Zymogen Activation: Effect of Peptides Sequentially Related to the Bovine P-trypsin N-terminus on Kazal Inhibitor and Benzamidine Binding to Bovine Trypsinogen

Paolo Ascenzi, Massimo Coletta and Gin0 Amiconit CNR Center for Molecular Biology, Department of Biochemical Sciences, University of Rome 'La Sapienza', Piazzale Aldo Moro 5 , 00185 Roma, Italy

Martino Bolognesi Department of Genetics and Microbiology, Section of Crystallography, University of Pavia, Via Tardmelli 16, 27100 Pavia, Italy

M a r i o Guarneri and Enea Menegatti Department of Pharmaceutical Sciences, University of Ferrara, Via Scandiana 21, 44100 Ferrara, Italy

The activating effect of peptides sequentially related to the Ile16-Va117-Gly18 N-terminus of bovine /?-trypsin (namely Ile-Val-Gly, Ile-Val, Ile-Leu, Ile-Ala, Val-Val, Leu-Val, and Val-Leu) on the thermodynamic parameters for the binding of the porcine pancreatic secretory trypsin inhibitor (Kazal inhibitor) and benzamidine to bovine trypsinogen was investigated a t pH 5.5 (Bis tris-HCI buffer, I=O.1 M) and T=21+0.5"C. Thermodynamic parameters for Kazal inhibitor and benzamidine association to the binary peptide/zymogen adducts are more favorable than those observed for ligand binding to the proenzyme alone, although never as much as those reported for the formation of bovine /?- trypsin/Kazal inhibitor and bovine /?-trypsinlbenzamidine adducts. Analogously, the affinity of activating peptides for the binary proenzyme/Kazal inhibitor and binary proenzyme/benzamidine complexes is higher than that observed for peptide binding to free bovine trypsinogen. Differences in affinity for ligand binding to free bovine trypsinogen, to its binary adducts and to bovine /?-trypsin suggest the presence of different activation levels of the proenzyme, none of which structurally coincide with that achieved in bovine /?-trypsin. The existence of different discrete states suggests that the zymogen-to-active enzyme transition should not be considered as a two-state process but as a multistep event.

INTRODUCTION

Control of biological functions by transmission of infor- mation within a given macromolecule is, in general, related to the ability to undergo conformational transi- tion(s). Thus, flexibility plays a central role as a regulatory

t Author to whom correspondence should be addressed

Abbreviations used Trypsinogen, bovine trypsinogen; trypsinogen:pGB, bovine trypsinogenpguanidinobenzoate acyl complex; trypsinogen:Z- ArgONp, bovine trypsinogen:N-a-carbobenzoxy-L-arginine-p-nitro- phenyl ester adduct; trypsinogen:Z-Arg, bovine trypsinogen:N-a-carbo- benzoxy-L-arginine acyl complex; P-trypsin, bovine P-trypsin; Kazal inhibitor, porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, type 1); Kunitz inhibitor, bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor); Ile-Val-Gly, N-a-isoleucyl-L-valylgly- cine; Ile-Val, N-a-L-isoleucyl-L-valine; Z-Ile-Leu, N-a-carbobenzoxy-L- isoleucyl-L-leucine; Ile-Leu, N-a-L-isoleucyl-L-leucine; Z-Ile-Ala, N-a- carbobenzoxy-~-isoleucyl-~-alanine; Ile-Ala, N-a-L-isoleucyl-L-alanine; Val-Val, N-a-L-valyl-L-valine; Leu-Val, N-a-L-leucyl-L-valine; Val-Leu, N-a-L-valyl-L-leucine; pNpGB, p-nitrophenyl-p-guanidinobenzoate; Z-ArgONp, N-a-carbobenzoxy-L-arginine p-nitrophenyl ester; Z- LysONp, N-a-carbobenzoxy-L-lysine p-nitrophenyl ester; Bis tris, bis[2- hydroxyethyll-imino-tris-[hydroxymethyl]methane.

property and is considered a determinant factor in allowing an inactive zymogen to turn into a fully active enzyme (Huber and Bennett, 1983; Amiconi et al., 1988; Brunger et al., 1987).

The bovine trypsinogen-to-B-trypsin activation is accomplished through the rigidification of three flexible loop segments (Gly142 through Pro152, GlyA184 through Gly193, and Gly216 through Thr222) and of the N-terminal sequence (all forming the so-called activation domain) in a well defined and ordered structure (Bode and Schwager, 1975; Bode and Huber, 1976; Bode et al., 1978,1984; Huber and Bode, 1978; Bode, 1979; Bolognesi et al., 1982, 1988; Huber and Bennett, 1983). The first step of this transition is the cleavage of the zymogen N- terminal hexapeptide by an activating enzyme. The newly formed N-terminal residue Ilel6 can then bind to its specific pocket present on the surface of the (pro)enzyme (i.e. the Ile-Val binding pocket; see Bode and Huber, 1976), forming a salt bridge with the internal residue Asp194. (Aminoacid residues have been identified by their three-letter code and by their sequence number. The numbering of serine (pro)enzymes cited in the text refers to that of homologous bovine chymotrypsinogens A and B (Hartley and Shotton, 1971). For better discrimination, the usual residue numbers of macromolecular serine

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(pro)enzyme inhibitors are increased by 300 (Bode et al., 1984)). This occurrence triggers the conformational re- arrangement(s) which are mainly concerned with the structure of the enzyme recognition subsite(s) rather than with that of the catalytic center (Bode and Schwager, 1975; Bode and Huber, 1976; Bode et al., 1978, 1984; Huber and Bode, 1978; Bode, 1979; Bolognesi et al., 1982, 1988; Huber and Bennett, 1983). In this respect, the low intrinsic proteolytic activity as well as the weak ligand binding properties of serine zymogens may be essentially related to the poorly structured recognition subsites in comparison with those of the active enzymes (Gertler et al., 1974; Bode and Huber, 1976; Bode, 1979; Antonini et al., 1983, 1984; Ascenzi et al., 1985; Menegatti et al., 1985; Amiconi et al., 1987, 1988). On the other hand, residues forming the catalytic charge-relay system (i.e. Aspl02- His57-Ser195 triad) have almost identical conformations in the zymogen and in the corresponding active enzyme (Bode and Schwager, 1975; Bode and Huber, 1976; Bode et al., 1978, 1984; Huber and Bode, 1978; Bode, 1979; Bolognesi et ul., 1982, 1988; Huber and Bennett, 1983).

