guanidination of lysine-15 in the active site of the basic pancreatic trypsin inhibitor :...

Eur. J. Biochem. 42,505-510 (1974)

Guanidination of Lysine-15 in the Active Site of the Basic Pancreatic Trypsin Inhibitor Implications for Complex Formation with Trypsin and Chymotrypsin

Jean-Pierre VINCENT, Hugues SCHWEITZ, and Michel LAZDUNSKI Laboratoire de Biochimie de la Facult6 des Sciences, Nice

(Received July 27/November 26, 1973)

1. Selective modification of lysine-15 at the active site of the basic pancreatic trypsin inhibitor strongly alters its association with trypsin. Ka, the equilibrium constant for the association, and ka, the first-order rate constant for the dissociation are 20 nM-l and 1 .O x s-l for the trypsin - 15-guanidinated-inhibitor complex as compared to 16 pM-1 and 6.6 x lo-" s-l for the trypsin - native-inhibitor complex. Guanidination of lysine residues (Lys,,, Lys41 and Lys4J outside the active site of the inhibitor does not affect the stability of the binding with trypsin.

2. The selective guanidination of lysine-15 in the pancreatic inhibitor does not alters its association with chymotrypsin.

3. Reduction and carboxamidomethylation of the disulfide bridge Cys14-Cys,, in the selectively guanidinated inhibitor decreases Ka by a factor of 4.6 and increases kd by a factor of 55. The same chemical mofication of the bridge in the non-guanidinated inhibitor decreases Ka by a factor of 2700 and increases kd by a factor of 330.

The interaction between trypsin and the basic trypsin inhibitor of the pancreas is an excellent model to study the formation of heterologous protein-protein complexes. It is a very simple system described by the following equilibrium

E + I + E I .

The trypsin - inhibitor complex is extremely stable; AGao, the free energy of association of the two partners, is - 18.1 kcal x mol-l. The complex is characterized by a very low first-order rate constant

Abbreviations. The inhibitor, the basic pancreatic trypsin inhibitor (Kunitz inhibitor) ; carboxamidomethylated inhibitor, inhibitor selectively reduced and alkylated with iodoacetamide a t Cys,,-Cys,,; fully guanidinated inhibitor, inhibitor with 4 &-amino groups (lysines 15, 26, 41 and 46) guanidinated and 1 or-amino group (arginine 1) free; 15-guanidinated inhibitor, inhibitor selectively guanidinated a t Lys,,; 15-guanidinated-carboxamidomethylated inhibitor, inhibitor selectively reduced and alkylated with iodoacetamide at Cys,,-Cys,,, and selectively guanidinated a t Lys15 ; citraconylated inhibitor, inhibitor with 3 &-amino (lysines 26, 41 and 46) and 1 or-amino (arginine 1) citraconylated, and with the &-amino of Lys,, free; 15-guanidinated-citraconylated inhibitor, citroconylated inhibitor selectively guanidinated a t Lys,,; BzArgOEt, a-N-benzoyl-L-arginine ethyl ester; TosArgOMe, ptosyl-L-arginine methyl ester; AcTyrOEt, a-N-acetyl-L-tyrosine ethyl ester; Har, homoarginine.

Enzymes. Trypsin (EC 3.4.4.4); a-chymotrypsin (EC 3.4.4.5).

of dissociation, ka = 6.6 x s-l, corresponding to a half-life of more than 4 month [l]. The stereo- chemistry of the association is being studied by X-ray crystallography techniques and models of interactions have already been proposed [Z, 31. There are as many as 200 Van der Waals contacts between trypsin and the inhibitor but the critical part of the active-site area in the inhibitor is constitued by lysine-15, which forms a salt-bridge With Asp,,, in the specificity site of trypsin, and by the dkulfide bridge Cys14-Cys38 which is shielded in the complex [l, 3,41.

