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Mechanism of action of thrombin on fibrinogen
-4RCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
160, 333-339 (1974)
Mechanism of Action of Thrombin on Fibrinogen IV. Further Mapping of the Active Sites of Thrombin and Trypsin~ R O N A L D K. H. L I E M ~ AND H A R O L D A. S C H E R A G A s
Department of Chemistry, Cornell University, Ithaca, New York 14850 Received September 10, 1973 Four heptapeptides were synthesized in a continuing effort to map the active sites of thrombin and trypsin. One of these has the sequence Gly-Val-Arg-Gly-Pro-ArgLeu, similar to that of the a(A)-chain of bovine fibrinogen around the arg-gly bond which is hydrolyzed by thrombin. In two analogs, the amino acids proline in site Ps' and arginine in Ps' were replaced by glycine. A fourth peptide in which the C-terminus of this heptapeptide was blocked by the amide group was also synthesized. The rates of hydrolysis of these four peptides by thrombin and trypsin were measured, and values of k~t/K,~ were determined. The data show that the "native" heptapeptide is a poor substrate for thrombin compared to fibrinogen, indicating that some other factor(s) outside of these seven amino acids are necessary to account for the high specificity of thrombin for the arggly bond of the a(A)-ehain of fibrinogen. A comparison of the values of ko~t/K~ of these peptides indicates that subsites Ss' and SJ of thrombin are both involved in binding of the substrate; replacements by glycine in either of these two positions (in the substrate) reduces the values of k~t/ K~. Trypsin behaves in the opposite way; these substitutions (in the substrate) increase the values of k~t/K~. of different amino acids for the sequence g l y - v a l - a r g - g l y to learn something about the nature of the active sites of the two enzymes. This "mapping" procedure has been carried out by Berger and his coworkers (4-8) for a number of other enzymes. We have, however, not examined the amino acid sequence to the "right" of the hydrolyzed arg-gly linkage to any extent. I n this paper, we describe the synthesis of four heptapeptides, in wtfieh the amino acids to the "right" of the glyeine in the arginyl-glyeine linkage have been varied. To determine the importance of specific amino acids in this sequence we have com1 This work was supported by Grants from the pared the values of k~t/K,~, for the hydrolysis National Heart and Lung Institute of the National of these peptides b y thrombin and trypsin. Institutes of Health, U. S. Public Health Service I t is hoped that by this work we can find (HL-01662) and from the National Science Foundaa partial answer to the question of the high tion (GB-28469X2). specificity of thrombin, i.e., why thrombin 2 Todd Fellow, 1969-1973. To whom requests for reprints should be cleaves only four arginyl-glycine linkages in fibrinogen, whereas trypsin cleaves all addressed. 333
I n the first three papers of this series (1-3), we have described the action of thrombin (EC 3.4.4.13) and trypsin (EC 3.4.4.4) on a series of synthetic oligopeptide amides. These peptides contain the amino acid sequence similar to that of the a(A)chain of bovine fibrinogen around the arginyl-glyeine bond which is cleaved by thrombin. I n the first two papers (1, 2), we h a v e studied the effect of increasing length, and found that at least three residues to the "left" of the hydrolyzed arginyl-glycine linkage influenced the rates. I n the third paper, we studied the effect of substitutions
Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
334
LIEM AND SCHERAGA
376 peptide bonds in fibrinogen following arginine and lysine (9, 10). MATERIALS AND METHODS Materials. 4 DMF was freshly distilled under vacuum by using a water aspirator. Methanol was dried using Davison type 4A Molecular Sieves and distilled just before using. All Boc-amino acids were prepared according to the method of Schnabel (11). Aoc-Tosyl arginine was purchased from ]3eekman. The purity of this compound as well as of the Boo-amino acids was checked by thin layer chromatography. 1-Hydroxybenzotriazole was from Aldrich Chemical Co. Boc-Leu resin was prepared as described earlier (3) and contained 0.22 mequiv Leu/g resin. Sephadex G-15 and G-25 were from Pharmacia, and Carboxymethyl Cellulose CM-52 from Whatman (Reeve Angel, distributor). Leucine amino peptidase and bovine trypsin were purchased from Worthington Biochemicals, a.nd the trypsin was purified as described before (2). Aminopeptidase M was from gohm and Haas (Henley and Co., distributor). Bovine Prothrombin (Lot 112C-8190) was from Sigma. Bovine thrombin was prepared from this material as described in our earlier report (2), and had an activity of 2380 NIH U/mg or 605 TAME U/rag. Trypsin had an activity of 1850 TAME U/mg. General synthetic procedure. The solid phase technique (12, 13) was used for the synthesis of the following peptides: Gly-Val-Arg-Gly Pro-Arg-Leu-NH~ (I) Gly-Val-Arg-Gly-Pro-Arg-Leu-OH (II) Gly-Val-Arg-Gly-Gly-Arg-Leu-OH (III) Gly-Val-Arg-Gly-Pro-Gly- Leu-OH (IV) For the first two peptides Gly-Val--TosylArgGly-Pro TosylArg-Leu resin was synthesized, starting with 15 g of Boc-Leu resin. The final material was divided in two and taken off the resin as the amide and the free earboxyl, respectively. The next two peptides were made in two separate batches starting with 7.5 g of Boe-Leu resin each. The general procedure for making the peptide resins is as follows: The Boc-leueine resin was introduced into a solid phase reaction vessel and swollen in methylene chloride. To introduce each new amino acid residue, the following steps were performed: (1) three washings with methylene chloride; (2) removal of the Boe-group by treatment with 25% TFA in methylene chloride; (3) three washings with methylene chloride; (4) three
4 Abbreviations: Boc, t-butyloxycarbonyl; Aoc, amyloxycarbonyl; tosyl, p-toluenesulphonyl; TAME, tosylarginyl methyl ester; DCI, dicyclohexylcarbodiimide; TEA, triethylamine; TFA, trifluoroacetic acid; DMF, dimethylformamide. All amino acids except glycine are of the L form.
washings with chloroform; (5) neutralization of the trifluoroacetate with 10% v / v TEA in chloroform; (6) three washings with chloroform; (7} three washings with methylene chloride; (8) addition of a 2.5 M excess of the appropriate Bocamino acid in methylene chloride; (9) addition of 25% DCI in methylene chloride to give an equimolar amount to the Boc-amino acid just added, followed by a reaction time of 1 4 hr at room temperature. Aoc-Tosyl arginine was used instead of the Boc-derivative, since the former material is more soluble in methylene chloride. For the coupling of this derivative, the material was dissolved in small amount of DMF and diluted with methylene: chloride. For both this derivative and for Bocvaline, the coupling did not go to completion, unless repeated additions were performed. We therefore added an equimolar amount of 1-hydroxybenzotriazole (14), dissolved in DMF, to the reaction mixture as catalyst. This material was added between steps 8 and 9. After the coupling step, the reaction was tested for completion using the Kaiser-Ninhydrin test (15). Half of the Gly-Val-Tos ylArg-Gly-ProTosylArg-Leu resin was treated by ammonolysis as described before (2) to give 1.15 g of heptapeptide amide. The remaining peptide resin, as well as the other two peptide resins were treated with H B r / T F A to give the free acid (13). The tosyl groups of the first three peptides were removed by sodium i~ liquid ammonia, as described earlier (3). Endpoints of less than 5 sec were used, since otherwise the gly-pro bond was found to be destroyed to a large extent. The fourth peptide did not give reasonable yields o f deprotected beptapeptide when this procedure was used; therefore this peptide was deprotected with anhydrous HF (13). All the peptides were purified by chromatography on the following series of cohtmns: I. Sephadex G-15 with 50% HOAc as effluent for desalting (16); II. Ion exchange chromatography using Carboxymethyl cellulose CM-52 with a gradient of 1% HOAc-30% HOAe (30 ml each) ; III. Gel filtration on Sephadex G-25 with 0.2 N HOAc as effluent. All the columns were 2.5 X 50 cm in size. The purity of the peptides was checked by thin layer chromatography using Merck's Silica Gel F-254 plates, layer thickness, 0.25 ram, with the following systems: RI I n-BuOH-HOAc-water, 40:30:30; R} I n-BuOH-HOAc-water, 60:20:20; R~]~ n-BuOH-pyridine-HOAc-water, 30:20:6:24. All the purified peptides showed a single Ninhydrin-, Cl-, and Sakaguchi-positive spot in all three systems.
