Bovine trypsinogen activation. A thermodynamic study

The N-alpha-L-isoleucyl-L-valine (Ile-Val) activating dipeptide, sequentially homologous to the Ile 16-Val 17 N-terminus of bovine beta-trypsin, displays an activating effect on equilibria involved in the binding of strong ligands (i.e., n-butylamine and the porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, type I; PSTI)) to bovine trypsinogen. This property has been investigated between pH 3.0 and 9.0 (I = 0.1 M) at 21.0 degrees C. The thermodynamics for the interaction of strong ligands with bovine beta-trypsin has also been studied under the same experimental conditions. The equilibria involved in the binding of the Ile-Val activating dipeptide and/or inhibitors to bovine beta-trypsin and its zymogen are described according to linkage relationships, wherefore interaction(s) between different functional and structural domains of the (pro)enzyme (i.e., the so-called Ile-Val pocket and the primary and/or secondary recognition subsite(s)), possibly involved in the bovine trypsinogen-to-beta-trypsin activation pathway, are considered.     

Thermodynamic modeling of internal equilibria involved in the activation of trypsinogen

The effect of activating dipeptides, sequentially homologous to the Ile16-Val17N-terminus of bovine beta-trypsin (beta-trypsin), on equilibria involved in the binding of strong ligands (i.e., n-butylamine, the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor; BPTI) and the porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, type I; PSTI)) to bovine trypsinogen (trypsinogen) was investigated at pH 5.51 (I = 0.1 M) and T = 21.0 +/- 0.5 degrees C; under the same experimental conditions, thermodynamics for the binding of strong ligands to beta-trypsin was also obtained. The equilibria involved in the binding of activating dipeptides and/or inhibitors to beta-trypsin and to its zymogen are described according to an induced-fit formalism, taking into account ligand-linked interaction(s) between different functional and structural domains of the (pro)enzyme possibly involved in the trypsinogen-to-beta-trypsin activation pathway. The analysis of data is focussed on parameters describing interactions between the so-called Ile-Val pocket (where the Ile16-Val17 N-terminus of beta-trypsin or activating dipeptides bind) and the primary and/or secondary recognition subsite(s) (where strong ligands associate) present in the (pro)enzyme. Such an analysis allows to dissect the contributions due to the primary recognition subsite, where small mono-functional ligands (e.g., n-butylamine) bind, from those of the secondary subsite(s), which are additional recognition clefts for macromolecular inhibitors (e.g., BPTI and PSTI).     

Zymogen activation: effect of peptides sequentially related to the bovine beta-trypsin N-terminus on Kazal inhibitor and benzamidine binding to bovine trypsinogen

The activating effect of peptides sequentially related to the Ile 16-Val17-Gly18 N-terminus of bovine beta-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 at pH 5.5 (Bis tris-HCl buffer, I = 0.1 M) and T = 21 +/- 0.5 degrees 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 beta-trypsin/Kazal inhibitor and bovine beta-trypsin/benzamidine 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 beta-trypsin suggest the presence of different activation levels of the proenzyme, none of which structurally coincide with that achieved in bovine beta-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.     

Trypsin activation. Effect of the Ile-Val dipeptide concentration on Kazal inhibitor binding to bovine trypsinogen

The effect of Ile-Val concentration (up to 2.0 M) on the thermodynamic parameters for the binding of the porcine pancreatic secretory trypsin inhibitor (Kazal inhibitor) to trypsinogen has been investigated at pH 5.5 between 7 degrees C and 42 degrees C. Thermodynamic parameters for Kazal inhibitor binding to the Ile-Val:zymogen adduct are more favorable than those observed for inhibitor association to the free proenzyme, but less so than those reported for beta-trypsin:Kazal inhibitor adduct formation (even under saturating dipeptide concentrations), suggesting that the effector dipeptide does not induce a complete rigidification of the proenzyme's activation domain. Considering the dependence of the association equilibrium constant for Kazal inhibitor binding to trypsinogen from Ile-Val concentration, thermodynamic parameters for the effector dipeptide binding to the free proenzyme and to its binary complex with Kazal inhibitor have been obtained. Differences in affinity for Ile-Val binding to the free zymogen and its binary complexes with inhibitors and substrates are indicative of the presence of different activation levels of the proenzyme, none of them exactly coincident with that of beta-trypsin. Such different discrete states should correspond to those involved in the zymogen-to-active-enzyme transition which should not be considered as an all-or-nothing process, but as a multistep event.     

