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Inactivation of trypsin-like proteases by active-site-directed sulfonylation
ARCHIVES
OF
BIOCHEMISTRY
Inactivation
AND
of Trypsin-like Proteases Sulfonylation
Ability
of the Departing SHOW-CHU
Biology
176, 113-118 (1976)
BIOPHYSICS
Department,
Group to Confer Selectivity’*
WONG3
Brookhaven
by Active-Site-Directed
AND
National
ELLIOTT
Laboratory,
Received January
2
SHAW Upton, New York 11973
19, 1975
The p-nitrophenyl ester of p-amidinophenylmethanesulfonic acid had been found to inactivate thrombin by affinity labeling but did not have this action on other proteases of similar specificity such as trypsin, plasmin, or plasma kallikrein [Wong, S.-C., and Shaw, E., Arch. Biochem. Biophys. 161, 536 (1974)]. The ortho- and meta-nitrophenyl esters of this sulfonic acid have now been synthesized and shown to be less selective. In addition to thrombin, trypsin and plasma kallikrein are also inactivated. The o&o isomer is more effective than the meta. Plasmin is unaffected by all three esters. The results are interpreted to reflect geometrical differences in the first departing group subsite of these homologous active centers and to provide an additional structural basis for achieving selectivity of affinity labeling.
In the course of attempting to achieve the selective inactivation of a single member of a group of proteases of closely related, that is trypsin-like, specificity, we succeeded in obtaining a reagent, p-nitrophenyl-p’-amidinophenylmethanesulfonate (I) which inactivates thrombin but not trypsin, plasmin, or plasma kallikrein (1). Thrombin (2) and plasmin (3) have been demonstrated to contain a sequence of molecular weight about 25,000 that includes the active center and is homologous with trypsin. Although the primary structure of plasma kallikrein has not yet been determined, its enzymatic properties suggest that it also belongs to this family of pro-
teases (4). In view of the homology, the active center regions comprising the primary specificity site are likely to have a very similar geometric arrangement of the strictly conserved aspartate side chain which serves for substrate binding and a hydrolytic center composed of histidine and serine side chains at appropriate distances from this charge. The selective inactivation of thrombin by a small molecule such as I is thus remarkable and merits further investigation. CH,-SO,C,H,NO,-(
’ By acceptance of this article, the publisher and/ or recipient acknowledges the U. S. Government’s right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper. 2 Research carried out at Brookhaven National Laboratory under the auspices of the U. S. Energy Research and Development Administration with support from U. S. Public Health Service Grant 17849 from the National Institute of General Medical Sciences. 3 Present address: New York Blood Center, 310 East 67th Street, New York, N. Y. 10021.
The reagent structure is based on the fact that benzamidine is an analog of lysine and arginine side chains and competitively inhibits trypsin by binding in the primary specificity site (5, 6). This reversible inhibition is observed with trypsin-like enzymes in general (4, 7, 8). The sulfonate ester, I, was found (4) to complex reversibly with thrombin, trypsin, plasmin, and plasma kallikrein, be113
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
p)
114
WONG
AND
havior to be expected due to the benzamidine moiety of the structure. However, only in the case of thrombin did inactivation follow with displacement of p-nitrophenol from the sulfonate ester part of the structure. Since the possibility was recognized that this unusual selectivity was based on differences in the binding of the p-nitrophenol part of the ester to the active centers of the four trypsin-like enzymes, the ortho- and metcz-nitrophenyl esters were prepared and examined for their inhibitory properties. EXPERIMENTAL
PROCEDURES
Enzymes The preparations used have been described (1) or, in the case of human plasma kallikrein, obtained by a published procedure (4)
Enzymatic
Studies
Incubation mixtures containing the ester to be tested and protease in 0.1 M Tris-chloride at pH 7.4 were incubated at 25°C. Kallikrein concentration was 2.1 enzyme units/ml (4); thrombin, 6.5 x 10e7 M, P-trypsin, 3.6 x lo-’ M, and plasmin, 4.8 x 10e7 M, were established by titration (9). In the case of trypsin and thrombin, 0.02 M Ca’+ was included. At indicated times, aliquots (0.100 ml) were removed for assay by addition to 1.880 ml of 0.2 M sodium maleate, pH 6.0, followed by 20 pl of Z-Lys-ONp4 stock solution (lo-* M in 95% acetonitrile-5% water). Optical density change at 340 nm was measured. Controls without inhibitors were also examined; only in the case of plasmin was there a significant loss (about 10%/h). The results with inhibitors were based on the control value.
Sites of Modification A sample of thrombin which had been inactivated with p-nitrophenyl-p’-amidinophenylmethanesulfonate (1) was treated with mercaptoethylamine in 8 M urea as described by Gold (10). Following dialysis and lyophilization, the derivatized protein was analyzed in the usual way. The yield of aminoethylcysteine was calculated with the aid of an authentic sample. Unmodified thrombin was taken through the same procedure as a control. /3-Trypsin (3.4 mg) was inactivated in Tris-chloride buffer, pH 7.4 (200 ml, 0.02 M Ca2+), with methylp-amidinophenylmethanesulfonate (5 x 1Om4 M). After 6 h, the mixture was acidified, dialyzed, and lyophilized. 4 Abbreviation used: Z-Lys-ONp, ester of N”-benzyloxycarbonyl-lysine.