We have undertaken in previous papers an analysis of the relationships which exist between the number and the nature of the interactions which stabilize the complex and the values of the thermodynamic and kinetic parameters which characterize the association (Ka, AGao, ka and kd). We have already established for example that selective reduction of the Cys,,-Cy~,~ bridge [5] in the inhibitor considerably decreases the stability of the complex formed with trypsin [l]. The disconnection of Asp,,, in the specificity site of trypsin or the mere replace- ment of trypsin by chymotrypsin have a similar effect on the stability of the complex formed with the pancreatic inhibitor [l, 61.

We analyse in this paper the effect of the specific transformation of Lys,, in the active site of the in-

Em. J. Biochem. 42 (1974)

506 Guanidination of Lysine-15 in the Basic Trypsin Inhibitor

hibitor into a homoarginine residue. This chemical modification, although it preserves tfhe positive charge of the critical residue in the active site, considerably affects the thermodynamic and kinetic properties of the complex formed with trypsin.

MATERIALS AND METHODS

Materials The pancreatic trypsin inhibitor was a gift from

Choay Laboratories. The protein is pure [7] as judged by polyacrylamide gel electrophoresis, analytical centrifugation and stoichiometry of the inhibition with trypsin [l, 81. Commercial bovine trypsin (Worthington) and bovine a-chymotrypsin (Worth- ington or Sigma) were purified before use by affinity chromatography in a pancreatic-trypsin- inhibitor * Sepharose column prepared according to Cuatrecasas [9]. The details for a-chymotrypsin purification have already been described [lo]. The pure enzyme has a specific activity of 445 AcTyrOEt unitslmg protein a t 25 "C pH 8.0. The procedure for the purification of commercial trypsin was slightly different. Trypsin was charged on the pancreatic- trypsin-inhibitor * Sepharose column equilibrated at pH 8.0 with 50 mM Tris buffer containing 50 mM CaCl, and 0.1 M NaC1. Under these conditions, a single peak representing 15O/, of the initial charge was eluted in the void volume. This protein fraction was completely inactive. Active trypsin which could not be eluted from the affinity column a t pH 8.0 was eluted in the void volume a t pH 2 with a 10 M urea

Table 1. Preparation of the inhibitor selectively guanidinated at lysine-15

solution. After removal of urea, the specific activity of the purified trypsin was 50-51 BzArgOEt units/mg protein at 25 "C, pH 8.0.

Preparation of Inhibitor Derivatives The carboxamidomethylated inhibitor was ob-

tained by selective reduction of the Cys,,-Cys,, disulfide bridge of the inhibitor with sodium boro- hydride, followed by alkylation of the newly formed - SH groups with iodoacetamide, as previously described [8,11].

Total gnanidination of the inhibitor was carried out with 0-methylisourea hydrogen sulfate according to Chauvet and Acher 1121.

The procedure for the selective guanidination of the Lys,, side-chain of the inhibitor is summarized in Table 1.

It is well known that the e-amino group of LysIs in the inhibitor is masked toward acetylating rea- gents when the inhibitor is associated with trypsin [13] or with a-chymotrypsin [14]. In consequence, complex formation with either trypsin or chymotrypsin could have been used to selectively prot'ect Lys,, against citraconylation (step 2 in Table 1) . However, since the enzyme-inhibitor complex had to be dissociated in step 3 under experimental conditions which avoid decitraconylation, i.e. near neutral pH (decitraconylation spontaneously occurs a t acidic pH), we had to choose a-chymotrypsin as the protective enzyme rather than trypsin. The a-chymotrypsin - inhibitor complex is readily dissociated at neutral pH in 8 M urea solutions, while the trypsin

~ ~ _ _ _ _ _ _

Step Starting materiala Amino groups in inhibitor Treatment

Step 1 Inhibitor 4 free &-amino 1 free a-amino

a-Chymotrypsin in slight excess (1.2 mol/ mol), 25 "C, pH 8.0, 15 min

Step 2 a-Chymotrypsin * inhibitor 3 free &-amino Citraconic anhydride, 0 "C, pH 8.0, 30 min 1 free a-amino [221 1 masked &-amino (Lys,,)

