Use of monoclonal antibodies to demonstrate different sites with different functional characteristics in a bacterial lipase from Pseudomonas aeruginosa YS-7.

Structural and functional features of the extracellular lipase from the low-water-tolerant bacterium Pseudomonas aeruginosa YS-7 were studied immunochemically with the aid of monoclonal antibodies (MAbs) raised against the enzyme. Fourteen different MAbs were obtained, verified as immunoglobulin G types, and characterized by their interaction with the enzyme in relation to (i) inhibition of activity of free enzyme, (ii) inhibition of activity of adsorbed enzyme, (iii) interaction with the cell-bound enzyme, and (iv) inhibition of adherence to hexadecane droplets. Four of the MAbs exhibiting the highest binding constants (Kapp greater than 10(8) M-1) were selected for further study of the lipase. Their binding to the enzyme was assayed by means of adapted enzyme-linked immunosorbent assay techniques. Use of these MAbs in single or dual binding procedures made it possible to reveal several distinct sites on the lipase macromolecule. Two of these are functional sites, one for hydrophobic adhesion (binds MAb 5) and the other (binds MAb 1) for implementation of its hydrolytic activity. A third binding site (binds MAb 8) does not participate directly in either of the above functions. A fourth binding site (binds MAb 10) appears to be involved in the active expression of the enzyme. The cell-associated form of the lipase seems to be located on the external surface of the cells with its active site exposed. It appears to be anchored to the outer membrane of the cells by means of its hydrophobic region in a way that resembles its adherence to hydrophobic surfaces such as hexadecane droplets.     

Use of monoclonal antibodies to demonstrate different sites with different functional characteristics in a bacterial lipase from Pseudomonas aeruginosa YS-7.

Structural and functional features of the extracellular lipase from the low-water-tolerant bacterium Pseudomonas aeruginosa YS-7 were studied immunochemically with the aid of monoclonal antibodies (MAbs) raised against the enzyme. Fourteen different MAbs were obtained, verified as immunoglobulin G types, and characterized by their interaction with the enzyme in relation to (i) inhibition of activity of free enzyme, (ii) inhibition of activity of adsorbed enzyme, (iii) interaction with the cell-bound enzyme, and (iv) inhibition of adherence to hexadecane droplets. Four of the MAbs exhibiting the highest binding constants (Kapp greater than 10(8) M-1) were selected for further study of the lipase. Their binding to the enzyme was assayed by means of adapted enzyme-linked immunosorbent assay techniques. Use of these MAbs in single or dual binding procedures made it possible to reveal several distinct sites on the lipase macromolecule. Two of these are functional sites, one for hydrophobic adhesion (binds MAb 5) and the other (binds MAb 1) for implementation of its hydrolytic activity. A third binding site (binds MAb 8) does not participate directly in either of the above functions. A fourth binding site (binds MAb 10) appears to be involved in the active expression of the enzyme. The cell-associated form of the lipase seems to be located on the external surface of the cells with its active site exposed. It appears to be anchored to the outer membrane of the cells by means of its hydrophobic region in a way that resembles its adherence to hydrophobic surfaces such as hexadecane droplets.     

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor) to human and bovine factor Xa. A thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to human and bovine factor Xa (Stuart-Prower factor; EC 3.4.21.6) has been investigated. Under all the experimental conditions, values of Ka for BPTI binding to human and bovine factor Xa are identical. On lowering the pH from 9.5 to 4.5, values of Ka (at 21.0 degrees C) for BPTI binding to human and bovine factor Xa decrease, thus reflecting the acidic pK shift of the His57 catalytic residue from 7.1, in the free enzyme, to 5.2, in the proteinase-inhibitor complex. At pH 8.0, values of the apparent thermodynamic parameters for BPTI binding to human and bovine factor Xa are: Ka = 2.1 x 10(5)M-1 (at 21.0 degrees C), delta G degree = -29.7 kJ/mol (at 21.0 degrees C), delta S degree = +161 entropy units (at 21.0 degrees C), and delta H degree = +17.6 kJ/mol (temperature-independent over the explored range, from 5.0 degrees C to 45.0 degrees C). Thermodynamics of BPTI binding to human and bovine factor Xa have been analysed in parallel with those of related serine (pro)enzyme/Kazal- and /Kunitz-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of BPTI to human and bovine factor Xa was related to the inferred stereochemistry of the proteinase/inhibitor contact region.     