Nitrophenyl
SHAW
Syntheses5 p-Amidinophenylmethanesulfonyl chloride hydrochloride. Sodium p-cyanophenylmethanesulfonate (1) (11.0 g) in anhydrous tetrahydrofurane (130 ml) containing anhydrous methanol (3.2 g) was treated with hydrogen chloride for 1 h at 0°C then stirred ovenight at 4°C. After removal of the solvent under reduced pressure, the residue was extracted with dry methanol (100 ml) and the soluble fraction treated with additional methanolic ammonia until pink to phenol red. The mixture was stirred for 2.5 h at 50-55°C with addition of methanolic ammonia if needed to maintain the pH at 7-8 (external indicator used). Subsequently the suspension was kept at 0°C for 30 min to favor precipitation of the product which was then collected by filtration, washed with ethyl acetate, and dried in oacuo at 6o”C, prior to conversion to the acid chloride; 17 g of ammoniump-amidinophenylmethanesulfonate hydrochloride was obtained and used without purification. Anhydrous ammonium p-amidinophenylmethanesulfonate (12 g) was heated at 80°C overnight with phosphorus oxychloride (60 ml) protected from moisture. Volatile materials were removed under reduced pressure and the residue stirred with anhydrous ether for filtration with suction. The crude product was taken up in acetonitrile (300 ml) containing phosphorus oxychloride (1 ml) and concentrated under reduced pressure until a dense crop of crystals formed. This was filtered and washed with anhydrous acetonitrile and ether. A second crop was obtainable from the mother liquor for a total of 12.6 g. A sample was crystallized by solution in acetone, addition of two volumes of 1% HCl, and concentration under reduced pressure. The crystals were washed with methylene chloride and dried, mp 150°C dec. (C 8H 10N 20 2SC1,).6 Isomeric nitrophenyl esters. p-Amidinophenylmethanesulfonyl chloride hydrochloride (1.08 g) was stirred with the sodium salt of the nitrophenol (0.8 g) in acetone (40 ml) for at least 4 h. The mixture was acidified with methanolic hydrogen chloride and concentrated to a syrup under reduced pressure. The residue was triturated with anhydrous ether and extracted with methanol, which was filtered to remove inorganic salts. The p-nitrophenyl and onitrophenyl esters were crystallized directly as hydrochlorides from the residue obtained on removal of 5 Melting points were determined with a FisherJohns apparatus. Nmr spectra obtained with a Varian T-60 spectrometer were consistent with the assigned structures. Microanalyses were carried out at Brookhaven National Laboratory (Department of Applied Science). 6 C, H, and N analyses consistent with this formula were obtained. Data were submitted with manuscript for review.
INACTIVATION
OF PROTEASES
the methanol as described for the isomer in the earlier, alternate synthesis (1). The o-nitrophenyl ester had mp 93-95°C (C,,H,,N,,0,CIS).6 The mete isomer resisted direct crystallization and was purified through the picrate, mp 284-286°C; Anal. (CzOH,BNOO,ZS).L The hydrochloride obtained after extraction of the picric acid from a solution in hydrochloric acid (11) also failed to crystallize. Evaporation of an aqueous solution of the hydrochloride with added trifluoracetic acid provided a trifluoroacetate, mp 198-201°C (C,RH,,F,N,OrS).’ Phenyl p-amidinophenylmethanesulfonate hydrochloride. pCyanophenylmethanesulfony1 chloride (1) (6 g) in acetone (60 ml) at 10°C was gradually treated with sodium phenoxide (8 g) and stirred for 4 h. The solvent was removed under reduced pressure, and the dried residue was taken up in chloroform (100 ml) and extracted with 40-ml portions of water (2x), 1% HCI, and water. The dried organic layer was concentrated under reduced pressure and treated with ether (60 ml) for crystallization: 2.8 g, mp 134136°C (C,,H,,NO,S).” The nitrile (2.73 gl was converted to the methyl imidate as described earlier (1); treatment with methanolic ammonia was controlled with phenol red as an internal indicator to avoid a harmful excess. The solvent was removed under reduced pressure and replaced with methanol (40 ml) prior to heating for 4 h at 55-60°C. The product was purified through the picrate: 3.2 g, mp 211212°C; Anal. (C20H,,N5010S),6 from which the hydrochloride was obtained by treatment with Dowex lchloride (11). Crystals from methanol-ethyl acetate had mp 188-190°C (C,,H,,Cl Nz0,S).6 Alkyl esters ofp-amidinophenylmethanesuZfonate. Methyl ester: p-AmidinophenylmethanesuIfonyl chloride hydrochloride (1.34 g) was cooled and stirred with portions of a methanolic (30 ml) suspension of sodium methylate (2 g); the addition was externally controlled with moistened indicator paper until a persistent (20 min) pH in the range of 77.5 was attained, at which time the addition was stopped. Following acidification with methanolic HCl and filtration, the filtrate was taken to dryness. The residue was dissolved in a minimum of N HCI and filtered. Trifluoroacetic acid was added to the point of cloudiness and the mixture extracted with ethyl acetate (250 ml). The organic layer was washed with lo-ml portions of 10% aqueous trifluoroacetic acid (3 x ), concentrated with reduced pressure to a small volume below 3o”C, and precipitated with ether. The trifluoroacetate salt had mp 312315°C (C,,H,,N,O,F,S,.G Butyl ester: The sulfonyl chloride (540 mg) was stirred for 4 h at 0°C with n-butanol (2 ml) and 2,6lutidine (428 mg). The insoluble material obtained on addition of anhydrous ether was dissolved in ethyl acetate containing 20% triiluoroacetic acid and filtered. Concentration under reduced pressure induced crystallization of the trifluoroacetate which
115
BY SULFONYLATION
was completed by the addition of anhydrous This salt had mp 314-316°C (C,,H,,N,F,O,S).’ RESULTS
AND
ether.