Whenever strong ligands (i.e., substrates, activators and inhibitors) and/or activating peptides, sequentially related to the Ilel6-Vall7-Glyl8 N-terminus of P-trypsin (Hartley and Shotton, 1971), bind at their specific cooperatively-linked sites (i.e., the specificity center and the Ile-Val cleft, respectively), trypsinogen can assume various activation levels, characterized by different func- tional properties (Bode and Huber, 1976; Bode et al., 1978,1984; Huber and Bode, 1978; Bode, 1979; Bolognesi et al., 1982, 1988; Antonini et al., 1983, 1984; Huber and Bennett, 1983; Ascenzi et al., 1985, 1987; Menegatti et a/., 1985; Amiconi et al., 1987, 1988); these structures may be postulated as intermediates along the pathway of the transition between trypsinogen and p-trypsin (Ascenzi et al., 1985).

The present study reports the effect of activating peptide (i.e. Ile-Val-Gly, Ile-Val, Ile-Leu, Ile-Ala, Val- Val, Leu-Val and Val-Leu) concentration on values of the association equilibrium constant for Kazal inhibitor and benzamidine binding to trypsinogen at T= 21 i0 .5"C and pH 5.5 (Bis tris-HC1 buffer I=O.1 M; see Ascenzi et a/., 1985). The choice of the unusual pH conditions has been dictated by the low inhibitor affinity for trypsinogen at pH 5.5 (Antonini et al., 1983), which allows a direct evaluation of K, values over the whole activating peptide concentration range explored (Ascenzi et al., 1985). In fact, at higher pH values (such as 8.0, where generally data for trypsinogen activation are obtained; see text), the inhibitor affinity for the zymogen is such that the determination of K, values at peptide concentrations higher than 1 .O x M is prevented (Ascenzi et al., 1985). The results given here indicate that ligand binding properties of the activating peptide/trypsinogen adducts as well as of trypsinogen/Kazal inhibitor and trypsino- genibenzamidine complexes are more favourable than those of the free zymogen, but never reach the ligand affinity displayed by /I-trypsin. Most of the observed differences may be attributed to less structured states of the interacting site(s) of the zymogen with respect to the activated enzyme, and therefore to a decrease in molecu- lar specificity for recognition. Moreover, present data could also be relevant in assessing the functional proper- ties of intermediates involved in the multistep zymogen- to-fully active enzyme transition(s) (Huber and Bennett,

1983; Perkins and Wuthrich, 1983; Ascenzi et al., 1985; Menegatti et al., 1985).

MATERIALS AND METHODS

TRYPSINOGEN ACTIVATION

0 Heyden & Son Limited, 1988 JOURNAL OF MOLECULAR RECOGNITION, VOL. 1, No 3,1988 131

Crystalline trypsinogen was obtained from Merck AG (Darmstadt, FRG); zymogen preparations were found virtually free of trypsin, chymotrypsin, elastase and kallikrein activities (Antonini et al. 1984). Kazal inhibitor (type I) was prepared and purified as previously described (Menegatti et al., 1982). Kunitz inhibitor was obtained from Lepetit S.p.A. (Milano, Italy) and purified as reported elsewhere (Antonini et al., 1983).

The homogeneity of trypsinogen, Kazal and Kunitz inhibitors was checked by polyacrylamide gel electro- phoresis with sodium dodecylsulphate, in the presence and in the absence of P-mercaptoethanol, according to the procedure of Weber et al. (1972). The preparations used contained less than 3% of nonenzymatic and/or noninhibitory protein contaminants.

Trypsinogen concentration was determined (i) by titration with pNpGB (Gertler et al., 1974; Chase and Shaw, 1969; Hruska et al., 1969) and (ii) spectrophotome- trically at 280 nm (El&= 13.9) (Vincent and Lazdunski, 1976); the values obtained by the two methods agree very well (> 95%). Kazal and Kunitz inhibitor concentration was determined spectrophotometrically at 280 nm, E;:; values being 5.18 (Greene et al., 1968) and 8.3 (Kassel, 1970), respectively.

Ile-Val-Gly was kindly provided by Lepetit S.p.A. (Milano, Italy). Z-Ile-Leu, Z-Ile-Ala, Val-Val, Leu-Val, Val-Leu, benzamidine, pNpGB and Z-LysONp were purchased from Sigma Chemical Co. (St Louis, MO, USA).

Ile-Leu and Ile-Ala were obtained by hydrogenolytic splitting of the carbobenzoxy group from Z-Ile-Leu and Z-Ile-Ala (Wunsch, 1974); both activating peptides were further purified by recrystallization from isopropanol/ water (Bode, 1979). Ile-Val was synthesized as detailed in Antonini et al. (1983).

All other products were from Merck AG (Darmstadt, FRG).

All chemicals were of analytical grade and used without further purification.

Both in the absence and in the presence of activating peptides values of thermodynamic parameters for Kazal inhibitor binding to trypsinogen have been obtained by spectrophotometric titrations at 300 nm 0.5 mhr'cm-' ; see Antonini et al., 1983; Ascenzi e ta / . , 1985). The value of the intrinsic molar fraction of Kazal inhibitor-bound (activating peptide) trypsinogen ( To) was directly obtained, for every free ligand concentration, from the amplitude of spectral changes accompanying adduct formation, as compared to the total optical density variation (common to all effector peptides) taken as completion of the process (To= 1; see Bode, 1979).

Both in the absence and in the presence of activating peptides values of thermodynamic parameters for benza- midine binding to trypsinogen have been obtained by the inhibitory effect on the trypsinogen-catalyzed hydrolysis of Z-LysONp; the proenzyme-catalyzed cleavage of Z - LysONp was followed spectrophotometrically at 360 nm (A~360=4.5 mM-'cm-'; see Ascenzi et al., 1980; Antonini

et al., 1984). The value of the intrinsic molar fraction of the benzamidine-bound (activating peptide) trypsinogen (TO) was calculated, for every free ligand concentration, from the experimental value of the apparent molar fraction of the inhibited proenzyme (F) determined at a fixed substrate concentration (i.e. [Z-LysONp]), taking into account the simple competition of benzamidine with Z-LysONp for the zymogen (Antonini et af., 1984), according to the following expression (Eqn (1)):

where K , (ranging between 6.1 x lop4 M and 2.2 x lop3 M; see Antonini et af., 1984) is the Michaelis constant for the system considered (TO = 1 refers to the completion of the process). The validity of Eqn (1) has been verified at different substrate concentrations; thus, at fixed free benzamidine concentration, values of To turned out to be independent of [Z-LysONp].

