Eur. J. Biochem. 38,365-372 (1973)

The Interaction between dhymotrypsin and Pancreatic Trypsin Inhibitor (Kunitz Inhibit or)

Kinetic and Thermodynamic Properties

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

(Received April 24, 1973)

1. The association constant, Ka, of the 1 : i complex formed between a-chymotrypsin and the pancreatic trypsin inhibitor is 110 pM-l a t pH 8.0 and 25 "C. The second-order rate constant for the association, Ea, is 110 mM-l s-l and the first-order rate constant for the dissociation, E d , is lo-, s-l under the same conditions. Thermodynamic parameters for complex formation a t 25 "C, pH 8.0 are A GaO = - li .O kcal x mol-l, A Ha'' = 3 kcal x mol-l and A 8,'' = 48 cal. - mol-l * K-l.

2. Temperature and pH-dependences of the rate constants k, and k d have been studied. 3. The difference in stability between the a-chymotrypsin * inhibitor complex and the

trypsin - inhibitor complex (Ka = 16 pM-l, d Q$ = - 18.1 kcal x mol-l) is due essentially to differences of ka values. The dissociation of the a-chymotrypsin inhibitor complex is i .5 x 104 times faster than that of the trypsin - inhibitor complex. Differences in Ka and ka values are discussed in terms of differences in stabilizing interactions.

4. The Cys,,-Cys,, disulfide bridge of the inhibitor, which is highly succeptible to reduction when the inhibitor is free, is masked in the a-chymotrypsin - inhibitor complex. Reduction of the Cys,,-Cys,, bridge in the free inhibitor does not prevent association with a-chymotrypsin. Values of k , and Ed for the formation of the 1 : 1 a-chymotrypsin * reduced-inhibitor complex and for the complex formed with the native inhibitor are very similar. This situation differs from that observed with the trypsin * pancreatic-inhibitor complex. In that case, reduction of Cys,,-Cys,, bridge considerably decreases the stability of the complex formed with trypsin ; K , is decreased by a factor of 3 x lo4 and kd is increased by a factor of 8.6 x los.

I n spite of their importance in control systems, only a limited number of interactions between heterologous proteins or between peptides and pro- teins have been extensively studied. However it is well known that many such associations are very tight. Protein and peptide hormones such as insulin [l], the nervous growth hormone [2], oxytocin [3] and the thyroid-stimulating hormone [4] for example act a t very low doses. Dissociation constants for the complexes they form with their receptors are lower that 10 nM.

We reported previously the results obtained for the formation of the complex between trypsin and pancreatic trypsin inhibitor. This association is one

Abbreviations. Inhibitor, virgin basic pancreatic trypsin inhibitor (Kunitz inhibitor) ; reduced inhibitor, basic pan- creatic trypsin inhibitor selectively reduced at Cys14- Cys,,; BzArgOEt, a-N-benzoyl-L-arginine ethyl ester; AcTyrOEt, a-N-acetyl-L-tyrosine ethyl ester.

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

of the security devices which avoid accidental activation of trypsinogen in the pancreas. The asso- ciation between trypsin and the pancreatic trypsin inhibitor is unusually strong. The dissociation con- stant, Kd, of the 1 : 1 complex is 60 fM at pH 8.0, 25 "C [5].

We report here the results obtained for the formation of the complex between ol-chymotrypsin and pancreatic trypsin inhibitor. Each of the partners in the complex is well characterized. Both their covalent and three-dimensional structures in the crystalline state are available [6-111 and a considerable amount of work has been devoted to the analysis of the components of their active sites. The essential catalytic groups of ol-chymotrypsin are His-57 and Ser-195 [12]; the essential element in the active site of the inhibitor is Lys-15 [13-161. The analysis of the kinetic and thermodynamic properties of the a-chymotrypsin - inhibitor associa- tion presents a particular interest considering that

3 66 Interaction between a-Chymotrypsin and Pancreatic Trypsin Inhibitor Eur. J. Biochem.

a three-dimensional representation of this complex is already available [17], which allows one to locate the main stabilizing interactions.

