J. Mol. Biol. (1992) 225, 177-184
Binding
of Hirudin
A Comparative
to Human
Kinetic
a, p and y-Thrombin
and Thermodynamic
Study-f
Paolo Ascenzi of Pharmaceutical Chemistry and Technology Via Pie&o Giuria 9, 10125 Turin, Italy
Depwtment
University
of Turin,
Gino Amiconi CNR, Center for Molecular Biology, Department of Biochemical Sciences University of Rome “La Sapienxa”, Piaxzale Aldo Moro 5, 00185 Rome, Italy
Massimo
Coletta,
Giulio
Lupidi
Department of Molecular, Cellular and Animal Biology, University of Camerino Via Filippo Camerini 6, 63032 Camerino (MC), Italy
Enea Menegatti Department of Pharmaceutical Sciences, University of Ferrara Via Scandiana 21, 44100 Ferrara, Italy
Silvia Onesti Blackett Laboratory, Imperial College Prince Consort Road, London S W7 2BZ, England
and Martin0
Bolognesil
Department of Genetics and Microbiology Section of Crystallography, University of Pavia Via Abbiategrasso 207, 27100 Pavia, Italy (Received 6 May 1991; accepted 7 January
1992)
Thermodynamic parameters for the binding of hirudin to human c(, /I and y-thrombin have been determined between pH 5.0 and 9.0, and from 10°C to 40°C; kinetic data for the association and dissociation of the proteinase-inhibitor complex were obtained at pH 7.5 and 21 “C. These results have been analysed in parallel with the inhibitor-binding properties of human CE,j? and y-thrombin for the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor; BPTI). For the purpose of an homogeneous comparison, values of the apparent association equilibrium constant for BPTI binding to human y-thrombin have been determined between pH 5.0 and 9.0, at 21 “C. The different binding behaviour of hirudin and BPTI with respect to human CI, /l and y-thrombin has been related to the inferred stereochemistry of the proteinase-inhibitor contact regions. In particular, whereas the /I and y-loops play an appreciable role in the stabilization of the enzyme-hirudin complexes, they contribute to impairment of the adduct formation for the proteinase/BPTI system.
Keywords: human
i This paper on the occasion
is dedicated of his 70th
002%2836/92/090177-08
a, /I and y-thrombin; kinetics;
to Professor birthday.
$03.00/O
Mario
hirudin; proteinase-inhibitor thermodynamics
1 Author addressed.
Guarneri 177
to whom
complex
all correspondence
0
formation;
should
1992 Academic
be
Press Limited
178
1. Introduction Thrombin is a serine proteinase that plays a central role in the coagulation and thrombogenetic processes (see Fenton, 1986). Thus, besides catalysing the fibrinogen-to-fibrin conversion, thrombin activates (1) platelets, interacting with a receptor membrane-bound (see Harmon & Jamieson, 1985); (2) factor XIII, to stabilize fibrin polymers (see Janus et aZ.; 1983); and (3) protein C, to inhibit coagulation (see Esmon et al., 1982). Thrombin action is inhibited very strongly by hirudin, a highly specific protein proteinase inhibitor from the leech Hirudo medicinalis (see Seemiiller et al., 1986; Stone et al., 1987). Therefore, relevant information on the molecular mechanism of the proteinase inhibition, underlying the modulation of thrombin activity, can be gathered by a detailed analysis of the functional properties of the thrombin/hirudin system. Furthermore, the threedimensional structures of free human a-thrombin (a-thrombin’f) and of hirudin as well as of the proteinase-inhibitor complex have been solved (see Bode et al., 1989; Folkers et al., 1989; Griitter et al., 1990; Rydel et al., 1990; Bode & Huber, 1991), thus furnishing a molecular background for such a functional investigation. In order to gain better insight into the hirudin binding properties, the inhibitor interaction with a;, /3 and y-thrombin has been investigated from kinetic and thermodynamic viewpoints. The results have been analysed in parallel with the inhibitor-binding properties of serine (pro)enzymes, with particular reference to the a, p and y-thrombin/BPTI system (see Laskowski & Kato, 1980; Gebhard et al., 1986; Read & James, 1986; Amiconi et al.: 1988; Ascenzi et aZ., 1988, 1990; Bolognesi et al., 1988). For the purpose of an homogeneous comparison, values of thermodynamic parameters for BPTI binding to y-thrombin were obtained. Considering the known molecular models (see Huber & Bode, 1978; Boissel et al., 1984; Read & James, 1986; Bolognesi et al., 1988; Bode et al., 1989; Folkers et al., 1989; Griitter et al., 1990; Rydel et al., 1990: Bode & Huber, 1991), the different binding behaviour of hirudin and BPTI to CI, p and y-thrombin has been related to the inferred stereochemistry of the enzyme-inhibitor contact regions.
t Abbreviations used: ol-thrombin: human a-thrombin (the enzyme with the B chain intact (see Boissel et al.; 1984; Elion et d., 1986)); fir-thrombin, human Pr-thrombin (the enzyme cleaved at position 778-78 of the B chain (see Braun et al., 1988)); P-thrombin, human ,&thrombin (the enzyme cleaved at positions 67-68 and 778-78 of the B chain and lacking the /?-loop (see Boissel et al., 1984; Elion et al.; 1986)); y-thrombin, human y-thrombin (the enzyme cleaved at positions 67-68 and 778-78 as well as 126-127 and 149E-150 of the B chain and lacking both /I- and y-loops (see Boissel et al., 1984; Elion et al.; 1986)); BPTI, bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor); H-D-Phe-L-Pip-L-Arg-p-NA, n-phenylalanyl-n-pipecolylL-arginine p-nitroanilide (S-2238).
2. Materials and Methods Human a, ,!I and y-t,hrombin were prepared from commercial enzyme preparations (from Sigma Chemical Co., St. Louis, MO, U.S.A.) as described (see Elion et al., 1986). Hirudin was obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.) and further purified as reported elsewhere (see &one et aE., 1987). BPTI was purchased from Lepetit S.p.A. (Milano, I) and further purified as described (see Ascenzi et al.. 1988). The homogeneity of a: p and y-thrombin; hirudin and BPTI was checked by (1) SDS/polyacrylamide gel electrophoresis, in the absence and in the presence of /I-mercaptoethanol. and (2) N-terminal sequence determination (see Weber et al.; 1972: Elion et al., 1986; Stone et rcl.. 1987; Ascenzi et al.. 1988). The preparations used contained less than 30/b (w/v) of non-enzymic and/or non-inhibitory protein contaminants. The chromogenic substrate H-D-Phe-I-Pip-L%[N-morpholinolethanesulphonie Arg-p-NA (S-2238), acid, 1,3-bis[tris(hydroxymethyl)methylamino]propane and il:-tris[hydroxymet~hyl]methylglycine were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). All the other products were from Merck AG (Darmstadt, F.R,.G.). All chemicals were of analytical grade and used without further purification. The characterization of a; /? and y-thrombin, as well as of hirudin. BPTI and H-D-Phe-L-Pip-L-Arg-p-NA has been reported (see Elion et a,l.: 1986; Stone et al.. 1987; Ascenzi et al.. 198X). Values of the apparent second order combination rate constant (/c,,) and of the apparent dissociation rate constant (lc,,r) for the association and dissociation of the ry, /3 and y-thrombin-hirudin complexes were determined, at 21 “C, from the effect of the inhibitor concentration on the apparent first order rate constant of the enzymic hydrolysis of H-D-Phe-L-Pip-I-Arg-p-NA (see Stone & Hofsteenge, 1986; Stone et al.. 1987). Values of the apparent association equilibrium constant (K,) for hirudin binding to CI, p and y-t,hrombin, as well as for the y-thrombin-BPTI complex formation were determined, between 10°C and 40°C and at 21 “CT respectively, from t,he effect of t,he inhibitor concentration on the apparent steady-state velocity of the enzymic hydrolysis of H-D-Phe-L-Pip-L-Arg-p-i\;A (see Stone & Hofsteenge. 1986; Stone et al.. 1987; Ascenzi et al., 1988). Next,, values of K, for the CI. b and y-thrombin-hirudin complex formation were also calculated, at 21°C: from kinetic parameters (see Stone & Hofsteenge: 1986; Stone et al.. 1987). Values of the apparent free energy (AGO) for the proteinase-hirudin complex formation were calculated, at, 21 “C, from values of K, (see Keleti. 1983; Ascenzi et al., 1990). Values of the apparent enthalpy variation (AM’) accompanying the proteinase-hirudin complex formar?ion were determined from the linear dependence of log K, on T-’ by van’t Hoff plots; the temperature ranged between 10°C and 40°C. Values of AH0 were determined from K, values obtained at not, less t,han 10 temperatures between 10°C and 40°C (see Keleti, 1983; Ascenzi et aI.> 1990). Va,lues of the apparent entropy variation (AS’) for the proteina,seehirudin complex formation were calculated. at 21”C, from values of AC0 and AH0 (see Keleti, 1983; Ascenzi et al., 1990). A standard deviation of & 8 Y0 was evaluated for k,,, koff, K, (obtained experimentally) and AGo values, and of + 12% for K, (calculated from kinetic parameters), AH0 and AS0 values (see Ascenzi , 1988, 1990). Under all the experimental conditions, reagent concentration ranges were as follows: CI> p and y-thrombin, from 1.0 x IO-r2 M to 1.0 x IO-’ Y; hirudin, from 1.0 x lo- ‘i Y to 1.0 x 10m6 &I; BPTI, from 1.0 x 1O-5 M to 2.0 x lo-’ M;
Human
a, fl and y-Thrombin
and H-D-Phe-L-Pip-L-Arg-pNA, from 2.0 x 10m5 M to 1.0 x 10e3 M. For each experiment, at least 10 reagent (i.e. inhibitor) concentrations were considered. All data were obtained in 605 iv-%[N-morpholino] ethanesulphonic acid/NaOH buffer (pH 5.0 to 7.0), 005 Ml ,3-bis[tris[hydroxymethyl)methylamino]propane/HCl buffer (pH 6.0 to 9.0), and 0.05 M-N-tris[hydroxymethyl] methylglycine/HCl buffer (pH 7.0 to 9.0), in the presence of 91 M-NaCl and 0.1 y0 (w/v) polyethylene glycol 6000 (see Stone & Hofsteenge, 1986; Stone et al., 1987; Ascenzi et al., 1988). Control experiments with different buffers overlapping in pH showed no specific ion effects. Next, t(; fl and y-thrombin undergo completely reversible transition(s) over the pH range explored; indeed, the 3 enzyme species when brought back to neutral pH show complete recovery of their characteristic activity. Furthermore, a, p and y-thrombin were stable at the extremes of the pH range considered for a time longer than that necessary for the determination of the experimental quantities (145 min). Data
analysis
was
carried
out
on a Digital
PDP
to each
experimental
11/23
point.