~

step 3 Citraconylated 3 citraconylated &-amino Dissociation by 8 M urea, 25 "C, pH 7.2, a-chymotrypsin inhibitor 1 citraconylated a-amino 30 min. Separation by Sephadex G-75b,

citraconylated a-chymotrypsin discarded 1 masked &-amino (Lysl,)

step 4 Citraconylated inhibitor 3 citraconylated e-amino 0-Methylisourea, 4 "C, pH 11.0, 6 daysb 1 citraconylated a-amino [121 1 free e-amino (Lys,,)

Step 5 Citraconylated 15-guanidated 3 citraconylated &-amino Decitraconylation, 25 "C, pH 2.6,12hb [22] inhibitor 1 citraconylated a-amino

1 guanidinated e-amino (Lys,,)

Product 15-Guanidated inhibitor 3 free &-amino 1 free a-amino 1 guanidinated &-amino (LysI5)

a The starting material of steps 2, 3, 4 and 5 are the products of steps 1, 2, 3 and 4 respectively. Desalting or separations from excess reagent were carried out by chromatography in a Sephadex 6-25 column.

Eur. J. Biochem. 42 (1974)

J.-P. Vincent, H. Schweitz, and M. Lazdunski 507

Table 2. [14C]Acetic anhydride titration of the free amino groups in the inhibitor, the 15-guanidinated inhibitor and the inhibitor associated in a complex with chymotrypsin

Sample Expected num- Number of [14C] ber of free amino groups corporated into in the inhibitor inhibitor

acetyl groups in-

E O L

mol-I Inhibitor 4 1 4.95 Chymotrypsin . inhibi-

tor b 38 1 3.92 15-Guanidinated inhibi-

tor 3 1 3.80

a Assuming that lysine-15 is masked [2,3,13]; side- chains of lysines 26,41 and 46 are known to remain free [14].

b The number of [1*C]acetyl groups was measured in the inhibitor after acetylation of the complex, dissociation of the two partners by acidification a t pH 2 and separation of the inhibitor from the enzyme by chromatography in a Sephadex G-75 column.

* inhibitor complex is perfectly stable under the same conditions ([16], and our unpublished results). Acidification is the only way to dissociate the trypsin - inhibitor complex even in a 6 M guanidine-HC1 solution.

The number of free amino groups in the inhibitor, inhibitor derivatives or inhibitor associated with trypsin or chymotrypsin was measured by acetylation with [14C]acetic anhydride according to Chauvet and Acher [12]. Acetic anhydride acylates both amino groups and surface tyrosines. The latter residues were deacylated by treatment of the protein samples for 5 h in 0.5 M hydroxylamine, pH 7.0, 25 "C. The excess of hydroxylamine was then eli- minated by filtration on Sephadex G-25. The number of acetyl groups incorporated per mole of prot,ein was estimated from radioactivity measurements in a Packard Tricarb scintillation spectrometer model 3375. Some of the titration results are presented in Table 2.

Xtoichiometry of Complex Formation, Association and Dissociation Kinetics

Stoichiometry and kinetics of association of inhibitor with trypsin or chymotrypsin were followed by measuring the decrease of the enzyme activity after addition of the inhibitor. These techniques have already been described [l, 101. Substrates used to measure enzyme activities were BzArgOEt or TosArgOMe for trypsin and AcTyrOEt for or-chymotrypsin.

Dissociation kinetics were followed according to the procedure already described by Schweitz et al.

for the trypsin * Kazal-inhibitor complex [17] and by Vincent and Lazdunski for the or-chymotrypsin * Kunitz-inhibitor complex [lo]. The enzyme-inhibitor complexes were first prepared a t 25 "C, pH 8.0, by mixing the enzyme with a slight excess of the modified inhibitor, then incubated (at concentra- tions of 0.4 pM and 0.03 pM for the trypsin and chymotrypsin complexes, respectively) in 1 mM Tris buffer pH 8.0 containing 0.2 M NaCl and either 0.1 M BzArgOEt (trypsin - inhibitor complexes) or 10 mM AcTykOEt (or-chymotrypsin - inhibitor complexes). Under these conditions, the synthetic substrate reacted with the free enzyme and displaced the equilibrium E I + E + I in the direction of inhibitor liberation. The displacement was followed by increase in BzArgOEt or AcTyrOEt activity and was directly recorded in pH-stat.