MATERIALS AND METHODS Materials

The pancreatic trypsin inhibitor (hereafter called inhibitor) was a gift from Choay laboratories. The protein is pure [IS] as judged by polyacrylamide gel electrophoresis, analytical centrifugation, and stoichiometry of the inhibition with trypsin [5,19].

Commercial a-chymotrypsin (Sigma or Worthing- ton) was purified before use by affinity chromatog- raphy in an inhibitor-Sepharose column prepared according to Cuatrecasas [20]. a-Chymotrypsin was charged on the inhibitor-Sepharose column equili- brated a t pH 8.0 with 50 mM Tris buffer containing 50 mM CaC1, and 0.1 M NaC1. Under these conditions, a single peak representing about 1O0/, of the initial charge was eluted in the void volume. This protein fraction was completely devoid of activity towards AcTyrOEt. The active a-chymotrypsin which could not be eluted from the affinity column a t pH 8.0 was eluted in the void volume at pH 4.0 with 50 mM sodium acetate buffer containing 50 mM CaCl, and 0.5M NaC1. The purified enzyme had a specific activity of 445 AcTyrOEt units/mg protein at 25 "C, pH 8.0.

Trypsin with a specific activity of 50-51 BzArgOEt units/mg protein was obtained by activation of pure trypsinogen as previously de- scribed [21]. BzArgOEt and AcTyrOEt are Sigma products, iodo[14C]acetamide was obtained from the Radiochemical Centre (Amersham).

Prepration of Inhibitor Derivatives The &sulfide bridge Cys14 -Cys,, was selectively

reduced with sodium borohydride [22] and the reduced inhibitor obtained was reacted with iodo- acetamide to give the reduced and carboxamido- methylated inhibitor [19,23].

The reactivity of the Cys,,--Cys,, bridge of the inhibitor within the a-chymotrypsin * inhibitor com- plex was evaluated by using simultaneous reduction and alkylation, as previously described for the trypsin * inhibitor complex [5].

Isolation of the a- Chymotrypsin - Inhibitor Complex

a-Chymotrypsin (0.25 mM) was incubated a t 25°C) pH 8.0 with inhibitor (0.75mM) in 3 m l 50mM Tris buffer containing 50mM CaC1, and 0.1 M NaC1. After a reaction time of 1 h (this is much longer than that required for the formation of the complex which takes less than 1 min under

those conditions as will be seen later) the mixture was passed a t 4 "C through a Sephadex 0-75 column (3 x48 cm) equilibrated a t pH 8.0 with the Tris buffer just described. This chromatographic sys- tem separates well the complex (molecular weight 31 500) from excess free inhibitor (molecular weight 6500) but not free a-chymotrypsin (molecular weight 25000) from the a-chymotrypsin - inhibitor complex.

Xtoichiometry All stoichiometries of association involving a-chy-

motrypsin and the inhibitor or its derivatives were determined by measuring the decrease of AcTyrOEt activity which followed addition of the inhibitor. Determinations were essentially similar to those already reported for the trypsin * inhibitor association [5,19].

Association Kinetics The kinetics of association of a-chymotrypsin

with the inhibitor or its derivatives were evaluated by following the decrease of a-chymotrypsin activity. Enzymatic activities were determined in a pH-stat radiometer TTT 1C equipped with an SBR2C recorder. AcTyrOEt (10 mM) was the substrate. The mixture contained 0.2 M NaCl and 3OlO ethanol. All determinations (incubations and activity mea- surements) were carried out under nitrogen.

Dissociation Kinetics The a-chymotrypsin - inhibitor complex was first

isolated as described above, then incubated1 a t a concentration of 20pM a t pH 8.0 in 50mM Tris buffer containing 0.1 M NaCl and 50 mM CaC1,. The displacement was started by adding trypsin (final concentration 20pM) which forms a tighter asso- ciation with the inhibitor than a-chymotrypsin [24-261. Aliquots were taken a t different times and used to determine the amount of free trypsin. The replacement of a-chymotrypsin by trypsin within the enzyme * inhibitor complex was followed by measuring the loss of BzArgOEt activity. The BzArgOEt activity of free trypsin was measured in a pH-stat at 25 "C, pH 8.0, 0.2 M NaCl with 3 mM BzArgOEt. a-Chymotrypsin presented no activity for BzArgOEt under the same conditions.