An average error value of f 5 % was chosen as the standard deviation to 7 and 70 values (Antonini et af., 1983, 1984).

Values of the association equilibrium constant for Kazal inhibitor and benzamidine binding to the (pro) enzyme (K,), both in the presence and in the absence of activating peptides, were determined from the depen- dence of yo on the free [ligand] (1.e. log [Kazal inhibitor] or log [benzamidine]; see Figs 1 and 2), (Bode, 1979), according to the following equation (Eqn (2)):

Yo = F‘/{ 1 + K,.[Z-LysONp]} (1)

Yo = [ligand]/{[ligand] + K; ’ } (2) An average error value of *8% was ascribed as the

standard deviation to K, or K;‘ values (Antonini et al., 1983, 1984).

Under all experimental conditions the following free reagent concentrations were employed: trypsinogen con- centration ranged between 1 .O x M and 1 .O x lop4 M; Kazal inhibitor concentration ranged between 3.2 x l op8 M and 1.7 x 1fY’ M; benzamidine concentration ranged

1 .o

0.75 - yo

0.5

0.25

0.0 -8 -7 -6 -5 -4 -3 -2 -1

log[Kazal inhibitor] (MI

Figure 1. Intrinsic molar fraction of Kazal inhibitor bound to trypsinogen ( Y O ) versus the free ligand concentration (log [Kazal inhibitor]), in theabsence ( * ) and in the presenceof 1 .OxlO-’M Ile- Val-Gly ( A ) , Ile-Val (v), Ile-Leu (0). Ile-Ala (0). Val-Val (m), Leu- Val ( 0 ) and Val-Leu (+), at pH 5.5 (Bis tris-HCI buffer, I = O . l M, 2 . 0 ~ 1 O-’ M CaCI’) and T=21 “C. The continuous lines are the best fitting curves calculated according t o Eqn (2) wi th the following Ka-’ values: no peptides, 1.1 x I O - ~ M; Ile-Val-Gly and Ile-Val, 1 . 6 ~ 1 0 - ~ M; Ile-Leu, 1 . 8 ~ 1 o - ~ M; Ile-Ala, 1 ,I x 1 0-4 M; Val-Val, 2.5 x 1 0-5 M; Leu-Val, 1.7 x 1 0-4 M; Val-Leu, 4.8 x 1 0-4 M. Average error values of *5% (for ,‘,values) and *8% (forK,-’ values) were evaluated as the standard deviation (see text). For further details, see text.

P. ASCENZI ET A L .

132 JOURNAL O F MOLECULAR RECOGNITION. VOL 1, N O 3, 1988 0 Heyden & Son Limited, 1988

1.0

0.75 - yo

0.5

0.25

0.0 0 +1 -5 -4 -3 -2 -1

log[benzamidine] (MI

Figure 2. Intrinsic molar fraction of benzamidine bound to trypsino- gen ( Y O ) versus the free ligand concentration (log [benzamidine]) in the absence (*) and in the presence of 2.0 M He-Val-Gly ( A ) , He-Val (v), Ile-Leu (o), Ile-Ala (O), Val-Val (m), Leu-Val ( 0 ) and Val-Leu (+), at pH 5.5 (Bistris-HCI buffer, 1=0.1 M; 2.OX10-’ M CaC12) and T=21 “C. The continuous lines are the best fitting curves calculated according t o Eqn (2) wi th the following Ka-’ values: no peptides, 4.3~10-’ M; Ile-Val-Glyand Ile-Val, 2 . 6 ~ 1 0 - ~ M; Ile-Leu, 4 . 0 ~ 1 0 - ~ M; Ile-Ala, 1.1 x 1 0-’ M; Val-Val, 4.5x 1 0-3 M; Leu-Val, 1.6 x 1 0-’ M; Val-Leu, 2 .8x lW’ M. Average error values of *5% (for values) and *8% (for K i ’ values) were evaluated as standard deviation (see text). For further details, see text.

between 2.0 x M and 1.0 M; activating peptide concentration ranged between 8.0 x l op6 M and 2.0 M (the latter value being the upper solubility limit for activating peptides in the used solvent system; see Ascenzi et al., 1985; Menegatti et af., 1985); Z-LysONp concentration ranged between 1.0 x

In order to prevent any catalytic effect of /?-trypsin, possibly formed from autoactivation of trypsinogen during the reaction time, 0.1 mol Kunitz inhibitor/mol trypsinogen was always added in the reaction mixtures according to Bode ( 1 979).

Calcium (chloride) was always present in the system; the 2 . 0 ~ lo-* M calcium concentration used does not affect K, values for Kazal inhibitor and benzamidine binding to the zymogen (Antonini et af., 1984; Ascenzi et al., 1985). On the other hand, calcium indeed affects binding of activating peptides to trypsinogen and to its binary adducts with strong ligands, this effect being maxi- mal at calcium concentrations exceeding I .O x M (Bode, 1979).

M and 1.0 x lo-* M.

All measurements were performed at pH 5.5 (Bis tris- HCI, I=O.1 M; see Ascenzi et af., 1985) and T=21 k0.5”C.

The spectrophotometric measurements were carried out with double beam spectrophotometers Varian Cary 21 9 and Jasco J-5 10.

The atomic coordinates used for computer graphics modeling of the (pro)enzyme adducts discussed have been recovered from the Brookhaven Protein Data Bank distribution tape (Bernstein e f al., 1977).

RESULTS AND DISCUSSION

Over the whole range of activating peptide concentration (up to 2.0 M), Kazal inhibitor and benzamidine binding to trypsinogen conforms to simple equilibria, as indicated

TRYPSINOGEN ACTIVATION

-

by curve fitting of the experimental data according to Eqn (2), reported in Figs 1 and 2. Moreover, as expected for simple systems, values of K, are independent of the proenzyme concentration.