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human Lys77-plasmin

The effect of pH and temperature on the association equilibrium constant (Ka) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI Kunitz inhibitor) to human Lys77-plasmin has been investigated. Ka values decrease with decreasing pH, reflecting the acid-pK and -midpoint shifts, upon BPTI binding, of a single ionizable group, between pH 5 and 9, and of a three-proton transition, between pH 3 and 5. At pH 8.0, values of thermodynamic parameters for BPTI binding to human Lys77-plasmin are: Ka = 1.2 X 10(9) M-1, delta G degree = -12.2 kcal/mol, and delta S degree = +49 entropy units (at 21 degrees C); and delta H degree = +2.3 kcal/mol (temperature independent between 5 degrees C and 45 degrees C; 1 kcal = 4184 J). BPTI binding properties of human Lys77-plasmin have been analysed in parallel with those of serine (pro)enzymes acting on cationic and non-cationic substrates. Considering the known molecular structures of homologous serine (pro)enzymes, or Kunitz and Kazal-type inhibitors and of their complexes, the observed binding behaviour of BPTI to human Lys77-plasmin was related to the inferred stereochemistry of the enzyme-inhibitor contact region.     

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 bovine basic pancreatic trypsin inhibitor (Kunitz) to the 33,000 Mr and 54,000 Mr species of human urokinase: thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to the 33,000 Mr and 54,000 Mr species of human urokinase (EC 3.4.21.31) has been investigated. Under all the experimental conditions, values of Ka for BPTI binding to the 33,000 Mr and 54,000 Mr species of human urokinase are identical. On lowering the pH from 9.5 to 4.5, values of Ka (at 21.0 degrees C) for BPTI binding to human urokinase (33,000 Mr and 54,000 Mr species) decrease thus reflecting the acidic pK-shift of the His-57 catalytic residue from 6.9, in the free enzyme, to 5.1, in the proteinase:inhibitor complex. At pH 8.0, values of the apparent thermodynamic parameters for BPTI binding to human urokinase (33,000 Mr and 54,000 Mr species) are: Ka = 4.9 x 10(4) M-1, delta G degree = -6.3 kcal/mol, and delta S degree = -37 entropy units (all at 21.0 degrees C); and delta H degree = +4.6 kcal/mol (temperature independent over the explored range, from 5.0 degrees C to 45.0 degrees C). Thermodynamics of BPTI binding to human urokinase (33,000 Mr and 54,000 Mr species) have been analyzed in parallel with those of related serine (pro)enzyme Kazal- and /Kunitz-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of BPTI to human urokinase (33,000 Mr and 54,000 Mr species) was related to the inferred stereochemistry of the proteinase/inhibitor contact region.     

Structure-activity relationships for the interaction of bovine pancreatic trypsin inhibitor with an intracellular site on a large conductance Ca(2+)-activated K(+) channel

Large conductance Ca(2+)-activated K(+) channels (BK(Ca)) contain an intracellular binding site for bovine pancreatic trypsin inhibitor (BPTI), a well-known inhibitor of various serine proteinase (SerP) enzymes. To investigate the structural basis of this interaction, we examined the activity of 11 BPTI mutants using single BK(Ca) channels from rat skeletal muscle incorporated into planar lipid bilayers. All of the mutants induced discrete substate events at the single-channel level. The dwell time of the substate, which is inversely related to the dissociation rate constant of BPTI, exhibited relatively small changes (<9-fold) for the various mutants. However, the apparent association rate constant varied up to 190-fold and exhibited a positive correlation with the net charge of the molecule, suggesting the presence of a negative electrostatic surface potential in the vicinity of the binding site. The substate current level was unaffected by most of the mutations except for substitutions of Lys15. Different residues at this position were found to modulate the apparent conductance of the BPTI-induced substate to 0% (K15G), 10% (K15F), 30% (K15 wild-type), and 55% (K15V) of the open state at +20 mV. Lys15 is located on a loop of BPTI that forms the primary contact region for binding to many SerPs such as trypsin, chymotrypsin, and elastase. The finding that Lys15 is a determinant of the conductance behavior of the BK(Ca) channel when BPTI is bound implies that the same inhibitory loop that contacts SerP's is located close to the protein interface in the BK(Ca) channel complex. This supports the hypothesis that the C-terminal region of the BK(Ca) channel protein contains a domain homologous to SerP's. We propose a domain interaction model for the mechanism of substate production by Kunitz inhibitors based on current ideas for allosteric activation of BK(Ca) channels by voltage and Ca(2+).     