The a-chymotrypsin * reduced-inhibitor complex could not be isolated as described above for the a-chymotrypsin inhibitor complex as a slow reoxi- dation of reduced inhibitor within the complex occurs during Sephadex G-75 chromatography. This complex was therefore prepared by mixing a-chymo- trypsin and reduced inhibitor a t equimolecular concentration (30 pM), a t 25 "C, pH 8.0. The com- plex was then diluted to a final concentration of 30 nM in 1 mM Tris buffer pH 8.0 containing 0.2 M

Vo1.38, No.2,1973 J.-P. VINCENT and M. LAZDUNSKI 367

NaCl and 30mM AcTyrOEt. The mixture had a small AcTyrOEt activity during the first few seconds (due to the intrinsic catalytic activity of the a-chymotrypsin-reduced inhibitor complex) but esterolytic activity appeared and increased with time. This behavior was due to a displacement of a-chymotrypsin from the complex by AcTyrOEt. The reaction was carried out in a pH-stat which recorded automatically the appearance of free a-chymotrypsin. A similar approach was taken previously for the evaluation of the dissociation kinetics of the complexes formed between trypsin and chemically modified derivatives of the inhibitor

RESULTS Stoichiometries of Association

Fig. 1 A shows that a-chymotrypsin associates stoichiometrically, in a 1 : 1 ratio, with both native

~51.

100 L I

I - I T - I I

1 2 i i 5 10 15 Inhibi tor I Chymotrypsin (rnol/ mol)

Fig. 1. Stoichimtric inhibitim of a-chymtrypin by native or redwed inhibitor. (A) Inhibition of a-chymotrypsin (c = 2.5 pM) by native (0) and by reduced inhibitor (0). (B) Inhibition of a-chymotrypsin (c = 25 nM) by native inhibitor (0). Solid lines are calculated curves using the value of R. which give the best fit, i.e., K1 = 110 pM-l.

25 "C, pH 8.0, 0.2 M NaCl

A

T i m e ( m i n )

T i m e ( m i n )

T i m e ( m i n )

T i m e ( m i n )

and reduced inhibitor. The data in Fig.1B will serve later for a direct determination of the dissociation constant of the a-chymotrypsin - inhi- bitor complex.

As shown in Fig.1A these 1:l complexes are endowed with a residual activity for AcTyrOEt. This activity is about 5O/, that of free a-chymo- trypsin. The residual activity is much too high to be attributed to the very small quantities of free a-chymotrypsin in equilibrium with the complex. For example, with a two-fold molar excess of inhi- bitor a t 25 "C, pH 8.0, one should expect a residual activity of a-chymotrypsin (2.5 pM) of only 0.4O/, if the complex were totally inactive. Moreover, since a-chymotrypsin has been purified by affinity chromatography on an inhibitor-sepha- rose column, residual activity cannot be due to a small fraction of active a-chymotrypsin which would not associate with the inhibitor. A comparative kinetic analysis of the catalytic efficiency of a-chymotrypsin and of the 1 : 1 a-chymotrypsin inhibitor complex for AcTyrOEt hydrolysis has

given the following results: kcat = 185 s-l, Km = 1.0 mM for a-chymotrypsin a t pH 8.0, 25 "C and kcat = 10.4 s-~, Km = 1.2 mM for the complex under the same conditions. The values of kcat is decreased by a factor of 20, while Km remains pratically un- changed. In consequence the inhibitor appears as a non-competitive inhibitor of a-chymotrypsin in the 1 : 1 a-chymotrypsin * inhibitor complex. Fig.2 pre- sents typical kinetics of association of a-chymotrypsin with native (Fig.2A) and reduced (Fig.2B) inhibitor at 25 "C and pH 8.0. Reduced inhibitor was obtained by selective reduction of the Cys,,-Cys,, bridge of the inhibitor [22]. Under conditions described in Fig. 2B, the association of a-chymotrypsin with reduced inhibitor was nearly completed after 5 min.