Binding of the trypsin inhibitor from white mustard (Sinapis alba L.) seeds to bovine beta-trypsin: thermodynamic study

The effect of pH and temperature on the association equilibrium constant (Ka) for the binding of the trypsin inhibitor from white mustard (Sinapis alba L.) seeds (MTI) to bovine beta-trypsin (EC 3.4.21.4) has been investigated. On lowering the pH from 9 to 3, values of Ka for MTI binding to bovine beta-trypsin decrease thus reflecting the acid-pK and -midpoint shifts, upon inhibitor association, of two independent ionizable groups, and of a three-proton transition, respectively. At pH 8.0, values of thermodynamic parameters for MTI binding to bovine beta-trypsin are: Ka = 4.5 X 10(8)M-1, delta G0 = -11.6 kcal/mol, and delta S0 = +53 entropy units (all at 21 degrees C); and delta H0 = +4.1 kcal/mol (temperature independent between 5 degrees C and 45 degrees C). Binding properties of MTI to bovine beta-trypsin have been analyzed in parallel with those concerning macromolecular inhibitor association to serine (pro)enzymes.     

Catalytic and ligand binding properties of bovine trypsinogen and its complex with the effector dipeptide Ile-Val

Steady-state and pre-steady-state kinetic data for the trypsinogen catalyzed hydrolysis of a series of synthetic substrates (i.e. p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids) have been obtained as a function of pH (3.4-8). Moreover, the effect of ethylamine on the hydrolysis of a neutral substrate and benzamidine binding have been extensively studied. In order to obtain direct information on the transition of trypsinogen to a beta-trypsin-like structure, the role of the effector dipeptide Ile-Val on the catalytic and ligand binding properties of the zymogen has been investigated. Kinetic and thermodynamic data for beta-trypsin and alpha-chymotrypsin are also reported for the purpose of an homogeneous comparison of the various (pro)enzymes. Under all the experimental conditions, kinetic data for (pro)enzyme catalysis are consistent with the minimum three-step mechanism: (formula; see text) involving the acyl intermediate E X P. In the presence of Ile-Val dipeptide, trypsinogen assumes catalytic and ligand binding properties that are reminiscent of activated beta-trypsin. This is at variance with free trypsinogen, which shows a alpha-chymotrypsin-like behavior. The large differences in the results of kinetic and thermodynamic measurements for free trypsinogen, as compared to its binary adduct with Ile-Val, can be ascribed to the substantial differences in the two molecular species, which include the spatial orientation of Asp189.     

Binding of the Ile-Val and Val-Val effector dipeptides to the binary adducts of bovine trypsinogen with Kunitz and Kazal inhibitors as well as the acylating agent p-nitrophenyl p-guanidinobenzoate. A thermodynamic and kinetic study

Thermodynamics and kinetics of binding of the Ile-Val and Val-Val effector dipeptides to the binary adducts of bovine trypsinogen with the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor), the porcine pancreatic secretory inhibitor (PSTI, Kazal inhibitor) and the acylating agent p-nitrophenyl p-guanidinobenzoate have been investigated at pH 7.4 and 21(+/- 0.5) degrees C. The affinity of both effector dipeptides for bovine trypsinogen: BPTI and bovine trypsinogen: PSTI binary adducts is higher than that observed for the formation of the dipeptide: bovine trypsinogen: p-guanidinobenzoate ternary complexes; moreover, the affinity of Ile-Val for the zymogen binary adducts is higher than that observed for Val-Val association. Binding of Ile-Val and Val-Val to the bovine trypsinogen binary complexes conforms to the induced-fit model, which consists of a fast pre-equilibrium followed by intramolecular isomerization change(s), the latter fast pre-equilibrium followed by intramolecular isomerization change(s), the latter representing the rate-limiting first-order process. For the three bovine trypsinogen systems considered, the rate of the intramolecular isomerization change(s) is essentially independent of the nature of the dipeptide and of the proenzyme binary complex.     