RESULTS

Stoichiometries of Association Fig.l shows that both trypsin and or-chymo-

trypsin still associate in 1 : l ratio with the basic pancreatic inhibitor after selective guanidination of Lys,,. Trypsin also associates stoichiometrically with the fully guanidinated inhibitor and with the selectively guanidinated inhibitor reduced and carboxamidomethylated on the C y ~ ~ ~ - C y s ~ ~ disulfide bridge.

Kinetics of Association

Fig. 2 presents typical kinetics of association of trypsin and or-chymotrypsin with the 15-guanidinated inhibitor a t 25 "C and pH 8.0. The rate constants were calculated from the slopes of the linear plots presented in the inserts. The second-order rate constants of association are given in Table 3.

Kinetics of Complex Dissociation

The 15-guanidinated inhibitor, the fully guanidinated inhibitor and the 15-guanidinated-carboxamidomethylated inhibitor can be displaced from their association with trypsin and/or chymotrypsin by trypsin and chymotrypsin substrates. This displacement can be recorded directly in a pH-stat. Two typical experiments showing the reappearance of trypsin and chymotrypsin activity with time are presented in Fig. 3. Since the association of substrate with free trypsin or chymotrypsin is extremely rapid, the initial rates of the displacements (inserts of Fig. 3) are directly measured to give an easy evalua- tion of the Erst-order rate constants of dissociation of complexes. These first-order rate constants are given in Table 3.


50s Guanidination of Lysine-15 in the Basic nypsin Inhibitor

[I1 / [ E l ( M I M I

Fig. 1. Stoichimetric inhibition of trypsin or a-chymotrypsin by the 15-guanidinated inhibitor. Inhibition of trypsin (0.4 yM) by the Wguanidinated inhibitor (0). Inhibition of a-chymotrypsin (2.5 pM) by 'the 15-guanidinated inhibitor (0). 25 "C, pH 8.0, 0.2 M NaCl

Time ( rn in)

Fig.2. Kineties of association of trypsin and a-chymotrypsin with the 15-gmnidinated inhibitor. Kinetics of association were evaluated by following the decrease of trypsin activity for TosArgOMe (A) or of chymotrypsin activity for AcTyrOEt (B): Association between trypsin (1.5 nM) and 15-guanidinated inhibitor (3.2 nM) (A); between a-chymotrypsin (30 nM) and 15-guanidinated inhibitor (49 nM) (B). 25 "C, pH 8.0, 0.2 M NaCl. Inserts present linear plots demonstrating second-order kinetics. Classical second-order equation :

When k d and k, are known, the association constants, Ka = k, /kd, of complexes can be caIcuIated (Table 3).

DISCUSSION Previous data obtained by Fritz et al. [14] and

Rigbi [18] have shown that the chemical modification of the &-amino group in the active site of the inhibitor by carbamylation, acetylation or dansyla- tion (the &-amino groups which do not belong to the active site area being maleylated or acetylated) do not prevent association of the pancreatic inhibitor with trypsin. These chemical modifications, however, seemed t o considerably decrease the stability of the

0 1 2 3 4 5 6 7 Time (rnin)

01 I ' ' ' ' ' I J

['lo - lE = ([I],, - [Elo) k,t + log - [I10 [El0 log [El, - [E I]

(E for trypsin or a-chymotrypsin, I for the 15-guanidinated inhibitor)

['lo - [E was used to calculate k, values [El0 - [E I1 with y =

i:l complexes. The decrease of stability is most probably due in that case to the lack of possibility to form a salt bridge between the inhibitor active site and Asp,,, in the specificity site of trypsin and also to steric hindrance when the &-amino group of Lys,, is blocked by bulky substituents.