iiE 0.4 0.2

0 1 2 3 T i m e (rnin)

1 1 1 1 1 1 1 1 1 1 1

1 2 3 4 5 6 7 8 9 1 0 1 1 T i m e (min)

Fig. 2. Kinetica of association of a-chymtryp8in with native or reduced inhibitor. Kinetics of association were evaluated by following the decrease of chymotrypsin activity for AcTyrOEt. Association between (A) a-chymotrypsin (36 nM) and inhibitor (110 a) and (B) ~-chvmotrv~sin (32 nM)

kinetics. Classical second-order equation: [I10 [El0

[I1' - rE ' I] = ([I], - [El,) k. t + log - Log [El, - [E * I] where E = a-chymotrypsin and 1 = inhibitor, with

[I1' - [E * I' was used to calculate k. values and reduced inhibitor (I13 nM). 25 "C, PH 8.6,&0.2 M NaCl: Inserts present linear plots demonstrating second-order = [El, - [E * I]

368 Interaction between a-Chymotrypsin and Pancreatic Trypsin Inhibitor Eor. J. Biochem.

I

0.1 - - 'Y) L

5 -2,O.Ot: -

0 3 4 5 6 7 8 9 10

PH Fig.3. pH dependence of the second-order rate constant, k,, for the association of a-chymotrypsin with inhibitor. 25 "C, 0.2 M NaCl. Experimental points have been described by a calculated titration curve (solid line) for a single ionizable

group with a p K = 7.3

It has been checked that this time is short enough to prevent significant reoxidation of the reduced partner (90°/o of the modified inhibitor remained in the reduced state after 5 min) [5]. The rate con- stants were calculated from the slope of the linear plots presented in the inserts. The second-order rate constants of association of a-chymotrypsin with native and reduced inhibitor, kit, are very similar as shown in Table 1.

Carboxamidomethylation of both -SH groups of the reduced inhibitor gives a dicarboxamido- methylated derivative which does not associate with a-chymotrypsin a t pH 8.0 and 25 "C, even a t high a-chymotrypsin concentration (10 pM) and with a 20-molar excess of the dicarboxamido- methylated inhibitor. This latter result confirms previous data obtained by Kress et al. [23].

The pH-dependence of ka is presented in Fig.3. Maximal values for k, are slightly higher than 0.1 pM-l s-l, they are attained between pH 8.0 and 9.0. A very slow association is observed between pH 4.0 nd 5.0. No inhibition of a-chymotrypsin activity could be observed a t pH 2.

Fig. 4 shows an Arrhenius plot describing the temperature dependence of La. The activation energy for the association is 14 kcal x mol-I.

Kinetics of Complex Dissociation (ChTi ) The displacement of a-chymotrypsin (Ti) by

trypsin is schematized as follows :

ChTi * I ChTi + I 3 Ti I

The kinetics of the displacement are presented in Fig.5A. The half-life of the displacement is 12 min. This means that the formation of the trypsin * inhibitor complex from the a-chymotrypsin

3.2 3.3 3.4 3.5 3.6 lo3/ r (K- ' )

Pig.4. Arrhenius plot for the second-order rate constant, k,, of the association of a-chymotrypsin with inhibitor (0) , and for the first-order rate constant, kd of the dissociation of the complex (0). pH 8.0, 0.2 M NaCl. Temperatures were varied

from 5 "C to 35 "C. k, : M-ls-l; kd : s-l

Table 1. Kinetic and thermodynamic parameters for the asso- ciations between a-chymotrypsin, trypsin or pseudotrypsin and pancreatic trypsin inhibitor or its derivatives at 25 "C,

p H 8.0 ka and kd are the rate constants for association and disso- ciation, respectively. K , is the association constant of the complex. Results for trypsin and pseudotrypsin were taken

from Vincent and Lazdunski [5]