on the fitted parameters was search of the parameter space, allowing only one parameter to vary for any single search (see Bevington, 1969). The detailed biochemical procedures have been published (see Keleti, 1983; Stone & Hofsteenge, 1986; Stone et al., 1987; Ascenzi et al., 1988, 1990).
3. Results and Discussion Under all the and BPTI binding
experimental conditions, to CI, fi and y-thrombin
179
by Hirudin
to a simple process (see also Ascenzi et al., 1988), as indicated also by the observation that the timecourse for the enzyme-inhibitor complex formation corresponds, for over 95 %, to a single exponential process. Furthermore, the binding isotherm for the proteinase-inhibitor complex formation always displays a Hill coefficient (n) equal to 1.00+@02. Next, values of K, obtained experimentally are in excelIent agreement with those calcuIsted from kinetic quantities (see Table 1). Moreover, values of kinetic and thermodynamic parameters are independent of the enzyme, inhibitor or substrate concentration. Whenever the comparison was possible, values of kinetic and thermodynamic parameters for hirudin and/or BPTI binding to CI, fl and y-thrombin are in good agreement with those taken from the literature (see Stone & Hofsteenge, 1986; Stone et al., 1987; Ascenzi et al., 1988). Next, hirudin displays similar kinetic and thermodynamic parameters for &-thrombin (the enzyme cleaved at site 778-78 of the B chain only; Braun et al., 1988: see Stone et al., 1987) and /I-thrombin (the enzyme cleaved at sites 67-68 and 778-78 of the B chain; see Boissel et al., 1984; Elion et al., 1986: and see Table 1 and Fig. 1 for comparison). This behaviour can be interpreted in terms of the structural location of the 68-778 undecapeptide of a-thrombin, which is, for a good part, in loop structure (i.e. p-loop). Upon cleavage the Arg77A-Asn78 peptide bond (in it-thrombin), this short fragment of t’he polypeptide B chain, as well as the residues following Asn78: is likely to increase its solvent exposure and
computer employing an iterative non-linear least-squares curve fitting procedure according to the Marquart algorithm, giving equal weight The standard deviation obtained by a systematic
Inhibition
hirudin conforms
Table 1 Values of k,,, k,,,,
K,,
AGo, AH0 and AS0 for the binding to CI, p and y-thrombin at pH 7.5
k a,b (&Y
COIXpleX
s-1)
k
3
Off 11’ (SG
cc-Thrombin-hirudin
1.1 X 109
1.3 X 10-S
j-Thrombin-hirudin
1.7 x 10’
3.1 x 1o-5
y-Thrombinhirudin
1.3 x 104
1.6 x 10m4
a-Thrombin-BPTI
’
40 x 104
7.0 x 10’
/l-Thrombin-BPTI
e
50 x 104
3.0 x 10’
y-Thrombin-BPTI
e
8.5 x 10“
1.0 x 10’
Kaa,b CM-‘) 8.3 x (8.5 x 56 x (5-4 x 7.9 x (8.1 x 1.2 x (1.5 x 25 x (32 x 91 x (1.1 x
10’3 1013)d 10” 10l’)d lo7 107)d 103 103)’ 103 103)’ lo3 104)’
AGO a, b (kcal mall’)
of hirudin
and BPTI
AHO a, E (kcal
mol-‘)
ASO =. b (e.n.)
- 18.6
-3.1
153
- 15.7
- 2.0
+47
- 10.6
0.0
+36
-4.1
f5.5
f33
- 4.5
+ 5.4
+34
-5.3
+52
+36
a A standard deviation of +S% was evaluated for k,,, .k,“, Ii, (obtained experimentally) and AGo values and of f 12% for K, (calculated from kinetic parameters), AH’ and AS’ values according to Ascenzi et al. (1988, 1990) (1 cal = 4.184 J). For further details, see the text. bValues of k,,, k,,,, K,, AGO and AS’ were obtained at 21 “C according to Keleti (1983), Stone & Hofsteenge (1986), Hofsteenge et al. (1987) and Ascenzi et al. (1988, 1990). For further details, see the text. ‘Values of AH’ were obtained from the effect of temperature on values of K, according to Keleti (1983) and Ascenzi et al. (1990); the temperature ranged between 5°C and 45°C. For further details, see the text. ‘Values of K, were calculated from kinetic parameters according to Stone & Hofsteenge (1986) and Stone etal. (1987). For further details, see the text. eFrom Ascenzi et al. (1988). f Values of K, were calculated from kinetic parameters according to Ascenzi et al. (1988). For further details, see the text.
P. Ascemi
180
PH
Figure 1. pH dependence of the apparent association equilibrium constant (K,; M-I) for hirudin binding to (0) I, (0) /I, and (A) y-thrombin, as well as for (0, +) BPTI association to y-thrombin at 21 “C. (+) The value of K, for the y-thrombin-BPTI complex formation at pH 7.5 was obtained from Ascenzi et ccl. (1988). The unbroken lines fitting the effect of pH on K, values for the c(, p and y-thrombin-hirudin complex formation were generated from eqn (1) with sets of parameters (i.e. C: pK&, p&o, values) given in Table 2. The unbroken pK&,, and pR[‘, line fit,ting the effect’ of pH on K, values for the y-thrombin-BPTI complex formation was generated from eqn (2) with the set of parameters (i.e. 6’; pKU,, and pKL,, values) given in Table 2. The unbroken lines were obtained with an iterative non-linear least-squares curve fitting procedure. A st)andard deviation of + 12% was evaluated for IO’, KU,,, I&, KG,, and KL,o values (see Ascenzi et aE., 1990). For further details, see the text.
acquire conformational flexibility, yielding a molecule that is quite comparable to the doubly proteolized fi-thrombin in terms of its interactions with macromolecular inhibitors (see Bode et al., 1989; Griitter et al., 1990; Rydel et al., 1990; Bode & Huber, 1991). The affinity of hirudin for a-thrombin is about two and six orders of magnitude higher t,han that for /3 and y-thrombin, respectively (see Table 1 and Fig. 1). In parallel, values of k,, for the a, p and y-thrombin-hirudin complex formation change from 1.1 x 10’ ZV-’ s-l to 1.3 x IO4 1~~~ se1 (see Table 1). Next, values of koff for hirudin dissociation from the a, j? and y-thrombin-inhibitor complexes are of the same order of magnitude (see Table 1). Thus, differences in k,, between CI, p and y-t’hrombin account for
et al.