335
A C T I O N OF T H R O M B I N ON F I B R I N O G E N For amino acid analyses, both acid and enzymatic hydrolyses were performed. Acid hydrolyses were carried out in constant boiling, ~xygen-free IIC1, at 105~ for 24 hr in a sealed tube. Enzymatic digestions for peptides I, II, and IV were carried out by adding a solution of leucine amino peptidase (2.0 U / m l ) to an equal amount of 1% peptide solution in 0.05 ~ phosphate buffer, p H 8.0 at room temperature for several days. Peptide II1 was digested with 1 unit of Amino peptidase M in 0.05 M phosphate buffer, p H 8.0 at r o o m temperature for 24 hr, since the gly-gly bond was found to be quite resistant to hydrolysis by leueine amino peptidase. The amino acid compositions of the acid and enzymatic hydrolyzates were determined with a Technicon amino acid analyzer. Optical rotations were determined with a Perkin E l m e r model 141 Polarimeter at the sodium D line using a 1 dm cell at room temperature (23~ on a 1% peptide solution in water. Gly-Val-Arg-Gly-Pro-Arg-Leu-NH.~ . Yield, 65 rag. R/I 0.33; R} I, 0.09; f , 0.24. laiD = --91.0. Amino acid ratios in acid hydrolyzate: Gly, 1.94; Val, 1.01; Arg, 2.03; Pro, 1.00; Leu, 1.00; N H a , O.91; in enzymatic hydrolyzate: Gly, 1.96; Yal, 1.02; Arg, 1.93; Pro, 1.03; Leu, 1.00. Gly-Val-Arg-Gly-Pro-Arg-Leu-OH. Yield, 110 mg. R~t, 0.34; R} I, 0.09; R~ II, 0.29. laiD = --91.3. Amino acid ratios in acid hydrolyzates: Gly, 2.03; Yal, 1.00; Arg, 2.03; Pro, 1.00; Leu, 1.01; in enzymatic hydrolyzate: Gly, 1.96; Val, 1.01; Arg, 2.00; Pro, 1.00; Leu, 1.01. Gly- Val--Arg--Gly-Gly-Arg-Leu-OH. Yield, 195 mE. Rz I, 0.30; R~ I, 0.09; R~ II, 0.19. [a]D = --68.9. Amino acid ratios in acid hydrolyzate: Gly, 2.91; Val, 0.98; Arg, 2.10, Lea, 1.01; in enzymatic hydrolyzate: GIy, 2.94, Val, 1.10, Arg, 1.97; Lea, 0.95. Gly-Val-Arg-Gly-Pro-Gly-Leu-OH. Yield, 96 rag. R/I, 0.38; R~ l, 0.17; n I/I I , 0.30. [a]n = -70.0.
Amino acid ratios in acid hydrolyzate: Gly, 2.90; Val, 0.99; Arg, 1.05; Pro, 1.03; Lea, 1.03; in enzymatic hydrolyzate: Gly, 2.94; Val, 1.03; Arg, 1.01; Pro, 1.05; Leu, 1.00. Qualitative experiments. To determine whether or not the arg-leu bond was cleaved, in addition to the arg-gly bond, the following experiments were performed: Each peptide was dissolved in 0.05 M sodium phosphate buffer, p H 8.0 to give a 1% peptide solution. To 0.5 ml of each peptide solution was added 100/Alters of a thrombin solution (750 N I H U / m l ) . To another 0.5 ml was added 50 ~liters of a trypsin solution (900 T A M E U / m l ) . For the thrombic digests, aliquots were applied to a thin layer chromatography plate after 1 and 7 hr, respectively. For trypsin, aliquots were applied after 1 rain, 15 min, and 1 hr, respectively. The plates were developed in system I I I and sprayed with Ninhydrin to locate the peptides and their reaction products. Kinetic studies. The kinetic studies were carried out as described in our previous reports (2, 3). Enzyme concentrations of 3.96 X 10-s ~a for thrombin and 2.66 X 10-~ M for trypsin were used throughout. Substrate concentrations (determined by Nitrogen analysis by the Micro-Kjeldahl method) varied from 0.002 M to 0.013 M. Lineweaver-Burk plots were constructed on the basis of four determinations of initial rates for each substrate. Satisfactory linear plots of 1/v (initial velocity) vs 1/S (substrate concentration) were obtained from which the values of V and K,~ were estimated by the method of least squares. After kc~t was obtained [from k~t = V(E0)], values of kr were calculated. RESULTS
Qualitative
experiments. T h e a c t i o n of and trypsin on the peptides are
thrombin
TABLE
I
APPEARANCE OF REACTION PRODUCTS IN INCUBATION MIXTURES OF HEPTAPEPTIDGS WITH THROMBIN AND TRYPSIN AT 25~ AND pH 8.0 a
Action with trypsin
Action with thrombin Appearance of LeuNH2 or LeuOH Peptide I II III IV
1 hr
7 hr
Appearance of Gly-Val Arg 1 hr
7 hr
Appearance of LeuNH2 or LeuOH 1 min
5 min
Appearance of Gly-Val-Arg
1 hr
I rain
5 min
1 hr
-
+
+
+
+
+
+
-
+
+
-
-
+
+
-
--
+
-
+
+
--
-
-
+
-
-
q-
-
+
+
-
-
-
+
-
-
+
-
+
+
a + indicates a visible ninhydrin-positive spot; -
indicates no visible ninhydrin-positive spot.
336
LIEM AND SCHERAGA
summarized in Table I. It can be seen that thrombin hydrolyzes the arg-leu bond only if the carboxyl group of leucine is blocked by the amide. Even in this case, however, this bond is cleaved much more slowly than the arg-gly bond. Hence, the initial rate of hydrolysis for peptide I will reflect the hydrolysis of the arg-gly bond, and not of the arg-leu bond. For trypsin, however, the arg-leu bond is cleaved much faster in peptide I than the arg-gly bond; hence, no kinetic data were obtained for this peptide. For the other peptides, where the carboxyl group of leucine is not protected, the arg-leu bond is cleaved much more slowly than the arg-gly bond. Kinetic results. The values of k~=t, K,~ and k~t/K,~ are listed in Tables II and III for thrombin and trypsin, respectively. In addition, for comparative purposes, Table IV gives the values of kr for all the peptides studied in this series of papers.