Binding of the soybean Bowman-Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to bovine alpha-chymotrypsin and bovine beta-trypsin. A thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C(p), F-T(p) and F-T(t), respectively) to bovine alpha-chymotrypsin (alpha-chymotrypsin) and bovine beta-trypsin (beta-trypsin) has been investigated. On the basis of Ka values, the proteinase inhibitor affinity can be arranged as follows: alpha-chymotrypsin: BBI approximately beta-trypsin:BBI approximately beta-trypsin:F-T(t) approximately beta-trypsin:F-T(p) much greater than alpha-chymotrypsin:F-C(p). F-C(p), F-T(p) and F-T(t) do not inhibit beta-trypsin and alpha-chymotrypsin action, respectively. On lowering the pH from 9.5 to 4.5, values of Ka for BBI, F-C(p), F-T(p) and/or F-T(t) binding to alpha-chymotrypsin and beta-trypsin decrease, thus reflecting the acid-pK shift of the invariant His57 catalytic residue from 7.0, in the free enzymes, to 5.2, in the proteinase:inhibitor complexes. Considering the known molecular models, the observed binding behaviour of BBI, F-C(p), F-T(p) and F-T(t) was related to the inferred stereochemistry of the proteinase:inhibitor contact regions.     

Kinetics of binding of bovine trypsin-killikrein inhibitor (K unitz) in which the reactive-site peptide bond Lys-15--Ala-16 is cleaved, to alpha-chymotrypsin and beta-trypsin.

Equilibrium measurements of the binding of reactive-site-cleaved (modified) bovine trypsin-kallikrein inhibitor (Kunitz) to alpha-chymotrypsin and beta-trypsin show a stoichiometric 1:1 association with high binding constants. At least in the case of chymotrypsin much evidence is presented that the reaction with modified inhibitor leads to the same complex as the reaction with virgin inhibitor does. The association rate constant of modified inhibitor with chymotrypsin at pH 7, 22.5 degrees C is 15.8 M-1 S-1. This is about 2 x 10(4) times slower than the binding of virgin inhibitor to that enzyme. In the analogous reaction of modified inhibitor with beta-trypsin, however, the association rate constant (1.2 x 10(4) M-1 s-1 at pH 6.9, 22.5 degrees C) is of about the same order of magnitude as it is in the reaction of virgin inhibitor and trypsin. These and analogous phenomena observed in the reactions of virgin and modified soybean trypsin inhibitor (Kunitz) with alpha-chymotrypsin and beta-trypsin suggest that the specificity of both inhibitors to trypsin is strongly reflected in the association rate constants of the modified forms. The dissociation rate constants of the complexes of trypsin-kallikrein inhibitor with chymotrypsin or with trypsin towards the modified inhibitor are estimated to be unmeasurably slow (half-life times of 45 or 1.5 x 10(4) years, respectively).     

Binding of the Kunitz-type trypsin inhibitor DE-3 from Erythrina caffra seeds to serine proteinases: a comparative study.

The effect of pH and temperature on kinetic and thermodynamic parameters (i.e., k(on),k(off),Ka,delta G0, delta H0 and delta S0 values) for the binding of the Kunitz-type trypsin inhibitor DE-3 from Erythrina caffra seeds (ETI) to bovine beta-trypsin, bovine alpha-chymotrypsin, the human tissue plasminogen activator, human alpha-, beta- and gamma-thrombin, as well as the M(r) 33,000 and M(r) 54,000 species of the human urinary plasminogen activator (also named urokinase) has been investigated. At pH 8.0 and 21.0 degrees C: (i) values of the second-order rate constant (K(on)) for the proteinase:ETI complex formation vary between 8.7 x 10(5) and 1.4 x 10(7)/M/s; (ii) values of the dissociation rate constant (k(off)) for the proteinase: ETI complex destabilization range from 3.7 x 10(-5) to 1.4 x 10(-1)/s; and (iii) values of the association equilibrium constant (Ka) for the proteinase:ETI complexation change from < 1.0 x 10(4) to 3.8 x 10(11)/M. Thus, differences in k(off) values account mostly for the large changes in Ka values for ETI binding. The affinity of ETI for the serine proteinases considered can be arranged as follows: bovine beta-trypsin > human tissue plasminogen activator > bovine alpha-chymotrypsin > human alpha-, beta- and gamma-thrombin approximately M(r) 33,000 and M(r) 54,000 species of the human urinary plasminogen activator. Moreover, the serine proteinase:ETI complex formation is an endothermic, entropy-driven, process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamic and structural properties of the calcium binding site of bovine serine proteases and their zymogens. A multinuclear nuclear magnetic resonance and stopped-flow study