the large affinity changes of hirudin binding, expressed by K, values (see Table I). The differences observed in K, and k,, values for the CI, ,L?and y-thrombin-hirudin complex formation may be related to differences in the extent of t,he proteinase B and y-loops interacting with the inhibitor. Thus, (1) a-thrombin, containing both fi and y-loops, displays the highest affinity (as measured by K, values) and the highest specificity (as quantified by k,, values) for hirudin; (2) P-thrombin, endowed with the y-loop only, shows an intermediate inhibitor affinity and specificity; and (3) y-thrombin, which lacks both the fi and the y-loops, shows the lowest K, and k,, values for hirudin. This behaviour suggests that the cleavage and removal of /j’ and/or y-loops from ol-thrombin brings about a structural perturbation of the extended proteinase area responsible for hirudin recognition and binding. Next, the small difference of the dissociation rate constants (i.e. koff values) for the a, ;O and y-thrombin-hirudin complex destabilization (I) indicates that the stability of the proteinaseinhibitor adducts is not significantly affected by the presence of /I and/or y-loops; and (2) suggests that common conformational change(s) might, represent the rate-limiting step for the kinetic control of the enzyme-hirudin complex dissociation. These functional observations are in keeping with t,he structural data (Griitter et al.: 1990; Rydel et al., 1990; Bode & Huber, 1991). In fact, the C-terminal tail of hirudin, dominated by negatively charged amino acid side-chains, interacts with cationic and apolar residues on the thrombin p and y-loops. In particular, relative to E-thrombin, three salt-bridges (between Asp55, Glu57 and Glu58 of hirudin and, respectively, Arg73, Arg75 and Arg77A of the proteinase) are lost in the I-thrombin-inhibitor complex; another salt-bridge (between Asp55 of hirudin and Lys149E of the proteinase) disappears in the y-thrombin-hirudin adduct. Even though on a smaller scale, the affinity of KPTT for the proteinase pursues a trend, which can be arranged as follows: y-thrombin > P-thrombin > x-thrombin (see Table 1; data from Ascenzi et al., 1988), that is in opposite direction relative to the series observed for hirudin (see Table 1 and Fig. I ). In parallel; values of Scofffor the dissociation of the X, p and y-thrombin-BPTI complexes change from 7.0 x 10’ s-l to 1.0 x 10’ s-l (see Table 1; data from Ascenzi et al., 1988); whereas; values of k,, for BPTI binding to a, fi and y-thrombin are closely similar (see Table 1; data from Ascenzi et al., 1988). Thus, contrary to what was observed for the x, ,i3 and y-thrombinlhirudin system (see Table l), differences in koff values for a, p and y-thrombin-BPTI complex destabilization mostly account for the affnity changes of the inhibitor binding, expressed by K, values (see Table 1; data from Aseenzi et al.? 1988). Variations in the proteinase-inhibitor complex affinity could be explained partially by the limited accessibilit,y of BPTI to the active site of M, /? and y-thrombin, as a consequence of the steric hindrance exerted (to different extent(s)) by the
Human
a, fi and y-Thrombin
proteinase j? and y-loops (see Ascenzi et al., 1988). Such a view is supported by the observation that the affinity and specificity of BPTI for a-thrombin is lower than that for the fi and y-proteolytic derivatives, in which the j and/or y-loops are cleaved off (see Ascenzi et al., 1988). Moreover, as suggested by the results of crystallographic studies (see Bode et al., 1989; Griitter et al., 1990; Rydel et al., 1990; Bode & Huber, 1991), the destabilizing effect of residues of the 60A-60D loop region, which protrude into the active-site area of the enzyme and thus strongly contact the inhibitor, should be considered. Thus, whereas the fl and y-loops play an appreciable role in the stabilization of the CC,p and y-thrombin hirudin complexes, they contribute to impairment of the complex formation for the proteinase/BPTI system. lt is of interest to point out that the CI, /? and y-thrombin-hirudin adduct formation is characterized by an exothermic enthalpy contribution that becomes less and less negative going from CI to y-thrombin, whereas the proteinase-BPTI complex formation is always endothermic, with values that are essentially independent of the thrombin species (i.e. of the presence of p and/or y-loops in the enzyme: see Table 1). This completely different behaviour underlines substantial differences for the two proteinase/inhibitor interaction mechanisms. Indeed, in the case of the CI, fi and y-thrombinl hirudin system, the exothermicity of complex formation implies the formation of bond(s), which strengthens the binding free energy and which directly involves the p and y-loops, as indicated by the effect of their removal (see Table 1). On the other hand, the endothermicity of the LX, fl and y-thrombin-BPTI complex formation is clearly not related to the presence of j? and y-loops in the serine proteinase (see Table 1). In addition, the positive values of AX0 (see Table 1) could reflect the increased degrees of freedom of water molecules related to the removal of the ordered solvent from the proteinase and/or the inhibitor during complex formation (see Amiconi et al., 1987; Ascenzi et al., 1988). Between pH 5.0 and 7.0, the effect of pH on the affinity of hirudin (i.e. on K, values) for LX, fl and y-thrombin is reminiscent of that observed for the binding of macromolecular inhibitors to serine (pro)enzymes (see Amiconi et al., 1988; Ascenzi et al., 1990), i.e. for the y-thrombin-BPTI complex formation (see Fig. 1). On the other hand, between pH 7.0 and 9.0, a unique opposite pH-effect modulating hirudin binding to ~1, fl and y-thrombin is observed (see Fig. l), this contribution being absent in the case of the y-thrombin-BPTI complex formation (see Fig. 1). The simplest mechanism (i.e. with the fewest ionization(s)) accounting for the observed data implies that (1) on increasing pH from 50 to 7.0, the increase of hirudin and BPTI affinity for CI, /l and/or y-thrombin reflects the acidic perturbation of a single ionizing group, upon inhibitor association; and (2) on increasing pH from 7.0 to 9.0, the decrease of K, values for ~1, p and y-thrombin-
Inhibition
181
by Hirudin
hirudin complex formation implies the alkaline pK shift of an additional ionizing residue upon complex formation. According to linkage relations (see Wyman, 1964; Amiconi et al., 1988; Ascenzi et al., 1990), the model holding for the CC, /l and y-thrombinlhirudin system leads to the following expression:
NH’1 +Gd logKa=
‘-log
x NH+1+G,, 1
([H+]+K;,,)x([H+]+K;l,,f (1)
where C is a constant that corresponds to the alkaline asymptote of log K,, and pK&,, pK&,, p&o and pK& are the pK values of the apparent proton dissociation equilibrium constants for the inhibitorfree (KuNL and KIhNL) and the inhibitor-bound (h& and K&o) proteinase, respectively. Next, the model holding for the y-thrombin/BPTI system leads to the following expression:
log K.3= ‘-log
NH+1+ KLd ([H+]+K$,) 2 (2)
i.e. equation (1) reduces to equation (2) (see Amiconi et al., 1988; Ascenzi et al., 1990). Equations (1) and (2) have been used to generate the unbroken lines shown in Figure 1 with sets of parameters (i.e. C, pK&, p&o, pK’&, and/or pK!io VdUeS) given in Table 2; the agreement with the experimental data is fully satisfactory. The analysis of the molecular models of CI, p and y-thrombin, of their primary structure homology, and of hirudin and BPTI (see Huber & Bode, 1978; Boissel et al., 1984; Read & James, 1986; Bolognesi et al., 1988; Bode et al., 1989; Folkers et aZ., 1989; Griitter et al., 1990; Rydel et al., 1990; Bode & Huber, 1991) suggests the assignment of the ionization(s) to both the enzyme and the inhibitor sides. Some idea about the chemical indentity of the ionizable group(s) controlling hirudin and BPTI binding to LX, /? and y-thrombin may be gained Srom values of pKuNL, pZ&., pK& and PKL (see Table 2), which appear to be closely related to those calculated from the pH dependence of: (1) the spectral properties of the proteinase; (2) kinetics for the enzymic hydrolysis of substrates; and (3) thermodynamics and kinetics for inhibitor binding (see Ascenzi et al., 1982, 1985; Menegatti et al., 1987, 1988; Amiconi et al., 1988). On the basis of these considerations, the inspection of the different (re)active site residues capable of affecting inhibitor binding to LX, /3 and y-thrombin suggests that the His57 residue, involved in the catalytic triad of the proteinase, and the His51 amino acid side-chain, present in the hirudin reactive area, have pK values comparable with those of as #?;NL and PK~NL. in free CC,b and y-thrombin,
P. Ascenxi
182
Values of C, pK;,,,
pKh, BPTI
Table 2 pGNL and pKL,,
PK;M. b b ’ c,d
---
for the binding
to CL,j3 and y-thrombin,
Gomplex a-Thrombin-hirudin j%Thrombin-hirudin y-Thrombin-hirudin y-Thrombin-BPTI
et al.