Enzyme Inhibitor ks kd Ka

a-Chymotrypsin Native a-Chymotrypsin Reduced a-Chymotrypsin Carboxamido-

methylated Trypsin Native Trypsin Reduced Trypsin Carboxamido-

methylated Pseudotrypsin Native

vM-l. g-l s-l I*M-l

0.11 10-3 110 0.08 1.4. 57

0 1.1 6.6 * 1.6.10' 0.32 5 .7 . 560

0.13 2.2. 5900 0.07 6.3 * 110

- -

- inhibitor complex is limited by the rate of disso- ciation of the or-chymotrypsin - inhibitor complex and not by the much faster association of trypsin with inhibitor [5]. As expected the displacement is a first-order reaction (insert of Fig.5). The value of k d is

Fig. 6 shows that kd is pH-dependent. Dissocia- tion of the a-chymotrypsin * inhibitor complex is much slower a t alkaline than at acidic pH. Maximal values of 1/kd slightly higher than lo3 s are obtained between pH 7 and 9.

An Arrhenius plot describing the temperature dependence of the first-order dissociation constant is shown in Fig.4. The activation energy of the dissociation process is 11 kcal x mol-1 a t pH 8.0.

The technique used for the measurement of the rate of dissociation of the a-chymotrypsin - inhibitor

s-1 a t pH 8.0, 25 "C.

Vo1.38, N0.2,1973 J.-P. VINCENT and M. LAZDUNSEI 369

0 0 10 20 30 40 100 200

Time ( rn in ) Fig. 5. Dissociation kinetics of the Complexes of a-chymo- trypsin with native and reduced inhibitor. (A) Trypsin- induced displacement of a-chymotrypsin from the a-chymo- trypsin * inhibitor complex. 25 "C, pH 8.0, 0.1 M NaCl, 50mM CaCI,. (0) Time course of the dissociation; (0) pseudo-first-order representation. [E * I] = amount of

1.- 8 I

9 PH

Fig. 6 . pHdependence of the first-order rate constant, kd, for the dissociation of the a-chymtrypsin . inhibitor complex. 25 "C, 0.1 M NaCl, 50 mM CaCl,. Experimental points have been described by a calculated titration curve (solid

line) for a single ionizable group with a pK of 5.3

complex, i .e. the displacement of a-chymotrypsin by trypsin, can only be used with success when tryp- sin forms tighter complexes with the inhibitor or its derivatives than a-chymotrypsin. As will be discussed later, trypsin and a-chymotrypsin asso- ciate with reduced inhibitor to form complexes of similar stability. Therefore, another technique was chosen to evaluate ka for the a-chymotrypsin * reduc- ed-inhibitor complex.

As already indicated in Materials and Methods, or-chymotrypsin (E) was displaced from its associa- tion with reduced inhibitor (red-I) by its classical substrate AcTyrOEt (S) :

E * red-I red-I + E , AO T-OEt ( S ) \ ES --f E + p.

0

Time (rnin)

complex. (B) Displacement of reduced inhibitor from its a-chymotrypsin complex was achieved with AcTyrOEt (final concentration: 30 mM). 25 "C, pH 8.0, 0.2 M NaCl, 30/0 ethanol. (0) Time course of reappearance of a-chymo- trypsin activity; (0) pseudo-first-order representation of

the data. y = 100 - percent of AcTyrOEt activity

The displacement was recorded directly in a pH-stat, following the reappearance of a-chymo- trypsin activity with time (Fig.5B). The system evolves toward an equilibrium position where a-chymotrypsin has been liberated from its inter- action with reduced inhibitor. The association of substrate with free a-chymotrypsin being extremely rapid, measurement of the rate of the displacement gives an easy evaluation of the first order rate con- stant, ka, for the dissociation of the complex (insert of Fig.5B and Table 1).