The affinity of Kazal inhibitor and benzamidine for trypsinogen (as expressed in terms of K,) increases with activating peptide concentration (see Figs 3 and 4). According to the linkage relationship (Wyman, 1964), this finding means that the activating peptide(s) combin- ing site in the zymogen is functionally associated with Kazal inhibitor or benzamidine binding site (Ascenzi et al., 1985) in such a way that the affinity for one ligand is rendered stronger as a result of the binding of the other ligand. The simplest model (Wyman, 1964; Ascenzi et al., 1985) for such behaviour (see Figs 3 and 4) leads to the following expression (Eqn ( 3 ) ) :

log& = hi$- log {(KLIG (1 + KUNL [PI))/ (KIINL (1 +KLIG [Pl))J+log {KLIGIKUNLJ (3)

where C is a constant that corresponds to the K, value obtained in the absence of activating peptides, [PI is the free activating peptide concentration, and KuNL and K L ~ G are the apparent association equilibrium constants for activating peptide binding to the free trypsinogen and its adducts with Kazal inhibitor and/or benzamidine. Equa- tion (3) has been used to generate the curves shown in Figs 3 and 4 with the choice of parameters given in Tables 1 and 2; in spite of the uncertainty in the evaluation of the upper asymptotes, the agreement with experimental data is satisfactory (see Figs 3 and 4). Unfortunately, over the activating peptide concentration explored, the small effect of Val-Leu on K, values for benzamidine binding to trypsinogen impaired the fitting of data according to Eqn (3). and thus the calculation of corresponding KUNL and

+2

r" 1

-

4,-- " " " " " ' 1 -00 -5 -4 -3 -2 -1 0 +1

log[P] [M) Figure 3. Effect of Ile-Val-Gly ( A ) , Ile-Val (v, b; the latter from Ascenzi eta/ . (1 985)), Ile-Leu (0 ) . l ie-Ala (O),Val-Val (m), Leu-Val (a) and Val-Leu (+ ) concentration (log [P I ) on the association equilibrium constant (log Ka) for Kazal inhibitor binding to trypsino- gen. at pH 5.5 (Bistris-HCI buffer, I=o.1 M; 2.0X10-* M CaC12) and T=21"C. The symbol on the left ordinate ( * ) refers to the K, value obtained in the absence of peptides. The concentration of activating peptides is that of the free ligand (Ascenzi et a/. , 1985). The continuous lines are the best fitting curves calculated according to Eqn (3) wi th K U N L and KL~G values given in Table 1. The arrow indicates the endpoint approximating to the Ka value for Kazal inhibitor binding to the binary activating peptide/trypsinogen adduct; such a K, value is independent of the chemical sequence of the activating peptide (see Table 2 ) . Average error values of *8% (for K, values) and * lo% (for K U N L and KUG values) were evaluated as the standard deviation (see text as well as Antonini et a/. (1 983, 1984); Ascenzi eta / . (1 985)). For further details, see text.

+3 i"

Figure4 . Effect of Ile-Val-Gly (A), Ile-Val (v ) , Ile-Leu (0 ) . Ile-Ala (0), Val-Val (H), Leu-Val (a) and Val-Leu (+) concentration (log [PI) on the association equilibrium constant (log K,) for benzami- dine binding to trypsinogen, at pH 5.5 (Bistris-HCI buffer, I=O.l M,

2 . 0 ~ 10 M CaC12) and T=21"C. The symbol on the left ordinate (") refers t o the K, value obtained in the absence of peptides. The concentration of activating peptides is that of the free ligand (Ascenzi eta/., 1985). Thecontinuous linesarethe best fitting curves calculated according to Eqn (3) with KUNL and K L i ~ values given in Table 1 . The arrow indicates the endpoint approximating to the K, value for benzamidine binding to the binary activating peptide/ trypsinogen adduct; such a K, value is independent of the chemical sequence of the activating peptide (see Table 2). Because of the small effect of Val-Leu on K, values for benzamidine binding t o trypsinogen it was unreliable to fit adequately these data according to Eqn (3) and thus to evaluate the corresponding KUNL and KLIG values. Average error values of *8% (for K, values) and *lo% (for K U N L and K ~ G values) were evaluated as the standard deviation (see text as well as Antonini eta / . (1 983, 1984); Ascenzi et a/. (1985)). For further details, see text.

K L ~ G values (see Fig. 4). The simple behaviour of thermo- dynamic parameter profiles, shown in Figs 3 and 4, indicates the absence of competition, for the specificity binding site of the zymogen, between Kazal inhibitor, or benzamidine and the activating peptides, even at the highest concentrations (Antonini et al., 1984; Ascenzi et al., 1985; Menegatti et al., 1985).

As already reported in the literature (Bode and Huber, 1976; Bode, 1979; Ascenzi et a/., 1987), the overall activation of trypsinogen (as measured by the increase in affinity of Kazal inhibitor and benzamidine for the proenzyme, induced by Ile-Val-Gly, Ile-Val, Ile-Leu, Ile- Ala, Val-Val, Leu-Val and Val-Leu) is independent of the amino acid sequence of the activating peptide (see Figs 3 and 4). In accordance with this observation, and in spite of their different intrinsic affinities (see Table l), Ile-Val and Val-Val have been reported to bring about almost identical structural effects on binding to the zymogen binary adducts with the native and with the semisynthetic Arg3 15 Kunitz inhibitor respectively (Bode and Huber, 1976; Bode et al., 1978, 1984; Huber and Bode, 1978). This behaviour may be taken as an excellent molecular example of conformational change(s), which provides regulation through specific recognition. In fact, closely related peptides are much less efficient with respect to Ile- Val (-Gly) (see Table 1, and Figs 3 and 4). the difference being probably due to a cavity or cavities not adequately filled by the peptides because of the structural relaxation in the zymogen to fill the unoccupied hole (Bode et al., 1984) (see Fig. 5):

0 Heyden & Son Limited, 1988 JOURNAL OF MOLECULAR RECOGNITION, VOL. 1 , No 3,1988 133

P. ASCENZI ET AL.

Table 1. Thermodynamic parameters

Activating peptide Free proenzyme

Ile-Val-Gly Trypsinogend

Trypsinogen'

Trypsinogend Ile-Val

Trypsinogen' Trypsinogen'

He-Leu

He-Ala

Val-Val

Leu-Val

Trypsinogend

Trypsinogen' Trypsinogend

Trypsinogen'

Trypsinogend

Trypsinogen'