131 10.9 7.1 4.1
a
PG.,,
7.0 6.8 6.9 7.0
of hirudin
and
at 21°C
53 5.2 52 5.2
a
PGM.
a
6.9 7.0 7.0
PGG
a
8.4 e5 84
a A standard deviation of -t- 12% was evaluated for lo’, Ku,,, K&o, K’&,, and K;,, values according to Ascenzi et al. (1990). For further details, see the text. and pI&, were determined by curve fitting from eqn (1) bValues of C, pK;,,, pK’,,,, pK’& according to Ascenzi et al. (1990). Data in Fig. 1. For further details, see the text. ‘Values of C; pK&, and p&o were determined by curve fitting from eqn (2) according to Ascenzi et al. (1990). Data in Fig. 1. For further details, see the text. d Over the whole pH range explored (5.0 to 9.0), values of K, for BPTI binding to y-thrombin reflect the acidic perturbation of a single ionizing group, upon inhibitor association. For further details. see t,he text and Figure 1.
well as in free hirudin, respectively (see Table 2). In this respect, the calculated pK shift for the acidic proton binding group, modulating BPTI and hirudin binding to (x, p and/or y-thrombin, could be interpreted as reflecting: (1) the strengthening of the hydrogen bond between the His57 NE2 and the Ser195 OG atoms of y-thrombin, upon the proteinase-BPTI complex formation (see Amiconi et al., 1988; Bolognesi et al., 1988; Bode et al., 1989; Ascenzi et al., 1990); and (2) the burial of His57 in the enzyme-inhibitor complex with the concomitant formation of a hydrogen bond between the hirudin Ilel N atom and the proteinase His57 sidechain (see Griitter et al., 1990; Rydel et al., 1990; Bode & Huber, 1991). The Iv terminus of hirudin (the pK of which in the proteinase-free state is 2 8.4 (see Wallace et al., 1989)) is expected to be positively charged in the enzyme-inhibitor complexes, as observed in the a-thrombin-hirudin adduct by X-ray crystallography (see Bode & Huber, 1991). Therefore, His57 appears to be the only base available in the enzyme-hirudin adducts for the proton involved in the modulation of the proteinaseinhibitor interaction in the acidic pH region. However, due to the presence of the positive charge on the hirudin N-termina,l group in M, /? and y-thrombin-inhibitor complexes, the pK of His57 is expected to be lower than 5, the value observed in many ot’her serine proteinase-inhibit,or adducts (see Amiconi et al., 1988; Ascenzi et al., 1990) in which unfavourable electrical interaction(s) is absent. It follows that a local conformational change or insertion of water molecule(s) should occur at acidic pH in order to abolish or to minimize the electrical repulsion expected on the basis of the three-dimensional model of the a-thrombin-hirudin complex at neutral pH (see Griitter et al.> 1990; Rydel et al., 1990; Bode & Huber; 1991). Next, the calculated pK shift for the alkaline proton binding group, modulating hirudin binding to a, fl and y-thrombin, could be interpreted as reflecting the formation of the saltbridge between the hirudin His51 residue and the proteinase 61~39 carboxylate, upon complex formation (Griitter et al., 1990; Rydel et al., 1990; Bode &
Huber, 1991). However, a possible contribution to this effect from the ionization of the N-terminal group of hirudin cannot be excluded in the lower port,ion of the alkaline pH limb (see Fig. 1). The observed pH-effects on the a, p and y-thrombin-hirudin complex formation (see Fig. 1) are independent of the enzyme derivative (i.e. the tr, B and y-species). Indeed, the His57 residue and the Glu39 amino acid side-chain are present in both the proteolyzed derivatives of thrombin (see Boissel et al., 1984). In spite of the different inhibitor binding modes (see Bode et aZ., 1989; Griitter et al.; 1990; Rydel et al., 1990; Bode & Huber, 1991), the acidic pK shift of the His57 residue induced by hirudin association to LX, fl and y-thrombin is closely comparable with that observed for BPTI binding to homologous serine (pro)enzymes (see Amiconi et al., 1988; Ascenzi et al., 1990), as well as for the formation of the y-thrombin-BPTT complex (see Fig. I). This finding implies that equiva,lent functional effects (namely the pH-induced modulation of the proteinase-inhibitor affinity, expressed by K, may depend on different structural values) determinants. As a whole, the overall functional and structural differences observed in the interaction of a, a and y-Dhrombin with hirudin and BPTI are reflected in the inhibition mechanism, as suggested by the kinetic constants, which clearly indicate that the rate-determining factors in the (de)stabilizat,ion of the enzyme-hirudin and enzyme-BPTI complexes are different. This study has been supported of
by the Italian
Ministry
University,
Scientific Research and Technology (MURST), as well as by the Italian Xational Research Council (CNR): target oriented project Biotecno!ogie e Biostrumentazione and special project Peptidi Bioattivi.
References Amiconi, 6.. Aseenzi, I?.. Bolognesi, M., Guameri, M. Menegatti, E. (1987). Rnthalpy-entropy compensation phenomena in serine (pro)enzyme : inhibitor complex formation. Advan. Riosci. 65. 177-180.
bs
Human.
CI, /3 and y-Thrombin
Amiconi, G., Ascenzi, P., Bolognesi, M.: Menegatti, E. & Guameri, M. (1988). Interaction between serine (pro)inhibitors. enzymes and macromolecular Thermodynamic and kinetic aspects. In Macromolecular Biorecognition: Principles and Methods (Chaiken, I. M., Chiancone, E., Fontana, A. & Neri, P., eds), pp. 117-130, The Humana Press, Clifton. Ascenzi, I’., Menegatti, E., Guarneri, M., Bortolotti, F. & Antonini, E. (1982). Catalytic properties of serine proteases. 2. Comparison between human urinary kallikrein and human urokinase, bovine p-trypsin, bovine thrombin, and bovine a-chymotrypsin. Biochemistry, 21, 2483-2490. Ascenzi, P., Menegatti, E., Bolognesi, M., Guarneri, M. & Amiconi, G. (1985). Catalytic properties of bovine a-thrombin. A comparative steady-state and presteady-state study. Biochim. Biophys. Acta, 871, 319-323. Ascenzi, P., Coletta, M., Amiconi, G., De Cristofaro, R., Bolognesi, M., Guarneri, M. & Menegatti, E. (1988). Binding of the bovine basic: pancreatic trypsin inhibitor (Kunitz) to human CL,/I and y thrombin. A kinetic and thermodynamic study. Biochim. Biophys. Acta, 956, 156-161. Ascenzi, P., Coletta, M., Amiconi, G., Bolognesi, M., Menegatti, E. & Guarneri, M. (1990). Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor) to human and bovine factor Xa. A thermodynamic study. Biol. Chem. Hoppe Seyler, 371, 389-393. Bevington, P. R. (1969). Data Regulation and Error Analysis for the Physical Sciences, pp. 204-246, McGraw-Hill, New York. Bode, W. & Huber, R. (1991). Ligand binding. Proteinase-protein inhibitor interactions. Curr. Opirtion Struct. Biol. 1, 45-52. Bode, W., Mayr, I.: Baumann, U., Huber, R., Stone, S. R. & Hofsteenge, J. (1989). The refined 1.9 A crystal structure of human a-thrombin. Interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J. 8, 346773475. Boissel, J.-P., Le Bonniec, B., Rabiet, M.-J., Labie, D. & Elion, J. (1984). Covalent structures of @and y autolytic derivatives of human a-thrombin. J. Biol. Chem. 259, 5691-5697. Bolognesi, M., Ascenzi, P., Amiconi, G., Menegatti, E. & Guarneri, M. (1988). Structural bases for the recognition of inhibitors by serine proteinases and their zymogens. In Macromolecular Biorecognition: Principles and Methods (Chaiken, I. M., Chiancone, E., Fontana, A. & Neri, P., eds), pp. 81-100, The Humana Press, Clifton. Braun, P. J., Hofsteenge, J., Chang, J.-Y. & Stone, S. R. (1988). Preparation and characterization of proteolyzed forms of human a-thrombin. Thromb. Res. 50; 273-283. Elion, ,J., Boissel, J.-P., Le Bonniec, B.; Bezeaud, A., Jondrot-Perrus, M., Rabiet, M.-J. & Guillin, M.-C. (1986). Proteolytic derivatives of thrombin. Ann. N.Y. Aead. Sci. 485, 16-26. Esmon, N. L., Owen, W. G. & Esmon, C. T. (1982). Isolation of a membrane-bound cofactor for thrombin-catalyzed activation of protein C. J. Biol. Chem. 257: 859-864. Fenton, J. W., II (1986). Thrombin. Ann. N. Y. Acad. Sci. 485, 5-15. Folkers, P. J. M., Clore, G. M., Driscoll: P. C., Dodt, J.,
Inhibition
by Him&n
183