The combined use of 43Ca and 113Cd nuclear magnetic resonance (n.m.r.) has provided information on the structural and dynamic properties of the calcium binding site located in homologous positions in bovine beta-trypsin, alpha-chymotrypsin and their zymogens. The 43Ca and 113Cd n.m.r. chemical shifts are consistent with an octahedral symmetry of the binding site and with the substitution of one of the two carboxylate ligands present in trypsin(ogen) with a neutral ligand in chymotrypsin(ogen). The constancy of the 113Cd n.m.r. chemical shifts upon binding of the pancreatic trypsin inhibitor and/or the dipeptide Ile-Val to trypsinogen confirms that structural changes in the activation domain do not affect the calcium binding site. The exchange between bound and "free" (solvated) Ca2+ is slow on the 43Ca n.m.r. time-scale for trypsin(ogen), but falls in the intermediate exchange region for chymotrypsin(ogen). In trypsin, the Ca2+ off-rate was measured by stopped-flow making use of the calcium indicator 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid and was found to be 3(+/- 1) s-1. In chymotrypsin(ogen) the off-rates calculated from the 43Ca n.m.r. data are 70 s-1 and 350 s-1, respectively. The dynamic properties of the calcium binding site of serine (pro)enzymes have been related to the flexibility of the binding site itself and have been compared to those of other extracellular and intracellular calcium binding proteins.     

The transition of bovine trypsinogen to a trypsin-like state upon strong ligand binding. The refined crystal structures of the bovine trypsinogen-pancreatic trypsin inhibitor complex and of its ternary complex with Ile-Val at 1.9 A resolution

The complex formed by bovine trypsinogen and the pancreatic trypsin inhibitor crystallizes in large crystals isomorphous with trypsin-PTI² complex crystals Rühlmann et al. 1973. X-ray diffraction data to 1.9 Å resolution were collected in the absence and presence of Ile-Val dipeptide. Both trypsinogen complex structures have been crystallographically refined, using the refined trypsin-PTI complex Huber et al. 1974a as a starting model. The final R values are 0.25 and 0.26, respectively. The mean main-chain atom deviations between the three complex structures are about 0.15 Å. In contrast, the mean deviation between the complexed and the free trypsinogen Fehlhammer et al. 1977 is 0.28 Å, reflecting the influence of crystal packing and complexation. The trypsinogen component adopts a trypsin-like conformation upon PTI binding: The Asp194 side-chain turns around and the activation domain becomes rigid, forming the specificity pocket and the Ile16 binding cleft. The specific interactions between PTI and trypsin are also observed in the trypsinogen complex. As in free trypsinogen, the N-terminus including residues Val10 to Gly18 is mobile and sticks out into solution. Apart from the different arrangement of the N-termini in the two complexes, the only significant, but minor structural difference is the enhanced thermal mobility of the autolysis loop in the trypsinogen complex. Upon binding of the Ile-Val dipeptide, the autolysis loop becomes fixed as in the trypsin complex. The Ile-Val position is identical in the ternary and the trypsin complex.     

Protease inhibitors from the parasitic worm Parascaris equorum

Two proteic inhibitors (I and II) of serine proteases have been purified from the parasitic worm Parascaris equorum by affinity chromatography on immobilized trypsin followed by preparative electrophoresis. They have an apparent relative molecular mass of 9000 and 7000 as determined by gel filtration, a slightly acid isoelectric point (5.5 and 6.1) and a similar amino acid composition. Both inhibitors lack serine, methionine and tyrosine. They bind bovine trypsin extremely strongly with an association constant, Ka, larger than 10(9) M-1, and form a 1:1 complex with this protease. The Ka values for the binding to bovine chymotrypsin are approximately 3.3 X 10(8) M-1 (inhibitor I) and approximately 2 X 10(6) M-1 (inhibitor II). Inhibitor I interacts also with porcine elastase (Ka approximately 5 X 10(7) M-1), while inhibitor II is inactive towards this enzyme.     

