman LS250 scintillation system. Hence, the decrease in the concentration of acetylchymotrypsin and the increase in the concentration of acetate ion were measured concurrently and independently. Reactivation. The activity of aliquots of the reaction mixture against N-acetyl-L-tryptophan ethyl estert was determined spectrophotometrically at 25° by following the increase in absorbance (AE = 240) at 300 nm (11). Aliquots (50 Al) were taken at 8-min intervals and added to a spectrophotometer cell containing 3 ml of 0.05 M phosphate buffer (pH 6.02) and 100 Al of 32.4 mM N-acetyl-L-tryptophan ethyl ester [Mann Research Laboratories; melting point, 109.40-109.70; literature (11, 12) melting point, 110°-111.5°, 1090-109.5o] in acetonitrile. Under these conditions, with a mixing time of 10 sec, initial rates were linear, and an accurate estimate of the activity could be obtained within 30 sec. The a-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate was studied at 250 in 0.05 M phosphate buffer (pH 6.02) with [S]o > [E]o. Reaction mixtures contained p-nitrophenyl acetate in water (2 ml), 0.15 M phosphate buffer (1 ml), and a-chymotrypsin in 0.1 M KCI, pH 3.0 (100AId), giving a final pH of 6.02. In each run, the "burst" of p-nitrophenol corresponding to acylation and the turnover rate were both measured at 347.5 nm. Details of the turnover experiments in the presence of isopropanol are included in Table 1.
RESULTS AND DISCUSSION Fig. 1 shows first-order plots for the deacetylation of acetylchymotrypsin (measured both by the loss of enzyme-bound acetyl groups and by the formation of acetate ion) and for the reactivation (measured against N-acetyl-L-tryptophan ethyl ester as substrate). The first-order rate constants obtained from the data of Fig. 1 are listed in Table 1, which also includes the values of kob (i.e., kcat) determined in the steady-state (turnover) experiments. The following conclusions may be drawn from these results. (i) The rate constant obtained for the deacetylation of acetylchymotrypsin at pH 6.0 is in good agreement with the value (3.33 + 1.33 X 10-4 sect at 25) determined by Chibber et al. (7). (ii) The rate constant for return of activity toward N-acet
This ester was chosen because of its desirable spectral characteristics, of its availability in essentially pure form, of the difficulty experienced on many occasions in purifying N-acetyl-L-tyrosine ethyl ester from various commercial sources, and, finally, the evidence for existence of the proposed intermediate must be independent of the substrate used to assess reactivation.
&5.s
-a5
T~~~~~~~~~
8. ao1
-4.0
4
7.5 0
-4.5 40 60 80 TIME (min) FIG. 1. Deacetylation of [14C]acetylchymotrypsin as measured by loss of ['4C]acetylchymotrypsin (@), by formation of [14C]acetate (u), and by reactivation (A); solid lines were calculated by the method of least squares. Ct, cpm at time t; Rt, rate of hydrolysis of N-acetylL-tryptophan ethyl ester at time t. 20
1_
1
tyl-L-tryptophan ethyl ester is the same as the rate constant for deacetylation, indicating that a simple one-step process is sufficient to account for the data. (Mi) Similarly, kat (determined by measuring the zero-order rate immediately following the stoichiometric acylation reaction) agrees well with the deacetylation and reactivation rate constants. Given the mechanism proposed by Chibber et al. (7), the release of p-nitrophenol would be triphasic, with a rapid "burst" of p-nitrophenol corresponding to acylation ([P1]burst -. [E10), a slower release of p-nitrophenol corresponding to the proposed acetyl transfer reaction, and a still slower zero-order release of p-nitr6phenol. Thus, the behavior observed is consistent with a simple one-step deacylation and argues strongly against an intraenzyme acetyl transfer as proposed by Chibber et al. (7). We submit that the results of Chibber et al. (7) and earlier Table 1. Rate constants associated with the a-chymotrypsincatalyzed hydrolysis of p-nitrophenyl acetate* Reaction observed
Deacetylation: Loss of acetylchymotrypsin Formation of acetate
kobsq
104 X sec-1 3.44 3.27 3.59
Reactivation Turnover: Zero isopropanolt 3.75 10% (vol/vol) isopropanolt 11.3 10% (vol/vol) isopropanol§ 14.7 * First-order rate constants at 250 in 0.05 M phosphate buffer, pH 6.02. t [Slo varied from 63 to 135 AsM; [Elo = 3.77 AtM. At higher [S]o, marked substrate activation was observed as previously noted (13, 14), and this activation may explain the slightly higher kobs (kcat = 3.54 X 10-4 sec-l at pH 6.02, from the data of ref. 13). [Slo = 101 uM; pH 6.024. The pH was adjusted after addition of 1P% (vol/vol) redistilled reagent grade isopropanol. § [SIo = 99.4 uM; pH 6.21. The pH was not adjusted after addition of 10% (vol/vol) redistilled reagent grade isopropanol.