Kiihler, S. & Gronenborn, A. M. (1989). Solution structure of recombinant hirudin and the Lys47 + Glu mutant. A nuclear magnetic resonance and hybrid distance geometry dynamical simulated annealing study. Biochemistry, 28, 2601-2617. Gebhard, W.: Tschesche, H. & Fritz, H. (1986). Biochemistry of aprotinin and aprotinin-like inhibitors. In Proteinase Inhibitors (Barrett, A. J. & Salvesen, G., Elsevier, eds), PP. 375-388. Amsterdam, New York and Oxford. Griitter, M. G., Priestle, J. P., Rahuel, J.; Grossenbacher, H., Bode, W., Hofsteenge, J. & Stone, S. R. (1990). Crystal structure of the thrombin-hirudin complex. A novel mode of serine proteinase inhibition. EMBO J. 9; 2361-2365. Harmon, J. T. & Jamieson, G. A. (1985). Thrombin binds to a high-affinity 900000 dalton site on human platelets. Biochemistry, 24, 58-64. Huber, R. & Bode, W. (1978). Structural basis of the activation and action of trypsin. Act. Chem. Res. 11; 114-122. Janus, T. J., Lewis, S. D., Lorand, L. & Shafer, J. A. (1983). Promotion of thrombin-catalyzed act,ivation of factor XIII by fibrinogen. Biochemistry, 22, 626996272. Keleti, T. (1983). Errors in the evaluation of Arrhenius and van% Hoff plots. Biochem. J. 209, 277-280. Laskowski, M., Jr. & Kato, I. (1980). Protein inhibitors of proteinases. Annu. Rev. Biochem. 49, 593-626. Menegatti, E., Ferroni, R.; Scalia, S.; Guarneri, M., Bolognesi, M.; Ascenzi, P. & Amieoni, G. (1987). Inhibition of serine proteinases by tetra-p-amidinophenoxy-neo-pentane. Thermodynamic .and molecular modeling study. J. Enzyme Inhibit. 2, 23-30. Menegatti, E., Guarneri, M., Bolognesi, M., Aseenzi, I’. & Amiconi, G. (1988). Role of the primary specificity subsite on the interaction between serine proteinases and low molecular weight substrates and inhibitors. In Macromolecular Biorecognition: Principles and Methods (Chaiken, I. M., Chiancone, E., Fontana, A. &: Neri, P., eds), pp. lOlI115, The Humana Press, Clifton. R,ead, R. J. &James, M. N. G. (1986). Introduction to the protein inhibitors. X-ray crystallography. In Proteinase Inhibitors (Barrett, A. J. & Salvesen, G., eds), pp. 301-336, Elsevier, Amsterdam, New York and Oxford. Rydel, T. J., Ravichandran, K. G., Tulinsky, A.; Bode, W., Huber, R., Roitsch, C. & Fenton J. W., II (1990). The structure of a complex of recombinant hirudin and human a-thrombin. Science, 249, 277-280. Seemiiller, U., Dodt, J., Fink, E. & Fritz, H. (1986). Proteinase inhibitors of the leech Hirudo medicinalis (hirudins, bdellins, eglins). In Proteinase Inhibitors (Barrett; A. J. & Salvesen, G., eds), pp. 3377359, Elsevier, Amsterdam, New York and Oxford. Stone, S. R. & Hofsteenge, J. (1986). Kinetics of the inhibition of thrombin by hirudin. Biochemistry, 25, 4622-4628. Stone, S. R., Braun, P. J. & Hofsteenge, J. (1987). Identification of regions of a-thrombin involved in its Biochemistry, 26, with hirudin. interaction 4617-4624. Wallace, A., Dennis, S.; Hofsteenge, J. & Stone, S. R. (1989). Contribution of the N-terminal region of interaction with thrombin. hirudin to its Biochemistry, 28; 10079-10084.
184
P. Ascerk
Weber, K., Pringle, J. R. & Osborn, M. (1972). Measurements of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol. 26. 3-27. Edited
et al. Wyman, J. (1964). Linked functions a,nd reciprocal eiYects in hemoglobin. A second look. Advan. Protein Chem. 19, 223-286.
by R. Huber
Binding
of Hirudin
A Comparative
to Human
Kinetic
a, p and y-Thrombin
and Thermodynamic
Study-f
Paolo Ascenzi of Pharmaceutical Chemistry and Technology Via Pie&o Giuria 9, 10125 Turin, Italy
Depwtment
University
of Turin,
Gino Amiconi CNR, Center for Molecular Biology, Department of Biochemical Sciences University of Rome “La Sapienxa”, Piaxzale Aldo Moro 5, 00185 Rome, Italy
Massimo
Coletta,
Giulio
Lupidi
Department of Molecular, Cellular and Animal Biology, University of Camerino Via Filippo Camerini 6, 63032 Camerino (MC), Italy
Enea Menegatti Department of Pharmaceutical Sciences, University of Ferrara Via Scandiana 21, 44100 Ferrara, Italy
Silvia Onesti Blackett Laboratory, Imperial College Prince Consort Road, London S W7 2BZ, England
and Martin0
Bolognesil
Department of Genetics and Microbiology Section of Crystallography, University of Pavia Via Abbiategrasso 207, 27100 Pavia, Italy (Received 6 May 1991; accepted 7 January
1992)
Thermodynamic parameters for the binding of hirudin to human c(, /I and y-thrombin have been determined between pH 5.0 and 9.0, and from 10°C to 40°C; kinetic data for the association and dissociation of the proteinase-inhibitor complex were obtained at pH 7.5 and 21 “C. These results have been analysed in parallel with the inhibitor-binding properties of human CE,j? and y-thrombin for the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor; BPTI). For the purpose of an homogeneous comparison, values of the apparent association equilibrium constant for BPTI binding to human y-thrombin have been determined between pH 5.0 and 9.0, at 21 “C. The different binding behaviour of hirudin and BPTI with respect to human CI, /l and y-thrombin has been related to the inferred stereochemistry of the proteinase-inhibitor contact regions. In particular, whereas the /I and y-loops play an appreciable role in the stabilization of the enzyme-hirudin complexes, they contribute to impairment of the adduct formation for the proteinase/BPTI system.
Keywords: human
i This paper on the occasion
is dedicated of his 70th
002%2836/92/090177-08
a, /I and y-thrombin; kinetics;
to Professor birthday.
$03.00/O
Mario
hirudin; proteinase-inhibitor thermodynamics
1 Author addressed.
Guarneri 177
to whom
complex
all correspondence
0
formation;
should
1992 Academic
be
Press Limited
178
1. Introduction Thrombin is a serine proteinase that plays a central role in the coagulation and thrombogenetic processes (see Fenton, 1986). Thus, besides catalysing the fibrinogen-to-fibrin conversion, thrombin activates (1) platelets, interacting with a receptor membrane-bound (see Harmon & Jamieson, 1985); (2) factor XIII, to stabilize fibrin polymers (see Janus et aZ.; 1983); and (3) protein C, to inhibit coagulation (see Esmon et al., 1982). Thrombin action is inhibited very strongly by hirudin, a highly specific protein proteinase inhibitor from the leech Hirudo medicinalis (see Seemiiller et al., 1986; Stone et al., 1987). Therefore, relevant information on the molecular mechanism of the proteinase inhibition, underlying the modulation of thrombin activity, can be gathered by a detailed analysis of the functional properties of the thrombin/hirudin system. Furthermore, the threedimensional structures of free human a-thrombin (a-thrombin’f) and of hirudin as well as of the proteinase-inhibitor complex have been solved (see Bode et al., 1989; Folkers et al., 1989; Griitter et al., 1990; Rydel et al., 1990; Bode & Huber, 1991), thus furnishing a molecular background for such a functional investigation. In order to gain better insight into the hirudin binding properties, the inhibitor interaction with a;, /3 and y-thrombin has been investigated from kinetic and thermodynamic viewpoints. The results have been analysed in parallel with the inhibitor-binding properties of serine (pro)enzymes, with particular reference to the a, p and y-thrombin/BPTI system (see Laskowski & Kato, 1980; Gebhard et al., 1986; Read & James, 1986; Amiconi et al.: 1988; Ascenzi et aZ., 1988, 1990; Bolognesi et al., 1988). For the purpose of an homogeneous comparison, values of thermodynamic parameters for BPTI binding to y-thrombin were obtained. Considering the known molecular models (see Huber & Bode, 1978; Boissel et al., 1984; Read & James, 1986; Bolognesi et al., 1988; Bode et al., 1989; Folkers et al., 1989; Griitter et al., 1990; Rydel et al., 1990: Bode & Huber, 1991), the different binding behaviour of hirudin and BPTI to CI, p and y-thrombin has been related to the inferred stereochemistry of the enzyme-inhibitor contact regions.
t Abbreviations used: ol-thrombin: human a-thrombin (the enzyme with the B chain intact (see Boissel et al.; 1984; Elion et d., 1986)); fir-thrombin, human Pr-thrombin (the enzyme cleaved at position 778-78 of the B chain (see Braun et al., 1988)); P-thrombin, human ,&thrombin (the enzyme cleaved at positions 67-68 and 778-78 of the B chain and lacking the /?-loop (see Boissel et al., 1984; Elion et al.; 1986)); y-thrombin, human y-thrombin (the enzyme cleaved at positions 67-68 and 778-78 as well as 126-127 and 149E-150 of the B chain and lacking both /I- and y-loops (see Boissel et al., 1984; Elion et al.; 1986)); BPTI, bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor); H-D-Phe-L-Pip-L-Arg-p-NA, n-phenylalanyl-n-pipecolylL-arginine p-nitroanilide (S-2238).