This work indicates that the chemical transformation of Lys,, into Har,, (where Har = homoarginine), although i t preserves the positive charge at the active site of the inhibitor, has drastic effects on complex formation with trypsin. Table 3 indicates that chemical modification of Lys,, by 0-methylisourea has a minor effect on Ic, but a major effect on Ka, the association equilibrium constant, which is decreased by a factor of 800. Transformation of the inhibitor

Em. J. Biochem. 42 (1974)

J.-P. Vincent, H. Schweitz, and M. Lazdunski 509

50

,-.. ,-" - 40 x

z c ._ ._ 5 30 m

LU 0

c

20 N m

10

0 1 50 100 150 200

Time ( r n i n )

Fig. 3. Dissociation kinetics of the trypsin - 15-guanidinated- inhibitor and the a-chymotrypsin * 15-guanidinated-inhibitor complexes. (A) Displacement of the Wguanidinated inhibitor from the trypsin * 15-guanidinated-inhibitor complex was achieved with BzArgOEt (final concentration: 0.1 M). (0) Time course of reappearance of trypsin activity; (0) pseudo first-order representation of the data obtained during the first 20 min gave the initial rate of the displacement. y = 100- percent BzArgOEt activity. 25 "C, pH 8.0, 0.2 M NaCI.

0 2 4 6 8 1 0 Time ( r n i n )

(B) Displacement of the 15-guanidinated inhibitor from the a-chymotrypsin 15-guanidinated-inhibitor complex was achieved with AcTyrOEt (final concentration: 10 mM). (0) Time course of reappearance of chymotrypsin activity; (0) pseudo lirst-order representation of the data obtained during the first 3 min gave the initial rate of the displacement. y = 100-percent AcTyrOEt activity. 25 "C, pH 8.0, 0.2 M NaCl, 3O/, ethanol

Table 3. Kinetic a d thermodynamic parameters for the interaction of trypsin or a-chymotrypsin with the inhibitor or it8 derivatives ka and ka are the rate constants for association and dissociation, respectively. Ks is the association constant of the complex. 25 "C, pH 8.0

Enzyme Reference Inhibitor t kd KB

$v-l. 8-1 8-1 nRI-1

15-pan idinated 2.0 1.0 x 10-4 20 native 1.1 6 . 6 ~ 1 0 - ~ 16000

Tr ypsin This work Trypsin This work fully guanidinated 6.0 1.2 x 10-4 50

Trypsin This work

a-Chymotrypsin This work 15-guanidiated 0.19 2.0 x 10-3 0.095

Trypsin Ell

Trypsin r11 a-Chymotrypsin [lo] native 0.11 1.0 x 10-3 0.11

carboxamidomethylated 0.13 2.2 x 10-5 6.0 carboxamidomethylated-15-guanidinated 1.6 1.2 x 10-3 1.3

into the 15-guanidinated inhibitor increases k d from 6 . 6 ~ 1 0 - ~ s-1 (t./, > 4 months) to 1 . 0 ~ s-1

The inhibitor associates 20 times faster with trypsin than with chymotrypsin (Table 3). This remains true after selective guanidination of Lys,, (Table 3). However, whereas the trypsin - inhibitor complex dissociates 1.5 x lo4 time more slowly than the chymotrypsin * inhibitor complex, the ratio of kd values between the trypsin 15-guanidinated- inhibitor and the chymotrypsin - 15-guanidinated- inhibitor complexes is only about 20. I n fact, transformation of Lys,, into Har,, in the pancreatic inhibitor only affects very slightly the stability of the complex formed with a-chymotrypsin. Guanidina- tion of the active site of the inhibitor only increases

(h/, = 2 h).

k d by a factor of 2 and Ka, values remain practically unchanged. The homoarginine side-chain is as easily accommodated in the specificity pocket of chymotrypsin as the lysine side-chain of the native inhibitor.