Thermodynamic Modeling of Internal Equilibria Involved in the Activation of Trypsinogen

Abstract

The effect of activating dipeptides, sequentially homologous to the Ile 16-Val 17 N-terminus of bovine β-trypsin (β-trypsin), on equilibria involved in the binding of strong ligands (i.e., n-butylamine, the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor; BPTI) and the porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, type I; PSTI)) to bovine trypsinogen (trypsinogen) was investigated at pH 5.5 (I = 0.1 M) and T = 21.0 ± 0.5°C; under the same experimental conditions, thermodynamics for the binding of strong ligands to β-trypsin was also obtained. The equilibria involved in the binding of activating dipeptides and/or inhibitors to β-trypsin and to its zymogen are described according to an induced-fit formalism, taking into account ligand-linked interaction(s) between different functional and structural domains of the (pro)enzyme possibly involved in the trypsinogen-to-β-trypsin activation pathway. The analysis of data is focussed on parameters describing interactions between the so-called Ile-Val pocket (where the Ile16-Val17/V-terminus of β-trypsin or activating dipeptides bind) and the primary and/or secondary recognition subsite(s) (where strong ligands associate) present in the (pro)enzyme. Such an analysis allows to dissect the contributions due to the primary recognition subsite, where small mono-functional ligands (e.g., n-butylamine) bind, from those of the secondary subsite(s), which are additional recognition clefts for macromolecular inhibitors (e.g., BPTI and PSTI).     

Introduction of alpha-hydroxymethyamino acid residues in substrate specificity P1 position of trypsin inhibitor SFTI-1 from sunflower seeds retains its activity

In many complexes formed by serine proteinases and their inhibitors, the hydroxyl group provided by water molecule or by the inhibitor Ser residue is located close to the inhibitor P1-P1' reactive site. In order to investigate the role of this group, we synthesized analogues of trypsin inhibitor SFTI-1 isolated from the seeds of sunflower modified in P1 by alpha-hydroxymethylserine (HmSer) and both enantiomers of alpha-hydroxymethylvaline (HmVal). All the synthesized analogues inhibited bovine beta-trypsin and human leukocyte elastase. SFTI-1 analogues with HmVal and HmSer appear to be potent inhibitors of bovine beta-trypsin, whereas [Val5]SFTI-1 is practically inactive. Also trypsin inhibitory activity of [Ser5]SFTI-1 is significantly lower. Since the electrostatic interaction between protonated epsilon-NH2 group of the inhibitor P1 position and beta-carboxylate of trypsin Asp189 is the main driving force for interaction of both molecules, the results obtained are very interesting. We believe that these SFTI-1 analogues belong to a novel class of serine proteinase inhibitors.     

Heat stabilization produced by protein-protein association. A differential scanning calorimetric study of the heat denaturation of the trypsin-soybean trypsin inhibitor and trypsin-ovomucoid complexes.

The irreversible thermal denaturation of the association complexes of bovine beta-trypsin with soybean trypsin inhibitor or ovomucoid was observed with a differential scanning calorimeter. Association of trypsin with either inhibitor results in increased heat stability. The largest effect is observed with beta-trypsin and soybean trypsin inhibitor. At pH 6.7, first order rate constants (s-1) for denaturation at 72 degrees, determined at a heating rate of 10 degrees per min, are: beta-trypsin, 30 times 10-3; soybean trypsin inhibitor, 9 times 10-3; trypsin-soybean trypsin inhibitor complex, 0.4 times 10-3. Under equivalent conditions, rate constants for ovomucoid and trypsin-ovomucoid complex are 4 times 10-3 and 1 times 10-3 s-1, respectively. These changes in rate correspond to heat stabilization of trypsin equivalent to an increase of 16 and 9 degrees, respectively, in its observed denaturation temperature. Rate constants determined for beta-trypsin and trypsin-soybean trypsin inhibitor complex are independent of heating rate; those for soybean trypsin inhibitor and ovomucoid are a function of heating rate. This suggests that predenaturational conformational alterations may be important steps in the denaturation of the inhibitors. Activation energies for denaturation of the complexes and their components are all similar, averaging 70 kcal per mol. The large activation energies observed suggest that denaturation of the complexes is not rate-limited by their dissociation.     

Kinetic and thermodynamic analysis of leech-derived tryptase inhibitor interaction with bovine tryptase and bovine trypsin

The interaction of leech-derived tryptase inhibitor (LDTI) with bovine liver capsule tryptase (BLCT) and bovine trypsin has been studied using both thermodynamic and kinetic approaches. Several differences were detected: (i) the equilibrium affinity of LDTI for BLCT (Ka = 8.9 x 10(5) M(-1)) is about 600-fold lower than that for bovine trypsin (Ka = 5.1 x 10(8) M(-1)); (ii) LDTI behaves as a purely non-competitive inhibitor of BLCT, while it is a purely competitive inhibitor of bovine trypsin. These functional data are compared with those previously reported for the LDTI binding to human tryptase, where tight inhibition occurs at two of the four active sites of the tetramer (Ka = 7.1 x 10(8) M(-1)). Amino acid sequence alignment of BLCT, human betaII-tryptase and bovine trypsin allows us to infer some possible structural basis for the observed functional differences.     