2. Materials and Methods Human a, ,!I and y-t,hrombin were prepared from commercial enzyme preparations (from Sigma Chemical Co., St. Louis, MO, U.S.A.) as described (see Elion et al., 1986). Hirudin was obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.) and further purified as reported elsewhere (see &one et aE., 1987). BPTI was purchased from Lepetit S.p.A. (Milano, I) and further purified as described (see Ascenzi et al.. 1988). The homogeneity of a: p and y-thrombin; hirudin and BPTI was checked by (1) SDS/polyacrylamide gel electrophoresis, in the absence and in the presence of /I-mercaptoethanol. and (2) N-terminal sequence determination (see Weber et al.; 1972: Elion et al., 1986; Stone et rcl.. 1987; Ascenzi et al.. 1988). The preparations used contained less than 30/b (w/v) of non-enzymic and/or non-inhibitory protein contaminants. The chromogenic substrate H-D-Phe-I-Pip-L%[N-morpholinolethanesulphonie Arg-p-NA (S-2238), acid, 1,3-bis[tris(hydroxymethyl)methylamino]propane and il:-tris[hydroxymet~hyl]methylglycine were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). All the other products were from Merck AG (Darmstadt, F.R,.G.). All chemicals were of analytical grade and used without further purification. The characterization of a; /? and y-thrombin, as well as of hirudin. BPTI and H-D-Phe-L-Pip-L-Arg-p-NA has been reported (see Elion et a,l.: 1986; Stone et al.. 1987; Ascenzi et al.. 198X). Values of the apparent second order combination rate constant (/c,,) and of the apparent dissociation rate constant (lc,,r) for the association and dissociation of the ry, /3 and y-thrombin-hirudin complexes were determined, at 21 “C, from the effect of the inhibitor concentration on the apparent first order rate constant of the enzymic hydrolysis of H-D-Phe-L-Pip-I-Arg-p-NA (see Stone & Hofsteenge, 1986; Stone et al.. 1987). Values of the apparent association equilibrium constant (K,) for hirudin binding to CI, p and y-t,hrombin, as well as for the y-thrombin-BPTI complex formation were determined, between 10°C and 40°C and at 21 “CT respectively, from t,he effect of t,he inhibitor concentration on the apparent steady-state velocity of the enzymic hydrolysis of H-D-Phe-L-Pip-L-Arg-p-i\;A (see Stone & Hofsteenge. 1986; Stone et al.. 1987; Ascenzi et al., 1988). Next,, values of K, for the CI. b and y-thrombin-hirudin complex formation were also calculated, at 21°C: from kinetic parameters (see Stone & Hofsteenge: 1986; Stone et al.. 1987). Values of the apparent free energy (AGO) for the proteinase-hirudin complex formation were calculated, at, 21 “C, from values of K, (see Keleti. 1983; Ascenzi et al., 1990). Values of the apparent enthalpy variation (AM’) accompanying the proteinase-hirudin complex formar?ion were determined from the linear dependence of log K, on T-’ by van’t Hoff plots; the temperature ranged between 10°C and 40°C. Values of AH0 were determined from K, values obtained at not, less t,han 10 temperatures between 10°C and 40°C (see Keleti, 1983; Ascenzi et aI.> 1990). Va,lues of the apparent entropy variation (AS’) for the proteina,seehirudin complex formation were calculated. at 21”C, from values of AC0 and AH0 (see Keleti, 1983; Ascenzi et al., 1990). A standard deviation of & 8 Y0 was evaluated for k,,, koff, K, (obtained experimentally) and AGo values, and of + 12% for K, (calculated from kinetic parameters), AH0 and AS0 values (see Ascenzi , 1988, 1990). Under all the experimental conditions, reagent concentration ranges were as follows: CI> p and y-thrombin, from 1.0 x IO-r2 M to 1.0 x IO-’ Y; hirudin, from 1.0 x lo- ‘i Y to 1.0 x 10m6 &I; BPTI, from 1.0 x 1O-5 M to 2.0 x lo-’ M;
Human
a, fl and y-Thrombin
and H-D-Phe-L-Pip-L-Arg-pNA, from 2.0 x 10m5 M to 1.0 x 10e3 M. For each experiment, at least 10 reagent (i.e. inhibitor) concentrations were considered. All data were obtained in 605 iv-%[N-morpholino] ethanesulphonic acid/NaOH buffer (pH 5.0 to 7.0), 005 Ml ,3-bis[tris[hydroxymethyl)methylamino]propane/HCl buffer (pH 6.0 to 9.0), and 0.05 M-N-tris[hydroxymethyl] methylglycine/HCl buffer (pH 7.0 to 9.0), in the presence of 91 M-NaCl and 0.1 y0 (w/v) polyethylene glycol 6000 (see Stone & Hofsteenge, 1986; Stone et al., 1987; Ascenzi et al., 1988). Control experiments with different buffers overlapping in pH showed no specific ion effects. Next, t(; fl and y-thrombin undergo completely reversible transition(s) over the pH range explored; indeed, the 3 enzyme species when brought back to neutral pH show complete recovery of their characteristic activity. Furthermore, a, p and y-thrombin were stable at the extremes of the pH range considered for a time longer than that necessary for the determination of the experimental quantities (145 min). Data
analysis
was
carried
out
on a Digital
PDP
to each
experimental
11/23
point.
on the fitted parameters was search of the parameter space, allowing only one parameter to vary for any single search (see Bevington, 1969). The detailed biochemical procedures have been published (see Keleti, 1983; Stone & Hofsteenge, 1986; Stone et al., 1987; Ascenzi et al., 1988, 1990).
3. Results and Discussion Under all the and BPTI binding
experimental conditions, to CI, fi and y-thrombin
179
by Hirudin
to a simple process (see also Ascenzi et al., 1988), as indicated also by the observation that the timecourse for the enzyme-inhibitor complex formation corresponds, for over 95 %, to a single exponential process. Furthermore, the binding isotherm for the proteinase-inhibitor complex formation always displays a Hill coefficient (n) equal to 1.00+@02. Next, values of K, obtained experimentally are in excelIent agreement with those calcuIsted from kinetic quantities (see Table 1). Moreover, values of kinetic and thermodynamic parameters are independent of the enzyme, inhibitor or substrate concentration. Whenever the comparison was possible, values of kinetic and thermodynamic parameters for hirudin and/or BPTI binding to CI, fl and y-thrombin are in good agreement with those taken from the literature (see Stone & Hofsteenge, 1986; Stone et al., 1987; Ascenzi et al., 1988). Next, hirudin displays similar kinetic and thermodynamic parameters for &-thrombin (the enzyme cleaved at site 778-78 of the B chain only; Braun et al., 1988: see Stone et al., 1987) and /I-thrombin (the enzyme cleaved at sites 67-68 and 778-78 of the B chain; see Boissel et al., 1984; Elion et al., 1986: and see Table 1 and Fig. 1 for comparison). This behaviour can be interpreted in terms of the structural location of the 68-778 undecapeptide of a-thrombin, which is, for a good part, in loop structure (i.e. p-loop). Upon cleavage the Arg77A-Asn78 peptide bond (in it-thrombin), this short fragment of t’he polypeptide B chain, as well as the residues following Asn78: is likely to increase its solvent exposure and
computer employing an iterative non-linear least-squares curve fitting procedure according to the Marquart algorithm, giving equal weight The standard deviation obtained by a systematic
Inhibition
hirudin conforms
Table 1 Values of k,,, k,,,,
K,,
AGo, AH0 and AS0 for the binding to CI, p and y-thrombin at pH 7.5
k a,b (&Y
COIXpleX
s-1)
k
3
Off 11’ (SG
cc-Thrombin-hirudin
1.1 X 109
1.3 X 10-S
j-Thrombin-hirudin
1.7 x 10’
3.1 x 1o-5
y-Thrombinhirudin
1.3 x 104
1.6 x 10m4
a-Thrombin-BPTI
’
40 x 104
7.0 x 10’
/l-Thrombin-BPTI
e
50 x 104
3.0 x 10’
y-Thrombin-BPTI
e
8.5 x 10“
1.0 x 10’
Kaa,b CM-‘) 8.3 x (8.5 x 56 x (5-4 x 7.9 x (8.1 x 1.2 x (1.5 x 25 x (32 x 91 x (1.1 x
10’3 1013)d 10” 10l’)d lo7 107)d 103 103)’ 103 103)’ lo3 104)’
AGO a, b (kcal mall’)
of hirudin
and BPTI
AHO a, E (kcal
mol-‘)
ASO =. b (e.n.)