Under our experimental conditions, reoxidation of reduced inhibitor was pratically completely pre- vented inside the complex even after fairly long periods of times (a reoxidation of loo/, of reduced inhibitor in the complex was observed after 40 min).

Association Constants of the Complexes Formed between a- Chyrnotrypsin and

Native or Reduced Inhibitor Association constants could be evaluated easily

when the rate constants for both association, k,, and dissociation, kd, were known (K, = k,/kd). Such values are reported in Table 1. The variation of Ka with pH is presented in Fig.7.

A direct determination of K, is also possible. Titrations of native a-chymotrypsin with the inhi- bitor under two widely different conditions of concentration have already been presented (Fig. 1).

Stoichiometry was easily obtained when the a-chymotrypsin concentration was much higher than the dissociation constant of the complex ( l /K , ) . The situation was markedly different when the a-chy- motrypsin concentration was of the same order of magnitude as the dissociation constant (Fig. 1 B). In the latter case, the experimental points could be

370 Interaction between a-Chymotrypsin and Pancreatic !Crypain Inhibitor Eur. J. Bioohem.

3 4 5 6 7 8 9 10 PH

Fig. 7. pH-dependence of the association constant, Ka, for the a-chymotrypsin - inhibitor complex at 25 "C

perfectly described by a calculated curve assuming a value of Ka, the association constant of the 1 : l complex, of 110 pM-l.

This value is identical to that obtained from the evaluation of ka/kd (Table 1), and in reasonably good agreement with the equilibrium constant (Ka = 35 pM-1) determined directly by another technique by Rigbi [16] at pH 8.0, 25 "C.

Values of d GaO ( A GaO = - R T log Ka), d Ha0 (AHaO = Ea - Ed where E a and Ed are activation energies for association and dissociation, respective- ly) and ASaO (ASaO = [ABaO - d Ga"]/T) have been calculated. A GaO = - 11 kcal x mol-l, A Ha0 = 3 kcal x mol-l, A 880 = 47 cal x mol-l x K-l a t 25 "C and pH 8.0.

Protection of the Disulfide Bridge of the Inhibitor in the Association

with a- Chymotrypsin It has been already demonstrated that the

Cys14 --Cys,, bridge of the inhibitor is protected against reduction with borohydride in the trypsin - inhibitor complex [5,27].

It was of interest to study the reactivity of this bridge in the complex formed between a-chymo- trypsin and the inhibitor. Whereas two [14C]carbox- amidomethyl groups were incorporated into the free inhibitor after reduction and alkylation with iodo[14C]acetamide, there was no incorporation, under the same conditions, into inhibitor associated with a-chymotrypsin in the complex. This is direct evidence that the bridge Cys14-Cys,, is mask- ed in the complex. This result is in agreement with the model of association proposed by Huber [28].

DISCUSSION The assembly of the three-dimensional structures

of or-chymotrypsin and inhibitor determined by

X-ray crystallography has given a model of the structure of their complex. The interactions which stabilize the complex are 7 hydrogen bonds ; about 200 Van der Waals contacts, and the probable forma- tion of a bond of the acyl-enzyme (or tetrahedral adduct) type which links lysine-15 of the inhibitor molecule (the or-carbonyl function) and serine-195 of the active site of or-chymotrypsin (the hydroxyl

The data presented in this paper indicate that all the interactions which stabilize the complex give an association constant, Ka, of about 100 pM-l a t pH 8.0 and 25 "C. Corresponding thermodynamic values are A GaO = - 11 kcal x mol-l, A Ha0 = + 3 kcal x mol-l and d SaO = 47 cal x mol-l x K-l. It is clear that complex formation is essentially entro- py driven. The fact that a considerable number of Van der Waals contacts is formed between the enzyme and inhibitor strongly suggests that the large value of ASaO is related, a t least in part, to expulsion of bound water molecules during complex formation. There is no fixed water observable in the contact area between inhibitor and enzyme [17].