DISCUSSION
It was shown earlier that the para-nitrophenyl ester of p-amidinophenylmethanesulfonic acid inactivated thrombin in a stoichiometric reaction as indicated by nitrophenol release (1). Trypsin, plasmin, and plasma kallikrein were not inactivated. The m-amidinophenylmethanesulfonic ester did not inactivate any of the four enzymes although competitively inhibiting e&erase action. It was expected that in the inactivation of thrombin by poru-nitrophenylp’ -amidinophenylmethanesulfonate the ‘active center serine was sulfonylated. Reaction at serine has now been confirmed since, after treatment of inactivated thrombin by aminoethylmercaptan in 8 M urea (lo), 0.70 residue of aminoethylcysteine was obtained. The inactivation is thus analogous to acyl-enzyme formation and, in the following discussion, differences in the susceptibility of the trypsin-like enzymes to inactivation by a given ester may be thought of as due to differences in acyl (sulfonyl)-enzyme formation In order to obtain other esters ofp-amidinophenylmethanesulfonate for enzymatic study, a synthetic method was developed using p-amidinophenylmethanesulfonyl chloride as an intermediate. This provided an alternate synthesis of the pnitrophenyl ester (I) as well as a convenient means of obtaining the m- and o-nitrophenyl esters and alkyl esters. When the m-nitrophenyl ester of p-amidinophenylmethanesulfonic acid was incubated at pH 7.4 with a number of trypsinlike enzymes, it was evident that not only thrombin but trypsin and, to a slight extent, human plasma kallikrein were susceptible (Fig. 1). Thus, a shift in the position of the nitro group in the departing phenol resulted in a loss of selectivity. In the case of the o-nitrophenyl ester, the differences in susceptibility of the enzymes to the reagent diminished further and the rates of inactivation increased (Fig. 2, note change in time scale). However plasmin was not affected by any isomer. Second-
116
WONG
AND
order rate constants for the inactivations are summarized in Table I. The effectiveness of the isomeric nitrophenyl esters is not correlated with chemical reactivity. The metu isomer, for example, is much less reactive than the ortho or
SHAW TABLE
I
RATES OF INACTIVATION OF PROTEASES BY ISOMERIC NITROPHENYL ESTERS OF pAMIIDINOPHENYLMETHANESULFONIC ACID AT pH l.4” Nitrophenyl es ter
Thbrtm-
Trypsin
Plasmin
kreid
para-
4620
0
meta-
390 1390
69.3 730
ortho-
Pi.sy
0 2.5 77
0 0 0
u Rates calculated using observed tllZ for inactivation to obtain k,,, which was divided by inhibitor concentration. Rates are in units of minutes-l molar-‘. b The values for t,,, were obtained by extrapolation (Figs. 1 and 2).
THROMBIN I
FIG. 1. Relative rates of inactivation of thrombin, trypsin, plasma kallikrein, and plasmin by metu-nitrophenyl-p-amidinophenylmethanesulfonate at pH 7.4.
FIG. 2. Relative rates of inactivation of thrombin, trypsin, plasma kallikrein, and plasmin by orrho-nitrophenyl-p-amidinophenylmethanesulfonate at pH 7.4.
para isomers.’ On the other hand, the reactivity of the departing group does influence the rate of inactivation since the phenyl ester is relatively inert. Thus at 2.5 x 10e4 M at pH 8.3, the phenyl ester inactivated thrombin only 22% in 21 h. The other enzymes were not clearly affected relative to control samples. These results concur with conclusions drawn earlier about affinity labeling (12) that geometric factors are more important than chemical reactivity in that they must be suitable for covalent-bond formation. When these conditions are satisfied, reactivity can be rate determining. Two alkyl esters were examined although these were expected to act by a different mechanism than aryl esters, since alkyl sulfonates undergo alkyl-oxygen cleavage and are consequently alkylating agents. The methylation of chymotrypsin by methylp-nitrobenzenesulfonate (13) and of trypsin by methyl m-quanidinobenzenesulfonate (14) are examples of this chemical behavior. The methyl ester at 2.5 x 10e4 M caused a progressive loss of activity in all four enzymes. This was not followed to conclusion; however it was of interest that the spectrum of susceptibility to this alkyl ester was in contrast to the foregoing group of aryl esters. Plasma kallikrein was the most rapidly inactivated ’ Unpublished observations. Note also from Table I that the rates for the inactivation of thrombin decrease in the order p > m > o, wheres those for trypsin and kallikrein increase; therefore chemical reactivity cannot account for the results.