Trypsinogend

Trypsinogen' Val-Leu Trypsinogend

for activating peptide association to free trypsinogen and proenzyme: strong ligand binary adducts" AG'UNL~ Proenzyme binary adduct AG'UNL'

K U N L ~ w - ' ) (kcal/rnol) (Trypsinogenllisted compound) K L I G ~ (M-') (kcallmol)

1 .ox 10' -1.3 Kazal in h i bitord 7 . 5 ~ l o 4 -6.5 pGBe 2.1 x 104 -5.8

1.2 x 10' -1.4 benzamidine' 2.0x102 -3.1 Kunitz inhibitoPh 1 . 3 ~ 1 O5 -6.8

1.1 x 10' -1.4 Kazal inhibitord 8 . 3 ~ 1 0 ~ -6.6 Kazal inhibitorh 9.1 x 104 -6.6 pGBe,h 2.0~104 -5.8

9.8 -1.3 benzamidine' 1 . 7 ~ 1 0 ~ -3.0 1.OxlO' -1.3 Z-ArgONp' 6 .3~10 ' -2.4

Z-Arg' 6 .3~10 ' -2.4 7.9~10- ' +1.4x lo- ' Kazal inhibitord 5.9~103 - 5.0

pG Be 1.3 x 1 O3 -4.2 7 .6~10- ' +1.6 x lo- ' benzamidine' 1 . 3 ~ 10' -1.5 1 . 3 ~ l o - ' +1.2 Kazal inhibitord 9 .8~10 ' -4.0

pG Be 1 . 3 ~ 1 0 ~ -2.9 1.1 x10-' +1.3 benzam id i ne' 1.9 -3.7 x l o - '

Kunitz inhibitoPh 7.1 xi03 -5.1

Kazal inhibitorh 6.2 x 103 -5.1 pGBe,h 7.1 x 10' -3.8

5 .6~10- ' +3.4x l o - ' benzam id i ne' 9.5 -1.3 Kunitz inhibitors 1 . 3 ~ 1 O3 -4.2

9.ox 10-2 +1.4 Kazal inhibitord 6.8 x 1 O2 -3.8 pG Be 1.1 x102 -2.7

6 . 0 ~ 1 O-' +1.6 benzamidine' 1 .o 0.0 2.0x 10-2 +2.3 Kazal inhibitord 1.5 x 1 0' -2.9

pGBe 2 .5~10 ' -1.9

6.1 x lo - ' +2 .9~10- ' Kazal inhibitord 4.6 x 1 O3 -4.9

a Conditions: 2.0x 1 o-' M CaC12. An average error value of * lo% was evaluated for KuNLand KLIG values as the standard deviation (for details, see text and Ascenzi eta/ . (1 985)).

Values of KUNL and K L ~ G were determined by curve fitting from Eqn (2) concerning the activating effect of peptides considered on K, values for

Values of K L ~ G were determined by spectrophotometric titrations at pH=7.5 (0.2 M Tris-HCI buffer) and T=20"C (from Bode, 1979).

A G " I J N L = - ~ . ~ ' ~ O ~ KuNL*RT; L\GD~~~=-2.3*10g KLIG'RT; (1 kcal=4184 J).

Kazal inhibitor binding to trypsinogen; data were obtained at pH=5.5 and T=21 "C (see Fig. 3).

' Values of KUNL and KLIG were determined by curve fitting from Eqn (2) concerning the activating effect of peptides considered on K, values for benzamidine binding to trypsinogen; data were obtained at pH 5.5 and T=21"C (see Fig. 4). g Values of KLIG were determined by competition experiments at pH=8.0 (0.1 M Tris-HCI buffer) and T=20"C (from Bode, 1979).

Values of K L ~ G weredetermined byspectrophotometric titrations at pH=7.4 ( I = O . l M, Tris-HCI buffer) and T=21 "C (from Ascenzi eta/ . , 1987). ' Values of KUNL and KLIG were determined by the activating effect of He-Val on the trypsinogen catalyzed hydrolysis of Z-ArgONp at pH=8.0 (I=O.l M Tris-HCI buffer) and T=21"C (from Menegatti eta / . , 1985).

Table 2. Thermodynamic parameters for Kazal inhibitor and benzamidine association to free trypsinogen, activating peptide: trypsinogen adducts and B-trypsin"

System Kab ( M - l ) AG",' (kcallmol)

Trypsinogen/Kazal inhibitor Activating peptide/trypsinogen/Kazal inhibitore fi-TrypsinlKazal inhibitor Trypsinogen/benzamidine Activating peptide/trypsinogen/benzamidinee /I-Trypsin/benzamidine

8.8 x 1 OZd 6.8 x 1 06d 1 . 8 ~ 1 08' 2.3 x 1 O l d 4.0 x 1 OZd 5.0 x 1 049

-3.9d -9.ld

-1 1.1' -1 .Sd -3.5d -6.39

a Conditions: I=O. l M, Bis tris-HCI buffer, pH=5.5; 2 . 0 ~ lo- ' M CaC12; T=21'C.

Ascenzi et a/. (1 985) ) . An average error value of *8% was evaluated for K, values as the standard deviation (for details, see text, as well as Antonini eta/ . (1 983,1984);

AGSa=-2.3.log Ka.RT (1 kcal=4184 J). Present paper. Values of K, and AG", have been obtained in the presence of saturating peptide concentrations, thermodynamic parameters being independent

of the chemical sequence of the activating peptide (for details, see text, Figs. 3 and 4, as well as Ascenzi eta / . (1 985)). ' From Antonini e t a / . (1983); Ascenzi e t a / . (1 985). 8 From Antonini et a/. (1984).