As can be seen immediately, all the values of kcat/Km for thrombin are quite low, compared to that of the a(A)-chain of fibrinogen, which we have previously estimated to be ~ 2 N 10 5 M-1 sec-1 (2). The primary sequence of these peptides is in itself, therefore, still not sufficient to explain the specificity of thrombin for the arg-gly bond in fibrinogen, and the efficiency with which thrombin cleaves this bond selectively. The data do suggest, however,
that the particular amino acids in this sequence play at least a partial role in explaining the specificity of thrombin. A comparison of the values of kc~t/K,~ for peptides II and III, show that proline in site P2' is preferred over glycine in this subsite. The values of kc~t/K~ differ by a factor of 5. For trypsin, however, peptide ]II is a better substrate than peptide II. Peptide II is also a better substrate for thrombin than peptide IV, showing the preference for the large charged residue, arginine, over that of the nonspecific residue, glycine. In our first series of peptides (i, 2), alanine occupied position Pa'. At that time, we considered the possibility that the replacement of alanine by arginine might be
DISCUSSION Tables II and I I I give the values of kr for the reaction of thrombin and trypsin, respectively, with the peptides. TABLE
II
KINETIC CONSTANTS FOR THE HYDROLYSIS OF FOUR FIBRINOGEN-LIKE HEPTAPEPTIDES BY THROMBIN AT 25~ AT p H 8.0
No.
Substrate P,
I II III IV
Gly Gly Gly Gly
P2
Val Val Val Val
P1
PI'
P2'
Pa'
P4'
Arg Arg Arg Arg
Gly Gly Gly Gly
Pro Pro Gly Pro
Arg Arg Arg Gly
Leu Leu Leu Leu
NH~ OH OH OH
Km X
kc~
10 3 M
sec -1
1.8 3.7 9.6 15.3
0.50 0.42 0.20 0.08
kc~t/K,~ M--t sec -1
270 115 20 6
(5=50) (4-35) (4-7) (=t=2)
TABLE III ] ~ I N E T I C C O N S T A N T S FOR THE H Y D R O L Y S I S OF F O U R F I B R I N O G E N - L I K E H E P T A P E P T I D E S BY T R Y P S I N AT 2 5 ~
No.
II III IV
AT
pH
8.0
Substrate Pa
P~
PI
Gly Gly Gly
Val Val Val
, Arg Arg Arg
Km X
P/
P2'
Pa'
P4'
Gly Gly Gly
Pro Gly Pro
Arg Arg Gly
Leu Leu Leu
OH OH OH
ko~t
ko~t/K,~
10 ~ M
sec -1
M-1 sec -1
2.0 4.9 0.93
1.24 8.64 1.83
612 (• 1775 (4-250) 2750 (4-360)
ACTION OF THROMBIN ON FIBRINOGEN
337
TABLE IV VALUES OF
]r
FOR THE HYDROLYSIS OF FIBRINOGEN-LIKE OLIGOPEPTIDES BY THROMBIN AND TRYPSIN AT 2 5 ~ AND pH 8.0
ke,t/K.,
Substrate
M-1 sec-I P5
A B C D E F G tI I J K L M N O P Q
Gly
1:14
Gly Gly
P3
Gly Gly Gly Gly Gly Gly Ala Leu Gly Gly Gly Gly Gly Gly
1)2
~)1
Pi t
NO~
Arg Arg Arg Arg Arg Arg Arg Orn Arg Arg Arg Arg Arg Arg Arg Arg Arg
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Leu Gly Gly Gly Gly
Val Val Val Val Val Val Gly Val Val Val Val Val Val Val Val
P2'
Pro Pro Pro Pro Pro Pro NH2 NH2 NH2 NH~. NH2 NH2 NH~ Pro Pro Gly Pro
Pa t
1)4'
Ala Ala Ala Ala Ala Ala
NH2 NH2 NH2 NH2 NH~ NH2
Arg Arg Arg Gly
Leu Leu Leu Leu
k~,/K.,
M-1 sec-1
(throm- (trypsin) bin)
NH2 OH OH OH
--" ~ 10 59 21 84 90 0 35 50 10 1650 110 270 115 20 6
0 0 25 670 360 680 4900 0 3500 18000 21600 25900 230000 __b 612 1775 2750
Too slow to measure. b Not measured (see text). sufficient to increase the rates of hydrolysis of the arg-gly bond b y several orders of magnitude, so as to approach the rate of hydrolysis of the a(A)-chain of fibrinogen. As can be seen clearly from the first three peptides (Table II), which have arginine in position P J , this is not the case. A comparison of peptide D (Table IV) with peptide N (Table IV) indicates a change in kcat/K,,~ of about a factor of 4.5. However, this increase m a y reflect the presence of leueine in subsite P4', as well as the substitution of arginine for alanine in site P J . Thus, the presence of arginine in subsite P J m a y be slightly preferred over alanine, and considerably more t h a n glycine, b u t it is not sufficient to explain the difference in the rates of hydrolysis by thrombin of these peptides compared to t h a t of fibrinogen. We should also mention here t h a t fibrinogen Detroit, which has serine in subsite P~' rather t h a n arginine, has been reported to be hydrolyzed at a comparable rate to fibrinogen (17, 18). Trypsin again shows the opposite effect from thrombin, preferring glyeine over arginine in site Ps'.