Conformational changes of bovine beta-trypsin and trypsinogen induced by divalent ions: an energy-dispersive X-ray diffraction and functional study

The radius of gyration (R(g)) of bovine trypsinogen and beta-trypsin was measured by an energy-dispersive X-ray technique as a function of Ca(2+) or SO(4)(2-) concentration; these results have been supplemented with measurements of association equilibrium constants of Ca(2+) to its binding site(s) on both serine proteases and some of their adducts (with an effector and/or an inhibitor). As a whole, all information reported in the present work demonstrates that: (i) the strains exerted by different ions on these proteases produce diverse structural modifications; and (ii) at least in the case of Ca(2+), the changes in R(g) can be ascribed to the direct interaction of the binding site(s) on the protein matrix with the cation.     

Characterization of five new low-molecular-mass trypsin inhibitors from white mustard (Sinapis alba L.) seed.

Five new low-molecular-mass trypsin inhibitors belonging to the RTI/MTI-2 family were identified from white mustard (Sinapis alba L. ; MTI-2) seed. Purified MTI-2 consisted of a peptide mixture, displaying Ile or Arg at position 43, Trp or kynurenine (Kyn) at position 44, and C-terminal ragged ends. The occurrence of Ile or Arg at position 43 suggested that MTI-2 inhibitors originated from different genes. The presence of 5-oxo-proline (pyroglutamic acid; 5-oxoPro1) and Kyn44 reflected post-translational processing of the serine proteinase inhibitor. MTI-2 showed approximately 70% amino-acid identity with low-molecular-mass trypsin inhibitors isolated from oil rape (Brassica napus var. oleifera; RTI-III) seed and with serine proteinase inhibitors mapped in Arabidopsis thaliana chromosome II (ATTI). Furthermore, MTI-2 was homologous to brazzein, the sweet-tasting protein from Pentadiplandra brazzeana Baillon fruit ( approximately 30% amino-acid identity). Although snake-venom toxins showed a low amino-acid identity (< 20%) with MTI-2, RTI-III, and ATTI, some structurally relevant residues were conserved. The disulfide bridge pattern of MTI-2 (Cys5-Cys27, Cys18-Cys31, Cys42-Cys52, and Cys54-Cys57) corresponded to that of RTI-III and of snake-venom toxins, being different from that of brazzein. Therefore, protein similarity might be attributable to the three-dimensional arrangement rather than to the amino-acid sequence. Values of Ka for MTI-2 binding to bovine beta-trypsin (trypsin) and bovine alpha-chymotrypsin were 6.3 x 109 M-1 and 2.0 x 106 M-1, respectively, at pH 8.0 and 21.0 degrees C. Moreover, values of kon for MTI-2 binding to trypsin and of koff for the dissociation of the serine proteinase:inhibitor complex were 5.6 x 105 M-1.s-1 and 8.9 x 10-5 M-1.s-1, respectively, at pH 8.0 and 21.0 degrees C. Despite the heterogeneity of the purified inhibitor peptide mixture, the inhibition properties of the different MTI-2 inhibitors were indistinguishable.     

Conformational study of proteins by SAXS and EDXD: the case of trypsin and trypsinogen

The radius of gyration (Rg) of bovine trypsinogen and beta-trypsin was measured by an energy-dispersive X-ray technique (EDXD) and by small-angle X-ray scattering (SAXS), under different solvent conditions. Both techniques gave superimposable results. The experimental evidence demonstrated that: (1) no structural modifications and/or damage occurred during the data acquisition by EDXD; (2) at pH 4 the active enzyme has one class of chloride binding sites in common with the zymogen, whereas the latter protease shows an additional class able to reverse the effects on Rg induced by chloride at low concentration; and (3) the pH profile of the Rg of both proteases does not resemble at all the pH effect on beta-trypsin activity, a result in line with the finding that the electrical potentials induced by surface charge are small in absolute magnitude and produce no gradient across the active site.