134 JOURNAL OF MOLECULAR RECOGNITION, VOL. 1, NO. 3,1988 0 Heyden & Son Limited, 1988

The isotherms shown in Figs 3 and 4 indeed put in evidence the different affinities of individual activating peptides (i.e., changes in KuNL and KLIG values; see Eqn (3)) for free trypsinogen as well as for its adducts with Kazal inhibitor and benzamidine (see Table 1); such affinities may be classified as follows: Ile-Val-Gly = Ile- Val > Ile - Leu > Val -Val > Ile - Ala > Leu - Val > Val - Leu (see Table I , and Figs 3 and 4). The identity of results obtained for Ile-Val-Gly and Ile-Val is in accordance with the absence of any interaction involving Gly18 of the activated enzyme and residues either of the Ile-Val binding pocket or of the proteinase surface (Bode and Schwager, 1975; Huber and Bode, 1978; Huber and Bennett, 1983) (Fig. 5). The preferential binding of activating peptides to the binary trypsinogen/inhibitor adducts (see Table 1) reflects: (i) the less structured state of the Ile-Val binding pocket in free trypsinogen as compared to its binary adducts with inhibitors and (ii) the lower degree of rigidity of the Ile-Val binding pocket

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TRYPSINOGEN ACTIVATION

0 Heyden & Son Limited, 1988 JOURNAL OF MOLECULAR RECOGNITION, VOL. 1, No. 3 , 1 9 8 8 135

Figure 5. Stereoviews of the Ile-Val binding pocket of ( B - ) trypsin(ogen) in the presence of the endogenous l lel6-Vall7-Glyl8 P-trypsin N-terminal polypeptide chain (a), as well as of the exogenous Ile-Val and Val-Val dipeptides (b and c, respectively). The Val-Val dipeptide has been identified as Val31 6-Val31 7 accord- ing to Bode eta/. (1 984). Atomic coordinates of the ( p - ) trypsin(o- gen) adducts shown were obtained from Bode and Schwager (1975), and Bodeeta/. (1978, 1984).

induced in the proenzyme by the arginine side chain of Z - Arg(0Np) as compared to that brought about by either benzamidine or p-guanidinobenzoate residue of pNpGB (all interacting at the primary specificity subsite (i.e., S,) of the zymogen), or else the multiple-subsite interactions of Kazal and Kunitz inhibitors (Huber and Bode, 1976; Bode et al., 1978, 1984; Huber and Bode, 1978; Bolognesi et al., 1982, 1988; Huber and Bennett, 1983).

In quantitative terms, the less favourable free energy of interaction of the other activating dipeptides with respect to Ile-Val-Gly and Ile-Val ranges between N 1.4 kcal/mol for Ile-Leu and N 3.9 kcal/mol for Val-Leu, as calculated from data reported in Table 1. However, on the basis of structural considerations (Bode and Huber, 1976; Bode et al., 1978, 1984; Huber and Bode, 1978), the more positive free energy for binding of other dipeptides as compared to Ile-Val(-Gly) does not find a fully satisfactory explana- tion in a smaller imperfect fitting to the hydrophobic contact area (into the Ile-Val cleft), but also in a less densely packed internal region. In this respect, the removal of a methyl group in the ternary adduct of trypsinogen with Val-Val (as compared to that with Ile- Val) and the semisynthetic Arg3 15 Kunitz inhibitor, leaves an empty cavity in the Ile-Val pocket only partially filled by slight rearrangements of neighbouring side chains, thus maintaining the overall structure of the binding site (Bode et al., 1984) (see Fig. 5).

As expected for simple systems, values of KUNL for activating peptide binding to free trypsinogen are very similar whether they have been obtained observing Kazal inhibitor or benzamidine binding (see Table 1). More- over, at least for some of the peptides investigated, values of K U N L and KLIG at pH 5.5 are in agreement with those previously obtained at different pH values (Menegatti et al., 1985; Ascenzi et al., 1987) (see Table 1). This evidence suggests that the activating peptides fit into the Ile-Val binding pocket making contacts with groups which do not ionize between pH 5.5 and 8.0. Further, it may be recalled that Ile-Val affinity for the trypsinogen/pGB adduct is also essentially pH-independent between pH 5.5 and 8.0 (Bode and Huber, 1976). This can be easily explained, as residues involved in controlling Ile-Val binding (i.e., Asp194 and the free amino group of the activating peptide) undergo the acid-base transition well outside this pH range (Bode and Huber, 1976).

Comparison of the peptide-linked increase of K, for Kazal inhibitor binding to trypsinogen (= 10 000-fold) with that of benzamidine (= 20-fold) (see Table 2) suggests, also on the basis of structural considerations (Bode and Schwager, 1975; Bode and Huber, 1976; Bode et al., 1978, 1984; Huber and Bode, 1978; Bode, 1979; Bolognesi et al., 1982, 1988; Huber and Bennett, 1983), that on peptide binding, trypsinogen undergoes acti- vation transition(s) which extends beyond the primary S, specificity subsite (the unique benzamidine binding site) to residues involved in interaction(s) with the Kazal inhibitor recognition loop. Thus, if one removes from the AGO terms in Table 2 the specific free energy contributions due to inhibitors (i.e., A (AGO) values calculated as AGO (activating peptide/trypsinogen/inhibitor) minus AGO (trypsinogen/inhibitor), the net peptide/trypsinogen free energies of interaction are obtained for the proenzyme/ Kazal inhibitor and proenzyme/benzamidine systems (- 5.2 kcal/mol and - 1.7 kcal/mol, respectively). The difference between these two values (n = - 3.5 kcal/mol)

P. ASCENZI ET A L

reflects the additional free energy gain associated with the specificity subsites (from SS to SZ and S; to S;) involved in macromolecular inhibitor recognition only. As expected from cooperatively-linked sites (Wyman, 1964), equiva- lent values of CJ can be obtained from data in Table 1, which refer to the reverse of the thermodynamic pro- cess(es) described above.

The overall energy balance for the association of the two kinds of ligands (i.e., activating peptides and Kazal inhibitor, Kunitz inhibitor, benzamidine, pNpGB or Z - Arg (ONp)) to trypsinogen allows us to calculate the so- called coupling free energy between binding free energies of activating peptides (or strong ligands) to the free zymogen and to its binary complexes with inhibitors or substrates. From values of AGO given in Tables 1 and 2 it is possible to evaluate the coupling free energy of the two trypsinogen ligands, which ranges between N - 5.8 and ‘v - 1 .O kcal/mol for the Leu-Val/zymogen/Kunitz in- hibitor and the Ile-Val/proenzyme/Z-Arg(0Np) system, respectively. The negative values indicate that the two ligands promote each other’s binding stabilizing equiva- lent conformation(s) (i.e. three-dimensional structures of trypsinogen reminiscent of that of P-trypsin; see Huber and Bode, 1978). Although typical values ofcoupling free energy are usually not larger than 2 kcal/mol (Weber, 1975) for small structural changes of protein conforma- tion hardly detectable by x-ray analysis, in the trypsino- gen system the largest values reflect remarkable structural changes on ligand binding (involving 15% of the whole molecule) (Bode and Huber, 1976; Bode et al., 1978, 1984; Huber and Bode, 1978; Bode, 1979; Bolognesi et al., 1982, 1988; Huber and Bennett, 1983).