INACTIVATION
OF PROTEASES
t l/2 = 37 min followed by P-trypsin, with and plasmin t 112 = 142 min. Thrombin were estimated to be an order of magnitude more resistant than plasma kallikrein. The butyl ester was less effective in all cases. After the complete inactivation of trypsin by methyl p-amidinophenylmethanesulfonate, the histidine content was found to be 1.96 * 0.06 residues instead of 3, indicating alkylation of 1 residue. The results with the isomeric nitrophenyl esters of p-amidinophenylmethanesulfonic acid indicate that the departing group is responsible in large part for the highly selective behavior of the p-nitrophenyl ester in inactivating thrombin. One interpretation of the results is that only in the case of thrombin does the departing group binding region of the active center accomodate the p-nitrophenyl moiety sufficiently to permit the active center serine to approach the sulfonate ester and form the acyl-enzyme analog. In the case of trypsin and kallikrein, conceivably the p-nitro group encounters steric hindrance in the binding site which is relieved on shifting the nitro group to the metu and ortho positions. Since the order of susceptibility remains the same, a simple diagram (Fig. 3) serves to convey this idea. Conceivably plasmin provides the most limited access to all nitrophenyl isomers. An alternate explanation of the selectiv-
PROTEASE
ACTIVE
CENTERS
s!
+ FIG. 3. Diagram suggesting differences in the S1’ subsite of the departing group region of a group of trypsin-like proteases that may account for the selectivity of thep-nitrophenyl ester as an inactivator of thrombin and for the action of the other isomers.
BY SULFONYLATION
117
ity, one still involving the S’ region, would attribute an activating effect of the p-nitrophenyl group on the active center serine resulting from contact with the disulfide bridge 42-48 (15, 16). In the case of trypsin, the departing group or S’ region has been delineated by crystallographic studies of a trypsin-peptide inhibitor complex (17). Since the sequences in this region of thrombin (2), plasmin (31, and many other trypsin homologs (18) are becoming available, it may be possible by model building to evaluate the above interpretation of the experimental results. Even more important would be the ability to predict differences in the shape of the S’ region and utilize them in the design of selective inhibitors. REFERENCES 1. WONG, S.-C., AND SHAW, E. (1974) Arch. Biothem. Biophys. 161, 536-543. 2. MAGNUSSON, S., PETERSEN, T. E., SOTTRUP-JENSEN, L., AND CLAEYS, H. (1975) in Proteases and Biological Control (Reich, E., Ritkin, D., and Shaw, E., eds.), pp. 123-150, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 3. ROBBINS, K. C., BERNABE, P., ARZADON, L., AND SUMMARIA, L. (1973) J. Biol. Chem. 248,72427246. 4. SAMPAIO, C., WONG, S.-C., AND SHAW, E. (1974) Arch. Biochem. Biophys. 165, 133-139. 5. MARES-GUIA, M., AND SHAW, E. (1965) J. Biol. Chem. 240, 1579-1585. 6. KRIEGER, M., KAY, L. M., AND STROUD, R. M. (1974) J. Mol. Biol. 83, 209-230. 1. GERATZ, J. D. (1971) Thrombosis Diathesis Huemorphagica, 25, 391-404. 8. MARKWARDT, F., LANDMANN, H., AND WALSMANN, P. (1968) EUF. J. Biochem. 6, 502-506. 9. CHASE, T., JR., AND SHAW, E., (1969) Biochemistry 8, 2212-2224. 10. GOLD, A. M. (1965) Biochemistry 4, 897-901. 11. MARES-GUIA, M., SHAW, E., AND COHEN, W. (1967) J. Biol. Chem. 242, 5777-5781. 12. SAW, E. (1970) Physiol. Reu. 50, 244-296. 13. NAKAGAWA, Y., AND BENDER, M. L. (1970) Biochemistry 9, 259-267. 14. JACKSON, M. B., AND BENDER, M. L. (1970) Biothem. Biophys. Res. Commun. 39, 1X7-1162. 15. FERSHT, A. R., BLOW, D. M., AND FASTREZ, J. (1973) Biochemistry 12, 2035-2041. 16. BLOW, D. M. (1974) in Proteinase Inhibitors (Fritz, H., Tschesche, H., Greene, L. J., and Truscheit, E., eds.), pp. 473-483, Springer/ Verlag, Berlin.
118
WONG
AND
17. JANIN, J., SWEET, R. M., AND BLOW, D. M. (1974) in Proteinase Inhibitors (Fritz, H., Tschesche, H., Greene, L. J., and Truscheit, E., eds.), pp. 473-483, Springer-Verlag, Berlin.