174
Biochemistry: Riddles et al.
observations along the same lines (15) are in part manifestations of the "indole effect" (16-18). Foster (16) showed that the presence of 1 mM indole increased kcat for the a-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate at pH 7.9 by a factor of 1.6. Bender et al. (19) observed that the rate of reactivation of acetylchymotrypsin is increased 5-fold at 250 and pH 8.6 by the presence of 2.33 mM N-acetyl-L-tyrosine ethyl ester and interpreted this result in terms of the indole effect. Analogous observations have been made with trypsin: the trypsin-catalyzed hydrolysis of N-acetylglycine ethyl ester is accelerated by alkylamines and alkylguanidines (20). Similarly, benzene (8.44 mM) causes a 2-fold increase in kcat for the pig liver carboxylesterase-catalyzed hydrolysis of phenyl butyrate (21). The conditions used by Chibber et al. (7) to study the reactivation of acetylchymotrypsin differ in two important aspects [the presence of 10 mM N-acetyl-L-tyrosine ethyl ester and simultaneously 10% (vol/vol) isopropanol throughout the reactivation] from the conditions used to study the deacetylation. As discussed above, the presence of 10 mM N-acetyl-Ltyrosipe ethyl ester would be expected to increase the rate of reactivation. Moreover, the results in Table 1 show that 10% isopropanol also causes a marked increase in the rate of deacylation of acetylchymotrypsin. Faller and Sturtervant (14) have'previously reported that, in the Tris-dependent deacylation of acetylchymotrypsin (0.4 M Tris-HCl/O. 1 M NaCl, pH 7, 25'), the presence of 10% (vol/vol) isopropanol produces a 2.78-fold increase in the'deacylation rate constant. The remaining observation of Chibber et al. (7)-that immediately after reactivation of acetyl-chymotrypsin, kcat for the hydrolysis of N.acetyl-L-tyrosine ethyl ester is 27% higher than that for the native enzyme-is not directly commented on in the present work. We note, however, that attempted repetition of their procedure for the preparation of acetylchymotrypsin repeatedly caused partial precipitation of the protein when the pH was adjusted from 3.0 to 6.0 and that the anomalous' results observed by them at short time intervals may be due to this precipitation. The fact that, according to their work, "deacetylation [decrease in acid-precipitable radioactivity] is slow during the first 5 min of exposure of the protein to pH 6.0 but occurs exponentially after that time interval" goes without further comment. Yet this is the critical time period on which their complex analysis is based. Furthermore, there is a possible difference in the purity of the enzymes (i.e., reactivated versus native) used in their two experiments (7), owing to the removal of contaminating peptides present in commercial a-chymotrypsin by the Sephadex G-25 chromatography used
Proc. Natl. Acad. Sci. USA 75 (1978)
to prepare acetylchymotrypsin (3). However, although this proposition is of importance because protein concentration was measured by absorbance at 280 nm, it could not account per se for the decrease in kat reported for N-acetyl-L-tyrosine ethyl ester. In conclusion, our experiments demonstrate that, although the observations by Chibber et al. (7) may be as reported, their explanation of them is untenable. Furthermore, the mechanism implied in Eq. 1 still provides an adequate account of a-chymotrypsin-catalyzed reactions. This work was supported in part by the Australian Research Grants Committee. 1. Zerner, B. & Bender, M. L. (1964) J. Am. Chem. Soc. 86, 3669-3674. 2. de Jersey, J. & Zerner, B. (1969) Biochemistry 8, 1975-1983. 3. de Jersey, J., Keough, D. T., Stoops, J. K. & Zerner, B. (1974) Eur. J. Biochem. 42, 237-243. 4. Gutfreund, H. (1971) Annu. Rev. Biochem. 40,315-341. 5. Himoe, A. & Hess, G. P. (1967) Biochem. Biophys. Res. Commun.
27,494-499. 6. Miller, C. G. & Bender, M. L. (1968) J. Am. Chem. Soc. 90,
6850-6852. 7. Chibber, B. A. K., Tomich, J. M., Mertz, E. T. & Viswanatha, T. (1977) Proc. Natl. Acad. Sci. USA 74,510-514. 8. Hirs, C. H. W. (1955) J. Am. Chem. Soc. 77,5743-5744. 9. Edelhoch, H. (1967) Biochemistry 6, 1948-1954. 10. Bender, M. L., Begue-Cant6n, M. L., Blakeley, R. L., Brubacher, L. J., Feder, J., Gunter, C. R., Kezdy, F. J., Killheffer, J. V., Marshall, T. H., Miller, C. G., Roeske, R. W. & Stoops, J. K. (1966) J. Am. Chem. Soc. 88,5880-5889. 11. Zerner, B., Bond, R. P. M. & Bender, M. L. (1964) J. Am. Chem. Soc. 86,3674-3679. 12. Spies, J. R. (1948) J. Am. Chem. Soc. 70,3717-3719. 13. Kezdy, F. J. & Bender, M. L. (1962) Biochemistry 1, 10971106. 14. Faller, L. & Sturtevant, J. M. (1966) J. Biol. Chem. 241, 48254834. 15. Viswanatha, T. & Lawson, W. B. (1961) Arch. Biochem. Biophys. 93, 128-134. 16. Foster, R. J. (1961) J. Biol. Chem. 236,2461-2466. 17. Cane, W. P. & Wetlaufer, D. (1966) Abstr. Annu. Meet. Amer. Chem. Soc. 152d, 110C. 18. Moore, L. & de Jersey, J. (1970) Aust. J. Biol. Sci. 23, 607615. 19. Bender, M. L., Kezdy, F. J. & Gunter, C. R. (1964) J. Am. Chem. Soc. 86,3714-3721. 20. Inagami, T. & York, S. S. (1968) Biochemistry 7,4045-4052. 21. Stoops, J. K., Horgan, D. J., Runnegar, M. T. C., de Jersey, J., Webb, E. C. & Zerner, B. (1969) Biochemistry 8,2026-2033.