Large values of ASaO have been found for other heterologous protein-protein interactions, e.g. the association of the pancreatic secretory trypsin inhibitor with trypsin (ASaO = 43.6 calxmol-l x K-l) [29] and the association of snake neurotoxins with the acetylcholine receptor (A&O = 74 cal x mol-1 x K-l) [30]. The high value of A SaO could also be due in part to structural changes affecting the inhibitor and/or enzyme conformations within the complex.

The value of ka at pH 8.0,25 "C is 0.11 pM-l* s-l; it is a least 1000-times lower than would be expected for a diffusion-controlled process [31]. The activa- tion energy for the association is also too high, 14 kcal x mol-l, as compared to 3 kcal x mol-l for a classical diffusion step [31]. The interaction of a-chymotrypsin with inhibitor to form the inactive complex is likely to occur in a t least two steps:

E + 1 % EI*% EI,

with a rapid second-order absorption of the inhibitor to the enzyme complementary area (E + I % EI*) followed by a slow first-order rearrangement of the complex EI* % EI. Similar proposals have been already presented for the interaction of trypsin with ovomucoid, lima bean and pancreatic secretory inhibitors [29,32]. Recent crystallographic data also indicate that structural alterations in or-chymo- trypsin and the pancreatic trypsin inhibitor occur during association [33].

The pH-dependence of ka is described by a titra- tion curve which indicates that an ionisable group with a pK of about 7.3 a t 25 "C is important in the basic form for the association process. The best candidate for this pK value is the imidazole side-

group) ~ 7 1 .

V01.38, No.2,1973 J.-P. VINCENT and M. LAZDUNSEI 371

chain of histidine-57 in the active site of a-chymo- trypsin.

The variation of kd with pH is apparently con- trolled by the ionisation of a single group with a pK of about 5.3. The rate of dissociation of the a-chymotrypsin - inhibitor complex is small a t neutral pH. Protonation of the complex considerably increases its rate of dissociation. The pK value of 5.3 may be again tentatively assigned to the imidazole of histidine-57 which is masked in the a-chymo- trypsin - inhibitor complex [17].

Since both the a-chymotrypsin - inhibitor and the trypsin * inhibitor complexes have been studied by X-ray crystallographic techniques it is of interest to compare the rate constants ka and ka and the association constant K,, which characterize the association of the inhibitor and enzyme partners.

The stabilities of the complexes are very different. The association constant Ka and A G$ for the trypsin * inhibitor association are 16 pM-l and - 18.1 kcal x mol-1 [5] as compared to only 110 pM-l and - 11.0 kcal x mol-l for the a-chymo- trypsin * inhibitor complex.

Differences between the two types of complexes reside both in ka and kd values. At pH 8.0, 25 "C, k, for the association of trypsin with inhibitor is 1.1 pM-l s-l [5] as compared to only 0.11 pM-l s-1 for the a-chymotrypsin * inhibitor complex ( E , values are 10.5 and 14.0 kcalxmol-l for the trypsin - inhibitor [5] and the a-chymotrypsin . in- hibitor complex, respectively). Differences on kd values are much more important in the same conditions of pH and temperature. kd = 6.6 x 10-8 s-1 (t,,, = 17 weeks) for the trypsin * inhibitor complex [5] as compared to only s-l (ti,, = 12 min) for the a-chymotrypsin * inhibitor complex. The kinetic stability of the trypsin - inhibitor complex is more that 104-fold higher than that of the association with 01 - chymotrypsin.

All the hydrogen bonds and Van der Weals contacts which have been found in the association of a-chymotrypsin with the inhibitor are also present in the trypsin * inhibitor complex [17]. Moreover both complexes are believed to be stabilized by the forma- tion of a bond between lysine-15 of the inhibitor and the serine residue in the active site of the enzyme receptor. However, there exists a very specific interaction which can be formed only in the trypsin * inhibitor complex; this is the salt bridge between the &-ammonium of lysine-15 on the inhibitor and the p-carboxylate of aspartate-177 of trypsin. Asp-177 is the essential element in the active site of trypsin which gives the specificity for basic side-chains [34-361. Asp-177 is replaced by a serine residue in the a-chymotrypsin sequence [37]. Therefore, the formation of the ion-pair Lys-15 of inhibitor with Asp-177 of trypsin is not possible in the complex with a-chymotrypsin. Moreover, to