SHAW 18. DARRIE, E. W., FUKIKAWA, K., LEGAZ, M. E., AND KATO, H. (1975) in Proteases and Biological Control (Reich, E., Rifkin, D., and Shaw, E., eds.), pp. 65-77, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
OF
BIOCHEMISTRY
Inactivation
AND
of Trypsin-like Proteases Sulfonylation
Ability
of the Departing SHOW-CHU
Biology
176, 113-118 (1976)
BIOPHYSICS
Department,
Group to Confer Selectivity’*
WONG3
Brookhaven
by Active-Site-Directed
AND
National
ELLIOTT
Laboratory,
Received January
2
SHAW Upton, New York 11973
19, 1975
The p-nitrophenyl ester of p-amidinophenylmethanesulfonic acid had been found to inactivate thrombin by affinity labeling but did not have this action on other proteases of similar specificity such as trypsin, plasmin, or plasma kallikrein [Wong, S.-C., and Shaw, E., Arch. Biochem. Biophys. 161, 536 (1974)]. The ortho- and meta-nitrophenyl esters of this sulfonic acid have now been synthesized and shown to be less selective. In addition to thrombin, trypsin and plasma kallikrein are also inactivated. The o&o isomer is more effective than the meta. Plasmin is unaffected by all three esters. The results are interpreted to reflect geometrical differences in the first departing group subsite of these homologous active centers and to provide an additional structural basis for achieving selectivity of affinity labeling.
In the course of attempting to achieve the selective inactivation of a single member of a group of proteases of closely related, that is trypsin-like, specificity, we succeeded in obtaining a reagent, p-nitrophenyl-p’-amidinophenylmethanesulfonate (I) which inactivates thrombin but not trypsin, plasmin, or plasma kallikrein (1). Thrombin (2) and plasmin (3) have been demonstrated to contain a sequence of molecular weight about 25,000 that includes the active center and is homologous with trypsin. Although the primary structure of plasma kallikrein has not yet been determined, its enzymatic properties suggest that it also belongs to this family of pro-
teases (4). In view of the homology, the active center regions comprising the primary specificity site are likely to have a very similar geometric arrangement of the strictly conserved aspartate side chain which serves for substrate binding and a hydrolytic center composed of histidine and serine side chains at appropriate distances from this charge. The selective inactivation of thrombin by a small molecule such as I is thus remarkable and merits further investigation. CH,-SO,C,H,NO,-(
’ By acceptance of this article, the publisher and/ or recipient acknowledges the U. S. Government’s right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper. 2 Research carried out at Brookhaven National Laboratory under the auspices of the U. S. Energy Research and Development Administration with support from U. S. Public Health Service Grant 17849 from the National Institute of General Medical Sciences. 3 Present address: New York Blood Center, 310 East 67th Street, New York, N. Y. 10021.
The reagent structure is based on the fact that benzamidine is an analog of lysine and arginine side chains and competitively inhibits trypsin by binding in the primary specificity site (5, 6). This reversible inhibition is observed with trypsin-like enzymes in general (4, 7, 8). The sulfonate ester, I, was found (4) to complex reversibly with thrombin, trypsin, plasmin, and plasma kallikrein, be113
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
p)
114
WONG
AND
havior to be expected due to the benzamidine moiety of the structure. However, only in the case of thrombin did inactivation follow with displacement of p-nitrophenol from the sulfonate ester part of the structure. Since the possibility was recognized that this unusual selectivity was based on differences in the binding of the p-nitrophenol part of the ester to the active centers of the four trypsin-like enzymes, the ortho- and metcz-nitrophenyl esters were prepared and examined for their inhibitory properties. EXPERIMENTAL
PROCEDURES
Enzymes The preparations used have been described (1) or, in the case of human plasma kallikrein, obtained by a published procedure (4)
Enzymatic
Studies
Incubation mixtures containing the ester to be tested and protease in 0.1 M Tris-chloride at pH 7.4 were incubated at 25°C. Kallikrein concentration was 2.1 enzyme units/ml (4); thrombin, 6.5 x 10e7 M, P-trypsin, 3.6 x lo-’ M, and plasmin, 4.8 x 10e7 M, were established by titration (9). In the case of trypsin and thrombin, 0.02 M Ca’+ was included. At indicated times, aliquots (0.100 ml) were removed for assay by addition to 1.880 ml of 0.2 M sodium maleate, pH 6.0, followed by 20 pl of Z-Lys-ONp4 stock solution (lo-* M in 95% acetonitrile-5% water). Optical density change at 340 nm was measured. Controls without inhibitors were also examined; only in the case of plasmin was there a significant loss (about 10%/h). The results with inhibitors were based on the control value.
Sites of Modification A sample of thrombin which had been inactivated with p-nitrophenyl-p’-amidinophenylmethanesulfonate (1) was treated with mercaptoethylamine in 8 M urea as described by Gold (10). Following dialysis and lyophilization, the derivatized protein was analyzed in the usual way. The yield of aminoethylcysteine was calculated with the aid of an authentic sample. Unmodified thrombin was taken through the same procedure as a control. /3-Trypsin (3.4 mg) was inactivated in Tris-chloride buffer, pH 7.4 (200 ml, 0.02 M Ca2+), with methylp-amidinophenylmethanesulfonate (5 x 1Om4 M). After 6 h, the mixture was acidified, dialyzed, and lyophilized. 4 Abbreviation used: Z-Lys-ONp, ester of N”-benzyloxycarbonyl-lysine.