We can at best m a k e only a tentative statement about site P4 r. I n h u m a n fibrinogen this site is occupied by valine (19). No published results are available for bovine fibrinogen. However, leucine and valine are probably not too different in eharaeter, in t h a t both have large nonpolar side chains. A comparison of feptides I and I I seems to indicate that LeuNH2 is preferred over L e u O H in subsite P4', suggesting that this subsite m a y also play a role in the specificity of thrombin. Clearly, however, the filling of this subsite is not sufficient to account for the efficient hydrolysis of the arg-gly bond in the a(A)-chain of fibrinogen. Our original purFose in synthesizing the amide as well as the free carboxyl was to m a k e sure that the free C-terminal carboxyl group does not inhibit binding of the substrate to the enzyme. This inhibition might have been serious if the binding of subsite S~' on the enzyme to subsite P3' on the substrate were indeed electrostatic. Although there is an effect of the charged C-terminus on the value of koa,/Km for thrombie hydrolysis, it is relatively minor compared to the
338
LIEM AND SCHERAGA
effect needed to approach the value of
kc~t/Km for the hydrolysis of the arg-gly linkage in the a(A)-chain of fibrinogen. The action of trypsin on peptide I agrees with our earlier observation (3), viz, that trypsin hydrolyzes an arg-leu bond much more efficiently than an arg-gly bond. The fact that the arg-leu bonds in peptides I I - I V are cleaved much more slowly than the arg-gly bond is probably caused by the free carboxyl group of leucine, which seems to have an inhibitory effect. Thrombin, however, seems to hydrolyze the arg-leu bond at a slower rate than the arg gly bond in peptide I. As can be seen from Table IV, the values of ]ccat/Krn for the hydrolysis of arg-gly (peptide G) and arg-leu (peptide M) are about equal; hence we might have expected two competing reactions in this peptide. F r o m the qualitative results (Table I), we found that this was not the case. The reason for this may be simply that the "natural" sequence can be more easily accommodated by thrombin. Thus, if argleu is the bond hydrolyzed, then by analogy with the arg gly bond, valine would occupy site P s , argilfine site P~ and proline would be in site P2. The presence of these amino acids in those subsites may inhibit the cleavage of the a r t leu bond in this peptide. Our results in this series of experiments are also consistent with those of Dorman et al. (20), who synthesized a number of fibrinogen-like peptide analogs, including two in which the amino acids in subsite P2' or P J were replaced b y glycine. However, they did not carry out any rate studies. Hence, it was impossible to determine from their study whether or not their peptides were hydrolyzed b y thrombin as rapidly as fibrinogen. They also noted that, in their analogs in which the amino acids of subsites P2' or P J were replaced by glycine, they could not detect any hydrolysis by thrombin. In our peptides we have found a drop in the values of ko~t/Km, but the bonds were still hydrolyzed by thrombin at a measurable rate. Table IV gives us an overview of the peptides that we have synthesized in this series of papers. Peptides A - F (1, 2) were synthesized to find out how many sub-
sites to the left of the arg-gly bond were necessary. We concluded that both thrombin and trypsin contain at least three subsites corresponding to the earboxyl side of the hydrolyzed linkage of the substrate, which are involved in substrate binding. These three subsites, as well as subsite $1', were mapped by peptides G - M (3). From this mapping procedure we found that arginine is preferred over ornithine in binding to subsite $1 of both enzymes; subsite $2 of thrombin and trypsin seems to be attracted to nonpolar residues. The two enzymes deviate, however, in subsite $3. The values of keat/Km for thrombic hydrolysis decreases as the amino acid corresponding to this subsite is increased in size. This result is consistent with the hypothesis that the active site of thrombin m a y involve a narrow cleft (1), hence the bigger the side chain, the poorer the substrate. Trypsin, on the other hand, shows a strong affinity for a large nonpolar residue in this subsite, the value of kc~t/Km for tryptic hydrolysis increasing with increasing size of the side chain. In mapping subsite $1', we found that thrombin prefers alanine over glycine, but further replacement by leucine reduces the rate of hydrolysis to the same value as for glycine. Trypsin, on the other hand, shows an increased preference for the larger nonpolar residue in this subsite. Peptides N-Q were synthesized to study the influence of subsites P2', P3', and P4'. In this study we found that for thrombin, proline in subsite P2' and arginine in subsite P3' were preferred over glycine, but the opposite was true for trypsin. Subsite $4' of thrombin may have some influence in binding to the substrate, but not sufficiently to increase the rate of hydrolysis of the substrate to be comparable to the rate of hydrolysis of fibrinogen. We have varied the amino acids of six subsites, three on each side of the arg-gly bond, and have found that all of them contribute to the specificity of thrombin for fibrinogen. However, even the sum of these contributions is not sufficient to explain this specificity completely. In addition, we have considered nine subsites in total, five to the left of the arg-gly bond
ACTION OF THROMBIN ON FIBRINOGEN
339
4. SCHECHTER, 2., AND BERGER, A. (1967) Bro. and four to the right; yet the values of chem. Biophys. Res. Commun. 27, 157. kcat/Km are still several orders of magnitude 5. ABRAMO~VITZ, N., SCHECHTER,I., ANDBERGER, below that for fibrinogen. For no other A. (1967) Biochem. Biophys. Res. Commun. enzyme studied previously (4-8) have more 29, 862. than six or seven subsites been found neces6. BERGEll, A., AND SCHECHTER, I. (1970) Phil. sary to obtain good values of l:o~#K~. Trans. Roy. Soc. London B257, 249. Thrombin is unique, therefore, in that it 7. ATLAS, D., LEVIT, S., SCttECttTER, I., AND seems to have a very extended active site, BERGER, A. (1972) FEBS Lett. 11, 281. or possibly even more than one binding 8. ATLAS,D., ANDBERGER, A. (1972) Biochemistry site. We are still interested in determining 11, 4719. 9. PECHET, L., AND ALF~XANDER, B. (1970) how much of the a(A)-chain is necessary to Biochemistry 1, 875. account for the specificity of thrombin. 10. MIHALYI, E., AND GODFREY, J. E. (1963) However, we do not believe that the answer Biochim. Biophys. Acla 67, 73. can be found very easily by synthesizing 11. SCnNABEL, E. (1967) Just~s Liebigs Ann. longer and longer peptides until a peptide Chem. 702, 188. is found that has a value of k~/Km com- 12. MERRIFIELD, ~. B. (1964) Biochemistry 3, parable to that of the a(A)-chain of fibrino1385. gen. Instead, we have decided to pursue a 13. STEWART, J. M., AND YOUNG, J. D., Solid Phase Peptide Synthesis, W. H. Freeman different approach, in which we isolate and Co., San Francisco 1969. progressively smaller pieces of the a(A)14. K/:iNIG, W., AND GEIGER, R.(1970) Chem. Ber. chain, obtained by chemical and enzy103, 788. matic cleavage of native fibrinogen (21). 15. KAISEn, E., COLESCOTT, R. L., BOSSINGER, B y examining the rates of hydrolyses of C. D.,AND COOK,P. I. (1970) Anal. Biochem. these peptides, we may be able to learn 34, 595 how much of the a(A)-chain is necessary to 16. MANNING, M., Wvu, T. C., AND BAXTER, J. W. M. (1968) Y. Chromatogr. 38, 396. account for efficient hydrolysis of fibrinogen 17. BLOMB~CK, M., BLOMB-~CK, B., MAMMEN, by thrombin. ACKNOWLEDGMENT
18.
We would like to thank Mr. H. T. Chan for performing the amino acid and nitrogen anMyses. 19. REFERENCES 1. ANDREATTA, R. I-l., LIEM, R. K. H., AND SCHERAGA, H. A. (1971) Proc. Nat. Acad. Sci. US 68, 253. 2. LIEM, ~:~. K. ~FI., ANDREATTA, R. ]yI., AND SCHERAGA, H. A. (1971) Arch. Biochem. Biophys. 147, 201. 3. LI~M, R. K. H., AND SCHnnAGA,H. A., (1973) Arch. Biochem. Biophys., 158, 387.
20.
21.
E. F., AND PRASAD, A. S. (1968) Nature 218, 134. MAMMEN, E. F., PRASAD, A. S., BARNHART, M. I., AND AU, C. C. (1969) J. Clin. Invest. 48, 235. BLOMBACK, B., BLOMBXCK, M., HESSEL, B., AND IWANAG~4., S. (1967) Nature (London) 215, 1445. DORMAN, L. D., CHOW, R. C. L., AND MARSHAL~, F. N. (1972) in Chemistry and Biology of Peptides, Proceedings of the 3rd American Peptide Symposium 1972, p. 445, Ann Arbor Science Publishers Inc., Ann Arbor, Michigan. HAGEMAN, T. C., AND SCHERAGA, I-I. A., in preparation.