In spite of the large values in the coupling free energy, binding of the activating peptide alone does not yield thermodynamic parameters for Kazal inhibitor or benza- midine binding as favourable as in the case of active P- trypsin (even under saturating conditions of peptides) (see Table 2). Such results find support in crystallographic observations which show that activating peptides bring about rigidification of the zymogen’s activation domain not as complete as that induced by the endogenous N- terminal of the enzyme (Bode and Schwager, 1975; Bode and Huber, 1976; Bode et al., 1978, 1984; Huber and Bode, 1978; Bode, 1979; Bolognesi et al., 1982, 1988; Huber and Bennett, 1983).

As a whole, data reported here allow the following considerations. (i) Binding of one or two ligands to the Ile-Val pocket and/or to the active center of the proen- zyme brings about different activation states in the zymogen, none of which are functionally fully equivalent to that of P-trypsin, even at saturating peptide concentra- tions. Such conformations, characterized by different flexibility degrees and activity levels, may indeed corres- pond to different (activation) states involved in the trypsinogen-to-P-trypsin transition, which should be con- sidered as a multistep rather than an ‘all or nothing’ process (Perkins and Wuthrich, 1980; Huber and Bennett, 1983; Ascenzi et al., 1985; Menegatti et al., 1985). (ii) The maximum change in the inhibitor affinity of trypsinogen, induced by the effector peptides, appears to be indepen- dent on their binding energy; this suggests the existence of a specific information relay that triggers the conforma- tional modification, a sort of a preferential propagation of the signal already observed in haemoglobin (Fermi and Perutz, 198 1). Such a finding agrees with the evidence that chemical links to the activation domain of trypsinogen bring about rigid P-trypsin-like structures (Butz et al., 1982). (iii) From the energetic viewpoint, specificity appears to be related to short-range interactions and therefore to depend upon close approach and molecular shape. Since no group in the trypsinogen/PSTI interface appears to be by itself overwhelmingly important, it comes out that other inhibitors of the same family differing by a single structural feature can also be bound (e.g. porcine versus bovine PSTI; Amiconi et al., 1988); therefore, from a thermodynamic standpoint, molecular recognition cannot be perfect mostly because of its physical origin.

Finally, these results may assume a general significance in the serine proenzyme activation mechanism(s). In fact, among other systems, present findings may be taken as representative of the molecular and functional model for the bovine chymotrypsinogen-to-cr-chymotrypsin and porcine pancreatic proelastase-to-elastase transitions in view of the high homology between the serine (pro)en- zymes considered and of the equivalence between the Ile- Val and Val-Val activating dipeptides and the Ilel6-Val17 and Val16-Val17 N-terminals of the active enzymes (Hartley and Shotton, 1971; Huber and Bode, 1978; Tsukada and Blow, 1985; Wang et al., 1985; Sawyer et al., 1978).

REFERENCES

Amiconi, G., Ascenzi, P., Bolognesi, M., Guarneri, M., and Mene- gatti, E. (1 987). Enthalpy-entropy compensation phenomena in serine (pro)enzyme: inhibitor complex formation. Adv. Bio- sciences 65, 177.

Amiconi, G., Ascenzi, P., Bolognesi, M., Menegatti, E., and Guarneri, M. (1 988). Interaction between serine (pro) enzymes and macromolecular inhibitors. Thermodynamic and kinetic aspects. In Macromolecular Biorecognition: Principles and Methods ed. by Chaiken, 1. M., Chiancone, E., Fontana, A,, and Neri, P. p. 11 7. The Humana Press, Clifton.

Antonini, E., Ascenzi, P., Bolognesi, M., Gatti, G., Guarneri, M., and Menegatti, E. (1 983). Interaction between serine (pro)enzymes and Kazal and Kunitz inhibitors. J. Mol. Biol. 165, 543.

Antonini, E., Ascenzi, P., Bolognesi, M., Guarneri, M., Menegatti, E., and Amiconi, G. (1 984). Catalytic and ligand binding properties of bovine trypsinogen and its complex with the effector dipep- tide He-Val. Mol. Cell. Biochem. 60, 163.

Ascenzi, P., Bertollini, A., Verzili, D., Brunori, M., and Antonini, E.

(1 980). The hydrolysis of cc-CBZ-L-lysine-p-nitrophenyl ester by two forms of human hurokinase. Anal. Biochem. 103, 235.

Ascenzi, P., Amiconi, G., Bolognesi, M., Menegatti, E., and Guarneri, M. (1985). Trypsin activation. Effect of the Ile-Val dipeptide concentration on Kazal inhibitor binding to bovine trypsinogen. Biochim. Biophys. Acta 832. 378.

Ascenzi, P., Amiconi, G., Bolognesi, M., Menegatti, E., and Guarneri, M. (1987). Binding of the Ile-Val and Val-Val effector dipep- tides to the binary adducts of bovine trypsinogen with Kunitz and Kazal inhibitors as well as the acylating agentp-nitrophenyl p-guanidinobenzoate. A thermodynamic and kinetic study. J. Mol. Biol. 194, 751.

Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meier jr, E. F., Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T., and Tasumi, M. (1977). The protein data bank: a computer-based archival file for macromolecular structures. J. Mol. Biol. 11 2, 535.

Bode, W. (1 979). The transition of bovine trypsinogen to a trypsin-

136 JOURNAL OF MOLECULAR RECOGNITION, VOL. 1, NO. 3,1988 0 Heyden & Son Limited, 1988

TRYPSINOGEN ACTIVATION

like state upon strong ligand binding. II. The binding of pancreatic trypsin inhibitor and of isoleucine-valine and of sequentially related peptides to trypsinogen and to p-guanidi- nobenzoate-trypsinogen. J. Mol. Biol. 127, 357.

Bode, W., and Schwager, P. (1 975). The refined crystal structure of bovine p-trypsin at 1.8 A resolution. I I . Crystallographic refine- ment, calcium binding site, benzamidine binding site and active site at pH 7.0. J. Mol. Biol. 98, 693.

Bode, W., and Huber, R. (1 976). Induction of the bovine trypsino- gen-trypsin transition by peptides sequentially similar to the N- terminus of trypsin. FEBS Lett. 68, 231.