Nitrophenyl
SHAW
Syntheses5 p-Amidinophenylmethanesulfonyl chloride hydrochloride. Sodium p-cyanophenylmethanesulfonate (1) (11.0 g) in anhydrous tetrahydrofurane (130 ml) containing anhydrous methanol (3.2 g) was treated with hydrogen chloride for 1 h at 0°C then stirred ovenight at 4°C. After removal of the solvent under reduced pressure, the residue was extracted with dry methanol (100 ml) and the soluble fraction treated with additional methanolic ammonia until pink to phenol red. The mixture was stirred for 2.5 h at 50-55°C with addition of methanolic ammonia if needed to maintain the pH at 7-8 (external indicator used). Subsequently the suspension was kept at 0°C for 30 min to favor precipitation of the product which was then collected by filtration, washed with ethyl acetate, and dried in oacuo at 6o”C, prior to conversion to the acid chloride; 17 g of ammoniump-amidinophenylmethanesulfonate hydrochloride was obtained and used without purification. Anhydrous ammonium p-amidinophenylmethanesulfonate (12 g) was heated at 80°C overnight with phosphorus oxychloride (60 ml) protected from moisture. Volatile materials were removed under reduced pressure and the residue stirred with anhydrous ether for filtration with suction. The crude product was taken up in acetonitrile (300 ml) containing phosphorus oxychloride (1 ml) and concentrated under reduced pressure until a dense crop of crystals formed. This was filtered and washed with anhydrous acetonitrile and ether. A second crop was obtainable from the mother liquor for a total of 12.6 g. A sample was crystallized by solution in acetone, addition of two volumes of 1% HCl, and concentration under reduced pressure. The crystals were washed with methylene chloride and dried, mp 150°C dec. (C 8H 10N 20 2SC1,).6 Isomeric nitrophenyl esters. p-Amidinophenylmethanesulfonyl chloride hydrochloride (1.08 g) was stirred with the sodium salt of the nitrophenol (0.8 g) in acetone (40 ml) for at least 4 h. The mixture was acidified with methanolic hydrogen chloride and concentrated to a syrup under reduced pressure. The residue was triturated with anhydrous ether and extracted with methanol, which was filtered to remove inorganic salts. The p-nitrophenyl and onitrophenyl esters were crystallized directly as hydrochlorides from the residue obtained on removal of 5 Melting points were determined with a FisherJohns apparatus. Nmr spectra obtained with a Varian T-60 spectrometer were consistent with the assigned structures. Microanalyses were carried out at Brookhaven National Laboratory (Department of Applied Science). 6 C, H, and N analyses consistent with this formula were obtained. Data were submitted with manuscript for review.
INACTIVATION
OF PROTEASES
the methanol as described for the isomer in the earlier, alternate synthesis (1). The o-nitrophenyl ester had mp 93-95°C (C,,H,,N,,0,CIS).6 The mete isomer resisted direct crystallization and was purified through the picrate, mp 284-286°C; Anal. (CzOH,BNOO,ZS).L The hydrochloride obtained after extraction of the picric acid from a solution in hydrochloric acid (11) also failed to crystallize. Evaporation of an aqueous solution of the hydrochloride with added trifluoracetic acid provided a trifluoroacetate, mp 198-201°C (C,RH,,F,N,OrS).’ Phenyl p-amidinophenylmethanesulfonate hydrochloride. pCyanophenylmethanesulfony1 chloride (1) (6 g) in acetone (60 ml) at 10°C was gradually treated with sodium phenoxide (8 g) and stirred for 4 h. The solvent was removed under reduced pressure, and the dried residue was taken up in chloroform (100 ml) and extracted with 40-ml portions of water (2x), 1% HCI, and water. The dried organic layer was concentrated under reduced pressure and treated with ether (60 ml) for crystallization: 2.8 g, mp 134136°C (C,,H,,NO,S).” The nitrile (2.73 gl was converted to the methyl imidate as described earlier (1); treatment with methanolic ammonia was controlled with phenol red as an internal indicator to avoid a harmful excess. The solvent was removed under reduced pressure and replaced with methanol (40 ml) prior to heating for 4 h at 55-60°C. The product was purified through the picrate: 3.2 g, mp 211212°C; Anal. (C20H,,N5010S),6 from which the hydrochloride was obtained by treatment with Dowex lchloride (11). Crystals from methanol-ethyl acetate had mp 188-190°C (C,,H,,Cl Nz0,S).6 Alkyl esters ofp-amidinophenylmethanesuZfonate. Methyl ester: p-AmidinophenylmethanesuIfonyl chloride hydrochloride (1.34 g) was cooled and stirred with portions of a methanolic (30 ml) suspension of sodium methylate (2 g); the addition was externally controlled with moistened indicator paper until a persistent (20 min) pH in the range of 77.5 was attained, at which time the addition was stopped. Following acidification with methanolic HCl and filtration, the filtrate was taken to dryness. The residue was dissolved in a minimum of N HCI and filtered. Trifluoroacetic acid was added to the point of cloudiness and the mixture extracted with ethyl acetate (250 ml). The organic layer was washed with lo-ml portions of 10% aqueous trifluoroacetic acid (3 x ), concentrated with reduced pressure to a small volume below 3o”C, and precipitated with ether. The trifluoroacetate salt had mp 312315°C (C,,H,,N,O,F,S,.G Butyl ester: The sulfonyl chloride (540 mg) was stirred for 4 h at 0°C with n-butanol (2 ml) and 2,6lutidine (428 mg). The insoluble material obtained on addition of anhydrous ether was dissolved in ethyl acetate containing 20% triiluoroacetic acid and filtered. Concentration under reduced pressure induced crystallization of the trifluoroacetate which
115
BY SULFONYLATION
was completed by the addition of anhydrous This salt had mp 314-316°C (C,,H,,N,F,O,S).’ RESULTS
AND
ether.