160, 333-339 (1974)
Mechanism of Action of Thrombin on Fibrinogen IV. Further Mapping of the Active Sites of Thrombin and Trypsin~ R O N A L D K. H. L I E M ~ AND H A R O L D A. S C H E R A G A s
Department of Chemistry, Cornell University, Ithaca, New York 14850 Received September 10, 1973 Four heptapeptides were synthesized in a continuing effort to map the active sites of thrombin and trypsin. One of these has the sequence Gly-Val-Arg-Gly-Pro-ArgLeu, similar to that of the a(A)-chain of bovine fibrinogen around the arg-gly bond which is hydrolyzed by thrombin. In two analogs, the amino acids proline in site Ps' and arginine in Ps' were replaced by glycine. A fourth peptide in which the C-terminus of this heptapeptide was blocked by the amide group was also synthesized. The rates of hydrolysis of these four peptides by thrombin and trypsin were measured, and values of k~t/K,~ were determined. The data show that the "native" heptapeptide is a poor substrate for thrombin compared to fibrinogen, indicating that some other factor(s) outside of these seven amino acids are necessary to account for the high specificity of thrombin for the arggly bond of the a(A)-ehain of fibrinogen. A comparison of the values of ko~t/K~ of these peptides indicates that subsites Ss' and SJ of thrombin are both involved in binding of the substrate; replacements by glycine in either of these two positions (in the substrate) reduces the values of k~t/ K~. Trypsin behaves in the opposite way; these substitutions (in the substrate) increase the values of k~t/K~. of different amino acids for the sequence g l y - v a l - a r g - g l y to learn something about the nature of the active sites of the two enzymes. This "mapping" procedure has been carried out by Berger and his coworkers (4-8) for a number of other enzymes. We have, however, not examined the amino acid sequence to the "right" of the hydrolyzed arg-gly linkage to any extent. I n this paper, we describe the synthesis of four heptapeptides, in wtfieh the amino acids to the "right" of the glyeine in the arginyl-glyeine linkage have been varied. To determine the importance of specific amino acids in this sequence we have com1 This work was supported by Grants from the pared the values of k~t/K,~, for the hydrolysis National Heart and Lung Institute of the National of these peptides b y thrombin and trypsin. Institutes of Health, U. S. Public Health Service I t is hoped that by this work we can find (HL-01662) and from the National Science Foundaa partial answer to the question of the high tion (GB-28469X2). specificity of thrombin, i.e., why thrombin 2 Todd Fellow, 1969-1973. To whom requests for reprints should be cleaves only four arginyl-glycine linkages in fibrinogen, whereas trypsin cleaves all addressed. 333
I n the first three papers of this series (1-3), we have described the action of thrombin (EC 3.4.4.13) and trypsin (EC 3.4.4.4) on a series of synthetic oligopeptide amides. These peptides contain the amino acid sequence similar to that of the a(A)chain of bovine fibrinogen around the arginyl-glyeine bond which is cleaved by thrombin. I n the first two papers (1, 2), we h a v e studied the effect of increasing length, and found that at least three residues to the "left" of the hydrolyzed arginyl-glycine linkage influenced the rates. I n the third paper, we studied the effect of substitutions
Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
334
LIEM AND SCHERAGA
376 peptide bonds in fibrinogen following arginine and lysine (9, 10). MATERIALS AND METHODS Materials. 4 DMF was freshly distilled under vacuum by using a water aspirator. Methanol was dried using Davison type 4A Molecular Sieves and distilled just before using. All Boc-amino acids were prepared according to the method of Schnabel (11). Aoc-Tosyl arginine was purchased from ]3eekman. The purity of this compound as well as of the Boo-amino acids was checked by thin layer chromatography. 1-Hydroxybenzotriazole was from Aldrich Chemical Co. Boc-Leu resin was prepared as described earlier (3) and contained 0.22 mequiv Leu/g resin. Sephadex G-15 and G-25 were from Pharmacia, and Carboxymethyl Cellulose CM-52 from Whatman (Reeve Angel, distributor). Leucine amino peptidase and bovine trypsin were purchased from Worthington Biochemicals, a.nd the trypsin was purified as described before (2). Aminopeptidase M was from gohm and Haas (Henley and Co., distributor). Bovine Prothrombin (Lot 112C-8190) was from Sigma. Bovine thrombin was prepared from this material as described in our earlier report (2), and had an activity of 2380 NIH U/mg or 605 TAME U/rag. Trypsin had an activity of 1850 TAME U/mg. General synthetic procedure. The solid phase technique (12, 13) was used for the synthesis of the following peptides: Gly-Val-Arg-Gly Pro-Arg-Leu-NH~ (I) Gly-Val-Arg-Gly-Pro-Arg-Leu-OH (II) Gly-Val-Arg-Gly-Gly-Arg-Leu-OH (III) Gly-Val-Arg-Gly-Pro-Gly- Leu-OH (IV) For the first two peptides Gly-Val--TosylArgGly-Pro TosylArg-Leu resin was synthesized, starting with 15 g of Boc-Leu resin. The final material was divided in two and taken off the resin as the amide and the free earboxyl, respectively. The next two peptides were made in two separate batches starting with 7.5 g of Boe-Leu resin each. The general procedure for making the peptide resins is as follows: The Boc-leueine resin was introduced into a solid phase reaction vessel and swollen in methylene chloride. To introduce each new amino acid residue, the following steps were performed: (1) three washings with methylene chloride; (2) removal of the Boe-group by treatment with 25% TFA in methylene chloride; (3) three washings with methylene chloride; (4) three
4 Abbreviations: Boc, t-butyloxycarbonyl; Aoc, amyloxycarbonyl; tosyl, p-toluenesulphonyl; TAME, tosylarginyl methyl ester; DCI, dicyclohexylcarbodiimide; TEA, triethylamine; TFA, trifluoroacetic acid; DMF, dimethylformamide. All amino acids except glycine are of the L form.
washings with chloroform; (5) neutralization of the trifluoroacetate with 10% v / v TEA in chloroform; (6) three washings with chloroform; (7} three washings with methylene chloride; (8) addition of a 2.5 M excess of the appropriate Bocamino acid in methylene chloride; (9) addition of 25% DCI in methylene chloride to give an equimolar amount to the Boc-amino acid just added, followed by a reaction time of 1 4 hr at room temperature. Aoc-Tosyl arginine was used instead of the Boc-derivative, since the former material is more soluble in methylene chloride. For the coupling of this derivative, the material was dissolved in small amount of DMF and diluted with methylene: chloride. For both this derivative and for Bocvaline, the coupling did not go to completion, unless repeated additions were performed. We therefore added an equimolar amount of 1-hydroxybenzotriazole (14), dissolved in DMF, to the reaction mixture as catalyst. This material was added between steps 8 and 9. After the coupling step, the reaction was tested for completion using the Kaiser-Ninhydrin test (15). Half of the Gly-Val-Tos ylArg-Gly-ProTosylArg-Leu resin was treated by ammonolysis as described before (2) to give 1.15 g of heptapeptide amide. The remaining peptide resin, as well as the other two peptide resins were treated with H B r / T F A to give the free acid (13). The tosyl groups of the first three peptides were removed by sodium i~ liquid ammonia, as described earlier (3). Endpoints of less than 5 sec were used, since otherwise the gly-pro bond was found to be destroyed to a large extent. The fourth peptide did not give reasonable yields o f deprotected beptapeptide when this procedure was used; therefore this peptide was deprotected with anhydrous HF (13). All the peptides were purified by chromatography on the following series of cohtmns: I. Sephadex G-15 with 50% HOAc as effluent for desalting (16); II. Ion exchange chromatography using Carboxymethyl cellulose CM-52 with a gradient of 1% HOAc-30% HOAe (30 ml each) ; III. Gel filtration on Sephadex G-25 with 0.2 N HOAc as effluent. All the columns were 2.5 X 50 cm in size. The purity of the peptides was checked by thin layer chromatography using Merck's Silica Gel F-254 plates, layer thickness, 0.25 ram, with the following systems: RI I n-BuOH-HOAc-water, 40:30:30; R} I n-BuOH-HOAc-water, 60:20:20; R~]~ n-BuOH-pyridine-HOAc-water, 30:20:6:24. All the purified peptides showed a single Ninhydrin-, Cl-, and Sakaguchi-positive spot in all three systems.