- 18.6
-3.1
153
- 15.7
- 2.0
+47
- 10.6
0.0
+36
-4.1
f5.5
f33
- 4.5
+ 5.4
+34
-5.3
+52
+36
a A standard deviation of +S% was evaluated for k,,, .k,“, Ii, (obtained experimentally) and AGo values and of f 12% for K, (calculated from kinetic parameters), AH’ and AS’ values according to Ascenzi et al. (1988, 1990) (1 cal = 4.184 J). For further details, see the text. bValues of k,,, k,,,, K,, AGO and AS’ were obtained at 21 “C according to Keleti (1983), Stone & Hofsteenge (1986), Hofsteenge et al. (1987) and Ascenzi et al. (1988, 1990). For further details, see the text. ‘Values of AH’ were obtained from the effect of temperature on values of K, according to Keleti (1983) and Ascenzi et al. (1990); the temperature ranged between 5°C and 45°C. For further details, see the text. ‘Values of K, were calculated from kinetic parameters according to Stone & Hofsteenge (1986) and Stone etal. (1987). For further details, see the text. eFrom Ascenzi et al. (1988). f Values of K, were calculated from kinetic parameters according to Ascenzi et al. (1988). For further details, see the text.
P. Ascemi
180
PH
Figure 1. pH dependence of the apparent association equilibrium constant (K,; M-I) for hirudin binding to (0) I, (0) /I, and (A) y-thrombin, as well as for (0, +) BPTI association to y-thrombin at 21 “C. (+) The value of K, for the y-thrombin-BPTI complex formation at pH 7.5 was obtained from Ascenzi et ccl. (1988). The unbroken lines fitting the effect of pH on K, values for the c(, p and y-thrombin-hirudin complex formation were generated from eqn (1) with sets of parameters (i.e. C: pK&, p&o, values) given in Table 2. The unbroken pK&,, and pR[‘, line fit,ting the effect’ of pH on K, values for the y-thrombin-BPTI complex formation was generated from eqn (2) with the set of parameters (i.e. 6’; pKU,, and pKL,, values) given in Table 2. The unbroken lines were obtained with an iterative non-linear least-squares curve fitting procedure. A st)andard deviation of + 12% was evaluated for IO’, KU,,, I&, KG,, and KL,o values (see Ascenzi et aE., 1990). For further details, see the text.
acquire conformational flexibility, yielding a molecule that is quite comparable to the doubly proteolized fi-thrombin in terms of its interactions with macromolecular inhibitors (see Bode et al., 1989; Griitter et al., 1990; Rydel et al., 1990; Bode & Huber, 1991). The affinity of hirudin for a-thrombin is about two and six orders of magnitude higher t,han that for /3 and y-thrombin, respectively (see Table 1 and Fig. 1). In parallel, values of k,, for the a, p and y-thrombin-hirudin complex formation change from 1.1 x 10’ ZV-’ s-l to 1.3 x IO4 1~~~ se1 (see Table 1). Next, values of koff for hirudin dissociation from the a, j? and y-thrombin-inhibitor complexes are of the same order of magnitude (see Table 1). Thus, differences in k,, between CI, p and y-t’hrombin account for
et al.
the large affinity changes of hirudin binding, expressed by K, values (see Table I). The differences observed in K, and k,, values for the CI, ,L?and y-thrombin-hirudin complex formation may be related to differences in the extent of t,he proteinase B and y-loops interacting with the inhibitor. Thus, (1) a-thrombin, containing both fi and y-loops, displays the highest affinity (as measured by K, values) and the highest specificity (as quantified by k,, values) for hirudin; (2) P-thrombin, endowed with the y-loop only, shows an intermediate inhibitor affinity and specificity; and (3) y-thrombin, which lacks both the fi and the y-loops, shows the lowest K, and k,, values for hirudin. This behaviour suggests that the cleavage and removal of /j’ and/or y-loops from ol-thrombin brings about a structural perturbation of the extended proteinase area responsible for hirudin recognition and binding. Next, the small difference of the dissociation rate constants (i.e. koff values) for the a, ;O and y-thrombin-hirudin complex destabilization (I) indicates that the stability of the proteinaseinhibitor adducts is not significantly affected by the presence of /I and/or y-loops; and (2) suggests that common conformational change(s) might, represent the rate-limiting step for the kinetic control of the enzyme-hirudin complex dissociation. These functional observations are in keeping with t,he structural data (Griitter et al.: 1990; Rydel et al., 1990; Bode & Huber, 1991). In fact, the C-terminal tail of hirudin, dominated by negatively charged amino acid side-chains, interacts with cationic and apolar residues on the thrombin p and y-loops. In particular, relative to E-thrombin, three salt-bridges (between Asp55, Glu57 and Glu58 of hirudin and, respectively, Arg73, Arg75 and Arg77A of the proteinase) are lost in the I-thrombin-inhibitor complex; another salt-bridge (between Asp55 of hirudin and Lys149E of the proteinase) disappears in the y-thrombin-hirudin adduct. Even though on a smaller scale, the affinity of KPTT for the proteinase pursues a trend, which can be arranged as follows: y-thrombin > P-thrombin > x-thrombin (see Table 1; data from Ascenzi et al., 1988), that is in opposite direction relative to the series observed for hirudin (see Table 1 and Fig. I ). In parallel; values of Scofffor the dissociation of the X, p and y-thrombin-BPTI complexes change from 7.0 x 10’ s-l to 1.0 x 10’ s-l (see Table 1; data from Ascenzi et al., 1988); whereas; values of k,, for BPTI binding to a, fi and y-thrombin are closely similar (see Table 1; data from Ascenzi et al., 1988). Thus, contrary to what was observed for the x, ,i3 and y-thrombinlhirudin system (see Table l), differences in koff values for a, p and y-thrombin-BPTI complex destabilization mostly account for the affnity changes of the inhibitor binding, expressed by K, values (see Table 1; data from Aseenzi et al.? 1988). Variations in the proteinase-inhibitor complex affinity could be explained partially by the limited accessibilit,y of BPTI to the active site of M, /? and y-thrombin, as a consequence of the steric hindrance exerted (to different extent(s)) by the
Human
a, fi and y-Thrombin
proteinase j? and y-loops (see Ascenzi et al., 1988). Such a view is supported by the observation that the affinity and specificity of BPTI for a-thrombin is lower than that for the fi and y-proteolytic derivatives, in which the j and/or y-loops are cleaved off (see Ascenzi et al., 1988). Moreover, as suggested by the results of crystallographic studies (see Bode et al., 1989; Griitter et al., 1990; Rydel et al., 1990; Bode & Huber, 1991), the destabilizing effect of residues of the 60A-60D loop region, which protrude into the active-site area of the enzyme and thus strongly contact the inhibitor, should be considered. Thus, whereas the fl and y-loops play an appreciable role in the stabilization of the CC,p and y-thrombin hirudin complexes, they contribute to impairment of the complex formation for the proteinase/BPTI system. lt is of interest to point out that the CI, /? and y-thrombin-hirudin adduct formation is characterized by an exothermic enthalpy contribution that becomes less and less negative going from CI to y-thrombin, whereas the proteinase-BPTI complex formation is always endothermic, with values that are essentially independent of the thrombin species (i.e. of the presence of p and/or y-loops in the enzyme: see Table 1). This completely different behaviour underlines substantial differences for the two proteinase/inhibitor interaction mechanisms. Indeed, in the case of the CI, fi and y-thrombinl hirudin system, the exothermicity of complex formation implies the formation of bond(s), which strengthens the binding free energy and which directly involves the p and y-loops, as indicated by the effect of their removal (see Table 1). On the other hand, the endothermicity of the LX, fl and y-thrombin-BPTI complex formation is clearly not related to the presence of j? and y-loops in the serine proteinase (see Table 1). In addition, the positive values of AX0 (see Table 1) could reflect the increased degrees of freedom of water molecules related to the removal of the ordered solvent from the proteinase and/or the inhibitor during complex formation (see Amiconi et al., 1987; Ascenzi et al., 1988). Between pH 5.0 and 7.0, the effect of pH on the affinity of hirudin (i.e. on K, values) for LX, fl and y-thrombin is reminiscent of that observed for the binding of macromolecular inhibitors to serine (pro)enzymes (see Amiconi et al., 1988; Ascenzi et al., 1990), i.e. for the y-thrombin-BPTI complex formation (see Fig. 1). On the other hand, between pH 7.0 and 9.0, a unique opposite pH-effect modulating hirudin binding to ~1, fl and y-thrombin is observed (see Fig. l), this contribution being absent in the case of the y-thrombin-BPTI complex formation (see Fig. 1). The simplest mechanism (i.e. with the fewest ionization(s)) accounting for the observed data implies that (1) on increasing pH from 50 to 7.0, the increase of hirudin and BPTI affinity for CI, /l and/or y-thrombin reflects the acidic perturbation of a single ionizing group, upon inhibitor association; and (2) on increasing pH from 7.0 to 9.0, the decrease of K, values for ~1, p and y-thrombin-