DISCUSSION
It was shown earlier that the para-nitrophenyl ester of p-amidinophenylmethanesulfonic acid inactivated thrombin in a stoichiometric reaction as indicated by nitrophenol release (1). Trypsin, plasmin, and plasma kallikrein were not inactivated. The m-amidinophenylmethanesulfonic ester did not inactivate any of the four enzymes although competitively inhibiting e&erase action. It was expected that in the inactivation of thrombin by poru-nitrophenylp’ -amidinophenylmethanesulfonate the ‘active center serine was sulfonylated. Reaction at serine has now been confirmed since, after treatment of inactivated thrombin by aminoethylmercaptan in 8 M urea (lo), 0.70 residue of aminoethylcysteine was obtained. The inactivation is thus analogous to acyl-enzyme formation and, in the following discussion, differences in the susceptibility of the trypsin-like enzymes to inactivation by a given ester may be thought of as due to differences in acyl (sulfonyl)-enzyme formation In order to obtain other esters ofp-amidinophenylmethanesulfonate for enzymatic study, a synthetic method was developed using p-amidinophenylmethanesulfonyl chloride as an intermediate. This provided an alternate synthesis of the pnitrophenyl ester (I) as well as a convenient means of obtaining the m- and o-nitrophenyl esters and alkyl esters. When the m-nitrophenyl ester of p-amidinophenylmethanesulfonic acid was incubated at pH 7.4 with a number of trypsinlike enzymes, it was evident that not only thrombin but trypsin and, to a slight extent, human plasma kallikrein were susceptible (Fig. 1). Thus, a shift in the position of the nitro group in the departing phenol resulted in a loss of selectivity. In the case of the o-nitrophenyl ester, the differences in susceptibility of the enzymes to the reagent diminished further and the rates of inactivation increased (Fig. 2, note change in time scale). However plasmin was not affected by any isomer. Second-
116
WONG
AND
order rate constants for the inactivations are summarized in Table I. The effectiveness of the isomeric nitrophenyl esters is not correlated with chemical reactivity. The metu isomer, for example, is much less reactive than the ortho or
SHAW TABLE
I
RATES OF INACTIVATION OF PROTEASES BY ISOMERIC NITROPHENYL ESTERS OF pAMIIDINOPHENYLMETHANESULFONIC ACID AT pH l.4” Nitrophenyl es ter
Thbrtm-
Trypsin
Plasmin
kreid
para-
4620
0
meta-
390 1390
69.3 730
ortho-
Pi.sy
0 2.5 77
0 0 0
u Rates calculated using observed tllZ for inactivation to obtain k,,, which was divided by inhibitor concentration. Rates are in units of minutes-l molar-‘. b The values for t,,, were obtained by extrapolation (Figs. 1 and 2).
THROMBIN I
FIG. 1. Relative rates of inactivation of thrombin, trypsin, plasma kallikrein, and plasmin by metu-nitrophenyl-p-amidinophenylmethanesulfonate at pH 7.4.
FIG. 2. Relative rates of inactivation of thrombin, trypsin, plasma kallikrein, and plasmin by orrho-nitrophenyl-p-amidinophenylmethanesulfonate at pH 7.4.
para isomers.’ On the other hand, the reactivity of the departing group does influence the rate of inactivation since the phenyl ester is relatively inert. Thus at 2.5 x 10e4 M at pH 8.3, the phenyl ester inactivated thrombin only 22% in 21 h. The other enzymes were not clearly affected relative to control samples. These results concur with conclusions drawn earlier about affinity labeling (12) that geometric factors are more important than chemical reactivity in that they must be suitable for covalent-bond formation. When these conditions are satisfied, reactivity can be rate determining. Two alkyl esters were examined although these were expected to act by a different mechanism than aryl esters, since alkyl sulfonates undergo alkyl-oxygen cleavage and are consequently alkylating agents. The methylation of chymotrypsin by methylp-nitrobenzenesulfonate (13) and of trypsin by methyl m-quanidinobenzenesulfonate (14) are examples of this chemical behavior. The methyl ester at 2.5 x 10e4 M caused a progressive loss of activity in all four enzymes. This was not followed to conclusion; however it was of interest that the spectrum of susceptibility to this alkyl ester was in contrast to the foregoing group of aryl esters. Plasma kallikrein was the most rapidly inactivated ’ Unpublished observations. Note also from Table I that the rates for the inactivation of thrombin decrease in the order p > m > o, wheres those for trypsin and kallikrein increase; therefore chemical reactivity cannot account for the results.