The stereochemical interpretation of the consider- able difference in stabilities of trypsin - inhibitor and trypsin - 15-guanidinated-inhibitor is not yet pos- sible. It will have to await a more detailed knowledge of the crystal structure of the trypsin-inhibitor complex.

This difference in stability may, however, in a certain way, be related to the known specificity of trypsin. Lysine and arginine side-chains are equally well recognized by trypsin; the two competitive inhibitors, n-butylamine and 1 -propylguanidine have very similar association constants for the


510 J.-P. Vincent, H. Schweitz, and M. Lazdunski: Guanidination of Lysine-15 in the Basic Trypsin Inhibitor

specificity site of the enzyme (Ki = 0.59 mM-l [19] and 1.89 mM-l [20], respectively). Conversely, substrates derived from arginine and homoarginine behave very differently. The K m and kcat values for a-N-toluene-p-sulphonyl-L-arginine methyl ester are 6.4 pM and 75 s-l, very different from K m and kcat values for a-N-toluene-p-sulphonyl-L-homoargine methyl ester, 332 pM and 4 s-1 [21]. The ratio of K , values, about 50, probably indicates a much better recognition of the arginine side-chain as compared to the homoarginine side-chain.

The analysis of the interaction of trypsin with the fully guanidinated inhibitor (Table 3) indicates that guanidination of lysines 26,41 and 46, which are not part of the active-site area of the inhibitor, does not affect greatly the association characteristics with trypsin. Stabilities of the trypsin - fully-guanidinated-inhibitor and trypsin - 15-guanidinated-inhibitor complexes are very similar.

Multiple chemical modifications in the active-site area of the pancreatic inhibitor do not produce quan- titatively cumulative effects. It has been shown before [ 11 that reduction and carboxamidomethylation of the Cys,,-Cys,, bridge, which is the nearest neighbour of Lys,, in the inhibitor, considerably decreases the stability of the complex formed with trypsin; Ka was decreases by a factor of 2700 and kd was increased by a factor of 330. If reduction and carboxamidomethylation of the Cy~,,-Cys,~ bridge are carried out on the pancreatic inhibitor in which Lysl, has already been transformed in Har,,, its effect on the association with trypsin is much less important. Ks, is decreased by a factor of only 4.6 and kd is increased by a factor of only 55 (comparison of the trypsin - 15-guanidinated-inhibitor and trypsin - 15 - guanidinated - carboxamidomethylated - inhibitor complexes, Table 3).

Finally, it may be of interest to remark that the chemical transformation of Lys,, into Har,, changes the basic pancreatic inhibitor (Kunitz inhibitor) into a Kazal-type inhibitor. It has been demon- strated previously [17] that the pancreatic secretory inhibitor, with an arginine (Arg,,) in its active site, forms complexes with trypsin which are much less stable than those formed by the Kunitz inhibitor with the same enzyme; Ka = 30 nM-l, ka = 6.8 pM-l s-l and k d = 2.2 x s-1. These thermodynamic and kinetic values are strikingly similar to those found for the trypsin-15-guanidinated inhibitor complex (Table 3). However, whereas the pancreatic secretory inhibitor is known to be a temporary inhibitor being slowly cleaved within the complex formed with trypsin [23], the 15-guanidinated in-

hibitor is not cleaved by trypsin within the trypsin - 15-guanidinated-inhibitor complex, even after long period of time (3 weeks) and a t pH values from 3.0 to 8.0.

The authors are very grateful to Choay Laboratories for their very generous gift of pure inhibitor. This work was supported by the DWigation Gdnirale de la Recherche Scienti- fipue et Technique, the Commissariat b Z’Energie Atinnique and the Fondation pour la Recherche Ndddicale.

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J.-P. Vincent, H. Schweitz, and M. Lazdunski, Institut de Biochimie, Faculte des Sciences, Universite de Nice, Parc Valrose, F-06034 Nice, France


guanidination of lysine-15 in the active site of the basic pancreatic trypsin inhibitor :...

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