335
A C T I O N OF T H R O M B I N ON F I B R I N O G E N For amino acid analyses, both acid and enzymatic hydrolyses were performed. Acid hydrolyses were carried out in constant boiling, ~xygen-free IIC1, at 105~ for 24 hr in a sealed tube. Enzymatic digestions for peptides I, II, and IV were carried out by adding a solution of leucine amino peptidase (2.0 U / m l ) to an equal amount of 1% peptide solution in 0.05 ~ phosphate buffer, p H 8.0 at room temperature for several days. Peptide II1 was digested with 1 unit of Amino peptidase M in 0.05 M phosphate buffer, p H 8.0 at r o o m temperature for 24 hr, since the gly-gly bond was found to be quite resistant to hydrolysis by leueine amino peptidase. The amino acid compositions of the acid and enzymatic hydrolyzates were determined with a Technicon amino acid analyzer. Optical rotations were determined with a Perkin E l m e r model 141 Polarimeter at the sodium D line using a 1 dm cell at room temperature (23~ on a 1% peptide solution in water. Gly-Val-Arg-Gly-Pro-Arg-Leu-NH.~ . Yield, 65 rag. R/I 0.33; R} I, 0.09; f , 0.24. laiD = --91.0. Amino acid ratios in acid hydrolyzate: Gly, 1.94; Val, 1.01; Arg, 2.03; Pro, 1.00; Leu, 1.00; N H a , O.91; in enzymatic hydrolyzate: Gly, 1.96; Yal, 1.02; Arg, 1.93; Pro, 1.03; Leu, 1.00. Gly-Val-Arg-Gly-Pro-Arg-Leu-OH. Yield, 110 mg. R~t, 0.34; R} I, 0.09; R~ II, 0.29. laiD = --91.3. Amino acid ratios in acid hydrolyzates: Gly, 2.03; Yal, 1.00; Arg, 2.03; Pro, 1.00; Leu, 1.01; in enzymatic hydrolyzate: Gly, 1.96; Val, 1.01; Arg, 2.00; Pro, 1.00; Leu, 1.01. Gly- Val--Arg--Gly-Gly-Arg-Leu-OH. Yield, 195 mE. Rz I, 0.30; R~ I, 0.09; R~ II, 0.19. [a]D = --68.9. Amino acid ratios in acid hydrolyzate: Gly, 2.91; Val, 0.98; Arg, 2.10, Lea, 1.01; in enzymatic hydrolyzate: GIy, 2.94, Val, 1.10, Arg, 1.97; Lea, 0.95. Gly-Val-Arg-Gly-Pro-Gly-Leu-OH. Yield, 96 rag. R/I, 0.38; R~ l, 0.17; n I/I I , 0.30. [a]n = -70.0.
Amino acid ratios in acid hydrolyzate: Gly, 2.90; Val, 0.99; Arg, 1.05; Pro, 1.03; Lea, 1.03; in enzymatic hydrolyzate: Gly, 2.94; Val, 1.03; Arg, 1.01; Pro, 1.05; Leu, 1.00. Qualitative experiments. To determine whether or not the arg-leu bond was cleaved, in addition to the arg-gly bond, the following experiments were performed: Each peptide was dissolved in 0.05 M sodium phosphate buffer, p H 8.0 to give a 1% peptide solution. To 0.5 ml of each peptide solution was added 100/Alters of a thrombin solution (750 N I H U / m l ) . To another 0.5 ml was added 50 ~liters of a trypsin solution (900 T A M E U / m l ) . For the thrombic digests, aliquots were applied to a thin layer chromatography plate after 1 and 7 hr, respectively. For trypsin, aliquots were applied after 1 rain, 15 min, and 1 hr, respectively. The plates were developed in system I I I and sprayed with Ninhydrin to locate the peptides and their reaction products. Kinetic studies. The kinetic studies were carried out as described in our previous reports (2, 3). Enzyme concentrations of 3.96 X 10-s ~a for thrombin and 2.66 X 10-~ M for trypsin were used throughout. Substrate concentrations (determined by Nitrogen analysis by the Micro-Kjeldahl method) varied from 0.002 M to 0.013 M. Lineweaver-Burk plots were constructed on the basis of four determinations of initial rates for each substrate. Satisfactory linear plots of 1/v (initial velocity) vs 1/S (substrate concentration) were obtained from which the values of V and K,~ were estimated by the method of least squares. After kc~t was obtained [from k~t = V(E0)], values of kr were calculated. RESULTS
Qualitative
experiments. T h e a c t i o n of and trypsin on the peptides are
thrombin
TABLE
I
APPEARANCE OF REACTION PRODUCTS IN INCUBATION MIXTURES OF HEPTAPEPTIDGS WITH THROMBIN AND TRYPSIN AT 25~ AND pH 8.0 a
Action with trypsin
Action with thrombin Appearance of LeuNH2 or LeuOH Peptide I II III IV
1 hr
7 hr
Appearance of Gly-Val Arg 1 hr
7 hr
Appearance of LeuNH2 or LeuOH 1 min
5 min
Appearance of Gly-Val-Arg
1 hr
I rain
5 min
1 hr
-
+
+
+
+
+
+
-
+
+
-
-
+
+
-
--
+
-
+
+
--
-
-
+
-
-
q-
-
+
+
-
-
-
+
-
-
+
-
+
+
a + indicates a visible ninhydrin-positive spot; -
indicates no visible ninhydrin-positive spot.
336
LIEM AND SCHERAGA
summarized in Table I. It can be seen that thrombin hydrolyzes the arg-leu bond only if the carboxyl group of leucine is blocked by the amide. Even in this case, however, this bond is cleaved much more slowly than the arg-gly bond. Hence, the initial rate of hydrolysis for peptide I will reflect the hydrolysis of the arg-gly bond, and not of the arg-leu bond. For trypsin, however, the arg-leu bond is cleaved much faster in peptide I than the arg-gly bond; hence, no kinetic data were obtained for this peptide. For the other peptides, where the carboxyl group of leucine is not protected, the arg-leu bond is cleaved much more slowly than the arg-gly bond. Kinetic results. The values of k~=t, K,~ and k~t/K,~ are listed in Tables II and III for thrombin and trypsin, respectively. In addition, for comparative purposes, Table IV gives the values of kr for all the peptides studied in this series of papers.