Inhibition
181
by Hirudin
hirudin complex formation implies the alkaline pK shift of an additional ionizing residue upon complex formation. According to linkage relations (see Wyman, 1964; Amiconi et al., 1988; Ascenzi et al., 1990), the model holding for the CC, /l and y-thrombinlhirudin system leads to the following expression:
NH’1 +Gd logKa=
‘-log
x NH+1+G,, 1
([H+]+K;,,)x([H+]+K;l,,f (1)
where C is a constant that corresponds to the alkaline asymptote of log K,, and pK&,, pK&,, p&o and pK& are the pK values of the apparent proton dissociation equilibrium constants for the inhibitorfree (KuNL and KIhNL) and the inhibitor-bound (h& and K&o) proteinase, respectively. Next, the model holding for the y-thrombin/BPTI system leads to the following expression:
log K.3= ‘-log
NH+1+ KLd ([H+]+K$,) 2 (2)
i.e. equation (1) reduces to equation (2) (see Amiconi et al., 1988; Ascenzi et al., 1990). Equations (1) and (2) have been used to generate the unbroken lines shown in Figure 1 with sets of parameters (i.e. C, pK&, p&o, pK’&, and/or pK!io VdUeS) given in Table 2; the agreement with the experimental data is fully satisfactory. The analysis of the molecular models of CI, p and y-thrombin, of their primary structure homology, and of hirudin and BPTI (see Huber & Bode, 1978; Boissel et al., 1984; Read & James, 1986; Bolognesi et al., 1988; Bode et al., 1989; Folkers et aZ., 1989; Griitter et al., 1990; Rydel et al., 1990; Bode & Huber, 1991) suggests the assignment of the ionization(s) to both the enzyme and the inhibitor sides. Some idea about the chemical indentity of the ionizable group(s) controlling hirudin and BPTI binding to LX, /? and y-thrombin may be gained Srom values of pKuNL, pZ&., pK& and PKL (see Table 2), which appear to be closely related to those calculated from the pH dependence of: (1) the spectral properties of the proteinase; (2) kinetics for the enzymic hydrolysis of substrates; and (3) thermodynamics and kinetics for inhibitor binding (see Ascenzi et al., 1982, 1985; Menegatti et al., 1987, 1988; Amiconi et al., 1988). On the basis of these considerations, the inspection of the different (re)active site residues capable of affecting inhibitor binding to LX, /3 and y-thrombin suggests that the His57 residue, involved in the catalytic triad of the proteinase, and the His51 amino acid side-chain, present in the hirudin reactive area, have pK values comparable with those of as #?;NL and PK~NL. in free CC,b and y-thrombin,
P. Ascenxi
182
Values of C, pK;,,,
pKh, BPTI
Table 2 pGNL and pKL,,
PK;M. b b ’ c,d
---
for the binding
to CL,j3 and y-thrombin,
Gomplex a-Thrombin-hirudin j%Thrombin-hirudin y-Thrombin-hirudin y-Thrombin-BPTI
et al.
131 10.9 7.1 4.1
a
PG.,,
7.0 6.8 6.9 7.0
of hirudin
and
at 21°C
53 5.2 52 5.2
a
PGM.
a
6.9 7.0 7.0
PGG
a
8.4 e5 84
a A standard deviation of -t- 12% was evaluated for lo’, Ku,,, K&o, K’&,, and K;,, values according to Ascenzi et al. (1990). For further details, see the text. and pI&, were determined by curve fitting from eqn (1) bValues of C, pK;,,, pK’,,,, pK’& according to Ascenzi et al. (1990). Data in Fig. 1. For further details, see the text. ‘Values of C; pK&, and p&o were determined by curve fitting from eqn (2) according to Ascenzi et al. (1990). Data in Fig. 1. For further details, see the text. d Over the whole pH range explored (5.0 to 9.0), values of K, for BPTI binding to y-thrombin reflect the acidic perturbation of a single ionizing group, upon inhibitor association. For further details. see t,he text and Figure 1.
well as in free hirudin, respectively (see Table 2). In this respect, the calculated pK shift for the acidic proton binding group, modulating BPTI and hirudin binding to (x, p and/or y-thrombin, could be interpreted as reflecting: (1) the strengthening of the hydrogen bond between the His57 NE2 and the Ser195 OG atoms of y-thrombin, upon the proteinase-BPTI complex formation (see Amiconi et al., 1988; Bolognesi et al., 1988; Bode et al., 1989; Ascenzi et al., 1990); and (2) the burial of His57 in the enzyme-inhibitor complex with the concomitant formation of a hydrogen bond between the hirudin Ilel N atom and the proteinase His57 sidechain (see Griitter et al., 1990; Rydel et al., 1990; Bode & Huber, 1991). The Iv terminus of hirudin (the pK of which in the proteinase-free state is 2 8.4 (see Wallace et al., 1989)) is expected to be positively charged in the enzyme-inhibitor complexes, as observed in the a-thrombin-hirudin adduct by X-ray crystallography (see Bode & Huber, 1991). Therefore, His57 appears to be the only base available in the enzyme-hirudin adducts for the proton involved in the modulation of the proteinaseinhibitor interaction in the acidic pH region. However, due to the presence of the positive charge on the hirudin N-termina,l group in M, /? and y-thrombin-inhibitor complexes, the pK of His57 is expected to be lower than 5, the value observed in many ot’her serine proteinase-inhibit,or adducts (see Amiconi et al., 1988; Ascenzi et al., 1990) in which unfavourable electrical interaction(s) is absent. It follows that a local conformational change or insertion of water molecule(s) should occur at acidic pH in order to abolish or to minimize the electrical repulsion expected on the basis of the three-dimensional model of the a-thrombin-hirudin complex at neutral pH (see Griitter et al.> 1990; Rydel et al., 1990; Bode & Huber; 1991). Next, the calculated pK shift for the alkaline proton binding group, modulating hirudin binding to a, fl and y-thrombin, could be interpreted as reflecting the formation of the saltbridge between the hirudin His51 residue and the proteinase 61~39 carboxylate, upon complex formation (Griitter et al., 1990; Rydel et al., 1990; Bode &
Huber, 1991). However, a possible contribution to this effect from the ionization of the N-terminal group of hirudin cannot be excluded in the lower port,ion of the alkaline pH limb (see Fig. 1). The observed pH-effects on the a, p and y-thrombin-hirudin complex formation (see Fig. 1) are independent of the enzyme derivative (i.e. the tr, B and y-species). Indeed, the His57 residue and the Glu39 amino acid side-chain are present in both the proteolyzed derivatives of thrombin (see Boissel et al., 1984). In spite of the different inhibitor binding modes (see Bode et aZ., 1989; Griitter et al.; 1990; Rydel et al., 1990; Bode & Huber, 1991), the acidic pK shift of the His57 residue induced by hirudin association to LX, fl and y-thrombin is closely comparable with that observed for BPTI binding to homologous serine (pro)enzymes (see Amiconi et al., 1988; Ascenzi et al., 1990), as well as for the formation of the y-thrombin-BPTT complex (see Fig. I). This finding implies that equiva,lent functional effects (namely the pH-induced modulation of the proteinase-inhibitor affinity, expressed by K, may depend on different structural values) determinants. As a whole, the overall functional and structural differences observed in the interaction of a, a and y-Dhrombin with hirudin and BPTI are reflected in the inhibition mechanism, as suggested by the kinetic constants, which clearly indicate that the rate-determining factors in the (de)stabilizat,ion of the enzyme-hirudin and enzyme-BPTI complexes are different. This study has been supported of
by the Italian
Ministry
University,
Scientific Research and Technology (MURST), as well as by the Italian Xational Research Council (CNR): target oriented project Biotecno!ogie e Biostrumentazione and special project Peptidi Bioattivi.
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by R. Huber