form this latter complex, it is necessary to bury the positive charge of the &-ammonium of Lys-15 in an hydrophobic environment; this event is not ther- modynamically favorable. In summary, the stabiliz- ing effect of the ion-pair Lys15 -Asp,,, in the trypsin - inhibitor complex disappears in the a-chymotrypsin . inhibitor complex and is replaced by a destablilizing effect due to the necessity of burying a charge in an apolar medium. It is of interest to note that ka, kd and Ka values for the a-chymotrypsin * inhibitor complex are very similar to those already deter- mined for the pseudotrypsin - inhibitor complex [5]. For the latter complex, ka = 70 mM-1 8-1, kd = 6.3 X lop4 s-l and Ka = 110 pM-l a t pH 8.0, 25 "C. In pseudotrypsin, the catalytic site is dis- connected from the specificity site upon specific hydrolysis of the Lys,,,-Asp,,, bond. This trans- formation of trypsin into pseudotrypsin abolishes all specificity for basic substrates or for classical inhibitors such as benzamidine [34]. Therefore, it is very likely that pseudotrypsin does not form the salt-bridge Lys,, (inhibitor) -Asp,,, (enzyme) [5] ; its mode of association with the inhibitor would then be nearly identical to that of a-chymotrypsin.

The disulfide bridge Cysl4--Cys3, of the inhibitor plays an important role in the association of trypsin with the inhibitor. Reduction of this bridge, as well as chemical modification of the -SH groups formed in this reduction greatly affects values of kd and Ka (variations of ka are less important) [5]. For example, kd and Ka are 5.7 X s-l and 560 pM-I, respec- tively for the trypsin * reduced-inhibitor association. Both crystallographic and chemical data [17,5,27] show that this disulfide bridge, which is extremely accessible at the surface of free inhibitor, becomes completely masked in the trypsin * inhibitor com- plex. It has been shown in this paper that the Cys14-Cys,, bridge is also masked in the a-chymo- trypsin - inhibitor complex. However, practically no difference has been noted between kinetic and thermodynamic parameters which characterize the association of a-chymotrypsin with native and reduced inhibitor (Table 1).

Huber and collaborators have postulated that the role of the bulky disulfide bridge Cys,,-Cys3, was to exclude water from the enzyme surface, within the complex, making the hydrogen bonds strong [28]. If the only role of the C y ~ , ~ - C y s ~ ~ bridge is to exclude water from the contact area between the inhibitor and its enzyme receptor, then it is presently not easy to understand why reduction of the bridge, which apparently brings no structural change in the inhibitor [19], produces such drastic effects in the association with trypsin and has practically no influence in the association with a-chymotrypsin.

Another difference between complexes formed with trypsin and those formed with a-chymotrypsin

372 VINCENT and LAZDTJNSKI: Interaction between a-Chymotrypsin and Pancreatic Trypsin Inhibitor Eur. J. Biochem.

remains unexplained. Carboxamidomethylation of the reduced inhibitor improves the affinity of the inhibitor for trypsin [5] whereas it prevents associa- tion with a-chymotrypsin (Table 1). A better knowledge of the stereochemical details of the tryp- sin - inhibitor and the a-chymotrypsin - inhibitor interactions would obviously be needed to under- stand all the kinetic and thermodynamic properties of the two types of complexes.

The authors are very grateful to Choay Laboratories for their very generous gift of pure inhibitor. They also thank G. Vaugoyau, G. Corthier, M. N. Marier and F. Sam- pieri for efficient help. This work was supported by the Diligation Cinirale de la Reherche Scientifique et Technique, the Cmmissariat it 1’Energie Atomique and the Fondation pour la Recherche Midicale.

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J.-P. Vincent and Ed. Lazdunski, Institut de Biochimie, Facult6 des Sciences, Universitb de Nice, Parc Valrose, F-06034 Nice, France