As can be seen immediately, all the values of kcat/Km for thrombin are quite low, compared to that of the a(A)-chain of fibrinogen, which we have previously estimated to be ~ 2 N 10 5 M-1 sec-1 (2). The primary sequence of these peptides is in itself, therefore, still not sufficient to explain the specificity of thrombin for the arg-gly bond in fibrinogen, and the efficiency with which thrombin cleaves this bond selectively. The data do suggest, however,
that the particular amino acids in this sequence play at least a partial role in explaining the specificity of thrombin. A comparison of the values of kc~t/K,~ for peptides II and III, show that proline in site P2' is preferred over glycine in this subsite. The values of kc~t/K~ differ by a factor of 5. For trypsin, however, peptide ]II is a better substrate than peptide II. Peptide II is also a better substrate for thrombin than peptide IV, showing the preference for the large charged residue, arginine, over that of the nonspecific residue, glycine. In our first series of peptides (i, 2), alanine occupied position Pa'. At that time, we considered the possibility that the replacement of alanine by arginine might be
DISCUSSION Tables II and I I I give the values of kr for the reaction of thrombin and trypsin, respectively, with the peptides. TABLE
II
KINETIC CONSTANTS FOR THE HYDROLYSIS OF FOUR FIBRINOGEN-LIKE HEPTAPEPTIDES BY THROMBIN AT 25~ AT p H 8.0
No.
Substrate P,
I II III IV
Gly Gly Gly Gly
P2
Val Val Val Val
P1
PI'
P2'
Pa'
P4'
Arg Arg Arg Arg
Gly Gly Gly Gly
Pro Pro Gly Pro
Arg Arg Arg Gly
Leu Leu Leu Leu
NH~ OH OH OH
Km X
kc~
10 3 M
sec -1
1.8 3.7 9.6 15.3
0.50 0.42 0.20 0.08
kc~t/K,~ M--t sec -1
270 115 20 6
(5=50) (4-35) (4-7) (=t=2)
TABLE III ] ~ I N E T I C C O N S T A N T S FOR THE H Y D R O L Y S I S OF F O U R F I B R I N O G E N - L I K E H E P T A P E P T I D E S BY T R Y P S I N AT 2 5 ~
No.
II III IV
AT
pH
8.0
Substrate Pa
P~
PI
Gly Gly Gly
Val Val Val
, Arg Arg Arg
Km X
P/
P2'
Pa'
P4'
Gly Gly Gly
Pro Gly Pro
Arg Arg Gly
Leu Leu Leu
OH OH OH
ko~t
ko~t/K,~
10 ~ M
sec -1
M-1 sec -1
2.0 4.9 0.93
1.24 8.64 1.83
612 (• 1775 (4-250) 2750 (4-360)
ACTION OF THROMBIN ON FIBRINOGEN
337
TABLE IV VALUES OF
]r
FOR THE HYDROLYSIS OF FIBRINOGEN-LIKE OLIGOPEPTIDES BY THROMBIN AND TRYPSIN AT 2 5 ~ AND pH 8.0
ke,t/K.,
Substrate
M-1 sec-I P5
A B C D E F G tI I J K L M N O P Q
Gly
1:14
Gly Gly
P3
Gly Gly Gly Gly Gly Gly Ala Leu Gly Gly Gly Gly Gly Gly
1)2
~)1
Pi t
NO~
Arg Arg Arg Arg Arg Arg Arg Orn Arg Arg Arg Arg Arg Arg Arg Arg Arg
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Leu Gly Gly Gly Gly
Val Val Val Val Val Val Gly Val Val Val Val Val Val Val Val
P2'
Pro Pro Pro Pro Pro Pro NH2 NH2 NH2 NH~. NH2 NH2 NH~ Pro Pro Gly Pro
Pa t
1)4'
Ala Ala Ala Ala Ala Ala
NH2 NH2 NH2 NH2 NH~ NH2
Arg Arg Arg Gly
Leu Leu Leu Leu
k~,/K.,
M-1 sec-1
(throm- (trypsin) bin)
NH2 OH OH OH
--" ~ 10 59 21 84 90 0 35 50 10 1650 110 270 115 20 6
0 0 25 670 360 680 4900 0 3500 18000 21600 25900 230000 __b 612 1775 2750
Too slow to measure. b Not measured (see text). sufficient to increase the rates of hydrolysis of the arg-gly bond b y several orders of magnitude, so as to approach the rate of hydrolysis of the a(A)-chain of fibrinogen. As can be seen clearly from the first three peptides (Table II), which have arginine in position P J , this is not the case. A comparison of peptide D (Table IV) with peptide N (Table IV) indicates a change in kcat/K,,~ of about a factor of 4.5. However, this increase m a y reflect the presence of leueine in subsite P4', as well as the substitution of arginine for alanine in site P J . Thus, the presence of arginine in subsite P J m a y be slightly preferred over alanine, and considerably more t h a n glycine, b u t it is not sufficient to explain the difference in the rates of hydrolysis by thrombin of these peptides compared to t h a t of fibrinogen. We should also mention here t h a t fibrinogen Detroit, which has serine in subsite P~' rather t h a n arginine, has been reported to be hydrolyzed at a comparable rate to fibrinogen (17, 18). Trypsin again shows the opposite effect from thrombin, preferring glyeine over arginine in site Ps'.