Bode, W., Schwager. P., and Huber, R. (1978). The transition of bovine trypsinogen to a trypsin-like state upon strong ligand binding. The refined crystal structures of the bovine trypsino- gen-pancreatic trypsin inhibitor complex and of its ternary complex with Ile-Val at 1.9 8, resolution. J. Mol. Biol. 118, 99.

Bode, W., Walter, J., Huber, R., Wenzel, H. R., and Tschesche, H. (1 984). The refined 2.2 A (0.22-nm) X-ray crystal structure of the ternary complex formed by bovine trypsinogen, valine- valine and the Argl5 analogue of bovine pancreatic trypsin inhibitor. Eur. J. Biochem. 144, 185.

Bolognesi, M.. Gatti. G.. Menegatti, E., Guarneri, M., Marquart, M., Papamokos, E., and Huber, R. (1 982). Three-dimensional structure of the complex between pancreatic secretory trypsin inhibitor (Kazal type) and trypsinogen at 1.8 8, resolution. Structure solution, crystallographic refinement and preliminary structural interpretation. J. Mol. Biol. 162, 839.

Bolognesi, M., Ascenzi, P., Amiconi, G., Menegatti, E., and Guarneri, M. (1988). Structural bases for the recognition of inhibitors by serine proteinases and their zymogens. In Macromolecular Biorecognition: Principles and Methods ed. by Chaiken, I. M., Chiancone, E., Fontana. A., and Neri. P. p. 81. The Humana Press, Clifton.

Brunger, A. T., Huber, R., and Karplus, M. (1987). Trypsinogen- trypsin transition: a molecular dynamics study of induced conformational change in the activation domain. Biochemistry 26, 51 53.

Butz, T., Lerf, A., and Huber, R. (1982). Intramolecular reorienta- tional motion in trypsinogen studied by perturbed angular correlation of 199mHg labels. Phys. Rev. Lett. 48, 890.

Chase jr, T., and Shaw, E. (1 969). p-Nitrophenyl-p'-guanidinoben- zoate HCI: a new active site titrant for trypsin. Biochem. Biophys. Res. Commun. 29, 508.

Fermi, G.. and Perutz, M. F. (1 981 ). In Atlas of MolecularStructures in Biology. Vol. 2: Hemoglobin and Myoglobin ed. by Phillips, D. S., and Richards, F. M. Clarendon Press, Oxford.

Gertler, A., Walsh, K. A,, and Neurath, H. (1974). Catalysis by chymotrypsinogen. Demonstration of an acyl-zymogen inter- mediate. Biochemistry 13, 1302.

Greene, L. J., Di Carlo, J. J., Sussman, A. J., Bartelt, D. C., and Roark, D. E. (1968). Two trypsin inhibitors from porcine pancreatic juice. J. Biol. Chem. 243, 1804.

Hartley, B. S., and Shotton, D. M. (1971). In The Enzymes Vol. 3:

Pancreatic Elastase ed. by Boyer, P. D. p. 323. Academic Press, New York and London.

Hruska, J . F., Law, J. H., and KBzdy, F. J. (1 969). Differential titration of trypsin-like enzymes. Biochem. Biophys. Res. Commun. 36, 272.

Huber, R., and Bode, W. (1978). Structural basis of the activation and action of trypsin. Acc. Chem. Res. 11, 1 14.

Huber, R., and Bennett jr, W. S. (1983). Functional significance of flexibility in proteins. Biopolymers 22, 261.

Kassel, B. (1 970). Bovine trypsin-kallikrein inhibitor (Kunitz inhibi- tor, basic pancreatic trypsin inhibitor, polyvalent inhibitor from bovine organs). Methods Enzymol. 19, 844.

Menegatti, E., Bortolotti, F., Minchiotti, L., and De Marco, A. (1 982). Isolation and characterization of a new form of the porcine pancreatic secretory trypsin inhibitor. Biochemical studies and high-resolution 'H-NMR. Biochim. Biophys. Acta 707, 50.

Menegatti, E., Guarneri, M., Bolognesi, M., Ascenzi, P., and Amiconi, G. (1985). Activating effect of the Ile-Val dipeptide on the catalytic properties of bovine trypsinogen. Biochim. Biophys. Acta 832, 1.

Nolte, H. J., and Neumann, E. (1 979). Kinetics and mechanism for the conformational transition in p-guanidinobenzoate bovine trypsinogen induced by the isoleucine-valine dipeptide. Bio- phys. Chem. 10.25:.

Perkins, S. J., and Wuthrich, K. (1 980). Conformational transition from trypsinogen to trypsin. 'H nuclear magnetic resonance at 360 MHz and ring current calculations. J. Mol. Biol. 138, 43.

Sawyer, L., Shotton, D. M., Campbell, J. W., Wendell, P. L., Muirhead, H., Watzon, H. C., Diamond, R., and Ladner, R. C. (1 978). The atomic structure of crystalline porcine pancreatic elastase at 2.5 8, resolution: comparison with the structure of a-chymotrypsin, J. Mol. Biol. 118, 137.

Tsukada, H., and Blow, D. M. (1985). Structure of r-chymotrypsin refined at 1.68 8, resolution. J. Mol. Biol. 184, 703.

Vincent, J.-P., and Lazdunski, M. (1 976). Pre-existenceof the active site in zymogens, the interaction of trypsinogen with the basic pancreatic trypsin inhibitor (Kunitz). FEBS Lett. 63, 240.

Wang, D., Bode, W.. and Huber. R. (1 985). Bovine chymotrypsino- gen. X-ray crystal structure analysis and refinement of a new crystal form at 1.8 8, resolution. J. Mol. Biol. 185, 595.

Weber, G. (1975). Energetics of ligand binding to proteins. Adv. Protein Chem. 29, 1.

Weber, K., Pringle, J. R., and Osborn, M. (1972). Measurements of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol. 26. 3.

Wunsch, E. (1974). In Houben- Weyl, Methoden der Organischen Chemie Vol. 1511 : Abspaltung der N-Benzyloxycarbonyl- Schutzgruppe durch Hydrogenolyse. p. 51. Georg Thieme Verlag, Stuttgart.

Wyman, J. (1964). Linked functions and reciprocal effects in hemoglobin: a second look. Adv. Protein Chem. 19, 223.

Received 9 April 1988; accepted 25 June 1988.

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