INACTIVATION
OF PROTEASES
t l/2 = 37 min followed by P-trypsin, with and plasmin t 112 = 142 min. Thrombin were estimated to be an order of magnitude more resistant than plasma kallikrein. The butyl ester was less effective in all cases. After the complete inactivation of trypsin by methyl p-amidinophenylmethanesulfonate, the histidine content was found to be 1.96 * 0.06 residues instead of 3, indicating alkylation of 1 residue. The results with the isomeric nitrophenyl esters of p-amidinophenylmethanesulfonic acid indicate that the departing group is responsible in large part for the highly selective behavior of the p-nitrophenyl ester in inactivating thrombin. One interpretation of the results is that only in the case of thrombin does the departing group binding region of the active center accomodate the p-nitrophenyl moiety sufficiently to permit the active center serine to approach the sulfonate ester and form the acyl-enzyme analog. In the case of trypsin and kallikrein, conceivably the p-nitro group encounters steric hindrance in the binding site which is relieved on shifting the nitro group to the metu and ortho positions. Since the order of susceptibility remains the same, a simple diagram (Fig. 3) serves to convey this idea. Conceivably plasmin provides the most limited access to all nitrophenyl isomers. An alternate explanation of the selectiv-
PROTEASE
ACTIVE
CENTERS
s!
+ FIG. 3. Diagram suggesting differences in the S1’ subsite of the departing group region of a group of trypsin-like proteases that may account for the selectivity of thep-nitrophenyl ester as an inactivator of thrombin and for the action of the other isomers.
BY SULFONYLATION
117
ity, one still involving the S’ region, would attribute an activating effect of the p-nitrophenyl group on the active center serine resulting from contact with the disulfide bridge 42-48 (15, 16). In the case of trypsin, the departing group or S’ region has been delineated by crystallographic studies of a trypsin-peptide inhibitor complex (17). Since the sequences in this region of thrombin (2), plasmin (31, and many other trypsin homologs (18) are becoming available, it may be possible by model building to evaluate the above interpretation of the experimental results. Even more important would be the ability to predict differences in the shape of the S’ region and utilize them in the design of selective inhibitors. REFERENCES 1. WONG, S.-C., AND SHAW, E. (1974) Arch. Biothem. Biophys. 161, 536-543. 2. MAGNUSSON, S., PETERSEN, T. E., SOTTRUP-JENSEN, L., AND CLAEYS, H. (1975) in Proteases and Biological Control (Reich, E., Ritkin, D., and Shaw, E., eds.), pp. 123-150, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 3. ROBBINS, K. C., BERNABE, P., ARZADON, L., AND SUMMARIA, L. (1973) J. Biol. Chem. 248,72427246. 4. SAMPAIO, C., WONG, S.-C., AND SHAW, E. (1974) Arch. Biochem. Biophys. 165, 133-139. 5. MARES-GUIA, M., AND SHAW, E. (1965) J. Biol. Chem. 240, 1579-1585. 6. KRIEGER, M., KAY, L. M., AND STROUD, R. M. (1974) J. Mol. Biol. 83, 209-230. 1. GERATZ, J. D. (1971) Thrombosis Diathesis Huemorphagica, 25, 391-404. 8. MARKWARDT, F., LANDMANN, H., AND WALSMANN, P. (1968) EUF. J. Biochem. 6, 502-506. 9. CHASE, T., JR., AND SHAW, E., (1969) Biochemistry 8, 2212-2224. 10. GOLD, A. M. (1965) Biochemistry 4, 897-901. 11. MARES-GUIA, M., SHAW, E., AND COHEN, W. (1967) J. Biol. Chem. 242, 5777-5781. 12. SAW, E. (1970) Physiol. Reu. 50, 244-296. 13. NAKAGAWA, Y., AND BENDER, M. L. (1970) Biochemistry 9, 259-267. 14. JACKSON, M. B., AND BENDER, M. L. (1970) Biothem. Biophys. Res. Commun. 39, 1X7-1162. 15. FERSHT, A. R., BLOW, D. M., AND FASTREZ, J. (1973) Biochemistry 12, 2035-2041. 16. BLOW, D. M. (1974) in Proteinase Inhibitors (Fritz, H., Tschesche, H., Greene, L. J., and Truscheit, E., eds.), pp. 473-483, Springer/ Verlag, Berlin.
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17. JANIN, J., SWEET, R. M., AND BLOW, D. M. (1974) in Proteinase Inhibitors (Fritz, H., Tschesche, H., Greene, L. J., and Truscheit, E., eds.), pp. 473-483, Springer-Verlag, Berlin.
SHAW 18. DARRIE, E. W., FUKIKAWA, K., LEGAZ, M. E., AND KATO, H. (1975) in Proteases and Biological Control (Reich, E., Rifkin, D., and Shaw, E., eds.), pp. 65-77, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.