We can at best m a k e only a tentative statement about site P4 r. I n h u m a n fibrinogen this site is occupied by valine (19). No published results are available for bovine fibrinogen. However, leucine and valine are probably not too different in eharaeter, in t h a t both have large nonpolar side chains. A comparison of feptides I and I I seems to indicate that LeuNH2 is preferred over L e u O H in subsite P4', suggesting that this subsite m a y also play a role in the specificity of thrombin. Clearly, however, the filling of this subsite is not sufficient to account for the efficient hydrolysis of the arg-gly bond in the a(A)-chain of fibrinogen. Our original purFose in synthesizing the amide as well as the free carboxyl was to m a k e sure that the free C-terminal carboxyl group does not inhibit binding of the substrate to the enzyme. This inhibition might have been serious if the binding of subsite S~' on the enzyme to subsite P3' on the substrate were indeed electrostatic. Although there is an effect of the charged C-terminus on the value of koa,/Km for thrombie hydrolysis, it is relatively minor compared to the
338
LIEM AND SCHERAGA
effect needed to approach the value of
kc~t/Km for the hydrolysis of the arg-gly linkage in the a(A)-chain of fibrinogen. The action of trypsin on peptide I agrees with our earlier observation (3), viz, that trypsin hydrolyzes an arg-leu bond much more efficiently than an arg-gly bond. The fact that the arg-leu bonds in peptides I I - I V are cleaved much more slowly than the arg-gly bond is probably caused by the free carboxyl group of leucine, which seems to have an inhibitory effect. Thrombin, however, seems to hydrolyze the arg-leu bond at a slower rate than the arg gly bond in peptide I. As can be seen from Table IV, the values of ]ccat/Krn for the hydrolysis of arg-gly (peptide G) and arg-leu (peptide M) are about equal; hence we might have expected two competing reactions in this peptide. F r o m the qualitative results (Table I), we found that this was not the case. The reason for this may be simply that the "natural" sequence can be more easily accommodated by thrombin. Thus, if argleu is the bond hydrolyzed, then by analogy with the arg gly bond, valine would occupy site P s , argilfine site P~ and proline would be in site P2. The presence of these amino acids in those subsites may inhibit the cleavage of the a r t leu bond in this peptide. Our results in this series of experiments are also consistent with those of Dorman et al. (20), who synthesized a number of fibrinogen-like peptide analogs, including two in which the amino acids in subsite P2' or P J were replaced b y glycine. However, they did not carry out any rate studies. Hence, it was impossible to determine from their study whether or not their peptides were hydrolyzed b y thrombin as rapidly as fibrinogen. They also noted that, in their analogs in which the amino acids of subsites P2' or P J were replaced by glycine, they could not detect any hydrolysis by thrombin. In our peptides we have found a drop in the values of ko~t/Km, but the bonds were still hydrolyzed by thrombin at a measurable rate. Table IV gives us an overview of the peptides that we have synthesized in this series of papers. Peptides A - F (1, 2) were synthesized to find out how many sub-
sites to the left of the arg-gly bond were necessary. We concluded that both thrombin and trypsin contain at least three subsites corresponding to the earboxyl side of the hydrolyzed linkage of the substrate, which are involved in substrate binding. These three subsites, as well as subsite $1', were mapped by peptides G - M (3). From this mapping procedure we found that arginine is preferred over ornithine in binding to subsite $1 of both enzymes; subsite $2 of thrombin and trypsin seems to be attracted to nonpolar residues. The two enzymes deviate, however, in subsite $3. The values of keat/Km for thrombic hydrolysis decreases as the amino acid corresponding to this subsite is increased in size. This result is consistent with the hypothesis that the active site of thrombin m a y involve a narrow cleft (1), hence the bigger the side chain, the poorer the substrate. Trypsin, on the other hand, shows a strong affinity for a large nonpolar residue in this subsite, the value of kc~t/Km for tryptic hydrolysis increasing with increasing size of the side chain. In mapping subsite $1', we found that thrombin prefers alanine over glycine, but further replacement by leucine reduces the rate of hydrolysis to the same value as for glycine. Trypsin, on the other hand, shows an increased preference for the larger nonpolar residue in this subsite. Peptides N-Q were synthesized to study the influence of subsites P2', P3', and P4'. In this study we found that for thrombin, proline in subsite P2' and arginine in subsite P3' were preferred over glycine, but the opposite was true for trypsin. Subsite $4' of thrombin may have some influence in binding to the substrate, but not sufficiently to increase the rate of hydrolysis of the substrate to be comparable to the rate of hydrolysis of fibrinogen. We have varied the amino acids of six subsites, three on each side of the arg-gly bond, and have found that all of them contribute to the specificity of thrombin for fibrinogen. However, even the sum of these contributions is not sufficient to explain this specificity completely. In addition, we have considered nine subsites in total, five to the left of the arg-gly bond
ACTION OF THROMBIN ON FIBRINOGEN
339
4. SCHECHTER, 2., AND BERGER, A. (1967) Bro. and four to the right; yet the values of chem. Biophys. Res. Commun. 27, 157. kcat/Km are still several orders of magnitude 5. ABRAMO~VITZ, N., SCHECHTER,I., ANDBERGER, below that for fibrinogen. For no other A. (1967) Biochem. Biophys. Res. Commun. enzyme studied previously (4-8) have more 29, 862. than six or seven subsites been found neces6. BERGEll, A., AND SCHECHTER, I. (1970) Phil. sary to obtain good values of l:o~#K~. Trans. Roy. Soc. London B257, 249. Thrombin is unique, therefore, in that it 7. ATLAS, D., LEVIT, S., SCttECttTER, I., AND seems to have a very extended active site, BERGER, A. (1972) FEBS Lett. 11, 281. or possibly even more than one binding 8. ATLAS,D., ANDBERGER, A. (1972) Biochemistry site. We are still interested in determining 11, 4719. 9. PECHET, L., AND ALF~XANDER, B. (1970) how much of the a(A)-chain is necessary to Biochemistry 1, 875. account for the specificity of thrombin. 10. MIHALYI, E., AND GODFREY, J. E. (1963) However, we do not believe that the answer Biochim. Biophys. Acla 67, 73. can be found very easily by synthesizing 11. SCnNABEL, E. (1967) Just~s Liebigs Ann. longer and longer peptides until a peptide Chem. 702, 188. is found that has a value of k~/Km com- 12. MERRIFIELD, ~. B. (1964) Biochemistry 3, parable to that of the a(A)-chain of fibrino1385. gen. Instead, we have decided to pursue a 13. STEWART, J. M., AND YOUNG, J. D., Solid Phase Peptide Synthesis, W. H. Freeman different approach, in which we isolate and Co., San Francisco 1969. progressively smaller pieces of the a(A)14. K/:iNIG, W., AND GEIGER, R.(1970) Chem. Ber. chain, obtained by chemical and enzy103, 788. matic cleavage of native fibrinogen (21). 15. KAISEn, E., COLESCOTT, R. L., BOSSINGER, B y examining the rates of hydrolyses of C. D.,AND COOK,P. I. (1970) Anal. Biochem. these peptides, we may be able to learn 34, 595 how much of the a(A)-chain is necessary to 16. MANNING, M., Wvu, T. C., AND BAXTER, J. W. M. (1968) Y. Chromatogr. 38, 396. account for efficient hydrolysis of fibrinogen 17. BLOMB~CK, M., BLOMB-~CK, B., MAMMEN, by thrombin. ACKNOWLEDGMENT
18.
We would like to thank Mr. H. T. Chan for performing the amino acid and nitrogen anMyses. 19. REFERENCES 1. ANDREATTA, R. I-l., LIEM, R. K. H., AND SCHERAGA, H. A. (1971) Proc. Nat. Acad. Sci. US 68, 253. 2. LIEM, ~:~. K. ~FI., ANDREATTA, R. ]yI., AND SCHERAGA, H. A. (1971) Arch. Biochem. Biophys. 147, 201. 3. LI~M, R. K. H., AND SCHnnAGA,H. A., (1973) Arch. Biochem. Biophys., 158, 387.
20.
21.
E. F., AND PRASAD, A. S. (1968) Nature 218, 134. MAMMEN, E. F., PRASAD, A. S., BARNHART, M. I., AND AU, C. C. (1969) J. Clin. Invest. 48, 235. BLOMBACK, B., BLOMBXCK, M., HESSEL, B., AND IWANAG~4., S. (1967) Nature (London) 215, 1445. DORMAN, L. D., CHOW, R. C. L., AND MARSHAL~, F. N. (1972) in Chemistry and Biology of Peptides, Proceedings of the 3rd American Peptide Symposium 1972, p. 445, Ann Arbor Science Publishers Inc., Ann Arbor, Michigan. HAGEMAN, T. C., AND SCHERAGA, I-I. A., in preparation.