Hypothesis for a serine proteinase-like domain at the COOH terminus of Slowpoke calcium-activated potassium channels

Bovine pancreatic trypsin inhibitor (BPTI) is a 58-residue protein with three disulfide bonds that belongs to the Kunitz family of serine proteinase inhibitors. BPTI is an extremely potent inhibitor of trypsin, but it also specifically binds to various active and inactive serine proteinase homologs with KD values that range over eight orders of magnitude. We previously described an interaction of BPTI at an intracellular site that results in the production of discrete subconductance events in large conductance Ca2+ activated K+ channels (Moss, G.W.J., and E. Moczydlowski. 1996, J. Gen. Physiol, 107:47-68). In this paper, we summarize a variety of accumulated evidence which suggests that BPTI binds to a site on the KCa channel protein that structurally resembles a serine proteinase. One line of evidence includes the finding that the complex of BPTI and trypsin, in which the inhibitory loop of BPTI is masked by interaction with trypsin, is completely ineffective in the production of substate events in the KCa channel. To further investigate this notion, we performed a sequence analysis of the alpha-subunit of cloned slowpoke KCa channels from Drosophila and mammals. This analysis suggests that a region of approximately 250 residues near the COOH terminus of the KCa channel is homologous to members of the serine proteinase family, but is catalytically inactive because of various substitutions of key catalytic residues. The sequence analysis also predicts the location of a Ca(2+)-binding loop that is found in many serine proteinase enzymes. We hypothesize that this COOH-terminal domain of the slowpoke KCa channel adopts the characteristic double-barrel fold of serine proteinases, is involved in Ca(2+)-activation of the channel, and may also bind other intracellular components that regulate KCa channel activity.     

Substitutions at the P(1) position in BPTI strongly affect the association energy with serine proteinases

The role of the S(1) subsite in trypsin, chymotrypsin and plasmin has been examined by measuring the association with seven different mutants of bovine pancreatic trypsin inhibitor (BPTI); the mutants contain Gly, Ala, Ser, Val, Leu, Arg, and Trp at the P(1) position of the reactive site. The effects of substitutions at the P(1) position on the association constants are very large, comprising seven orders of magnitude for trypsin and plasmin, and over five orders for chymotrypsin. All mutants showed a decrease of the association constant to the three proteinases in the same order: Ala>Gly>Ser>Arg>Val>Leu>Trp. Calorimetric and circular dichroism methods showed that none of the P1 substitutions, except the P1-Val mutant, lead to destabilisation of the binding loop conformation. The X-ray structure of the complex formed between bovine beta-trypsin and P(1)-Leu BPTI showed that the P(1)-Leu sterically conflicts with the side-chain of P(3)-Ile, which thereby is forced to rotate approximately 90 degrees. Ile18 (P(3)) in its new orientation, in turn interacts with the Tyr39 side-chain of trypsin. Introduction of a large side-chain at the P1' position apparently leads to a cascade of small alterations of the trypsin-BPTI interface that seem to destabilise the complex by it adopting a less optimized packing and by tilting the BPTI molecule up to 15 degrees compared to the native trypsin-BPTI complex.     

Crystal structures of five bovine chymotrypsin complexes with P1 BPTI variants

The bovine chymotrypsin-bovine pancreatic trypsin inhibitor (BPTI) interaction belongs to extensively studied models of protein-protein recognition. The accommodation of the inhibitor P1 residue in the S1 binding site of the enzyme forms the hot spot of this interaction. Mutations introduced at the P1 position of BPTI result in a more than five orders of magnitude difference of the association constant values with the protease. To elucidate the structural aspects of the discrimination between different P1 residues, crystal structures of five bovine chymotrypsin-P1 BPTI variant complexes have been determined at pH 7.8 to a resolution below 2 A. The set includes polar (Thr), ionizable (Glu, His), medium-sized aliphatic (Met) and large aromatic (Trp) P1 residues and complements our earlier studies of the interaction of different P1 side-chains with the S1 pocket of chymotrypsin. The structures have been compared to the complexes of proteases with similar and dissimilar P1 preferences, including Streptomyces griseus proteases B and E, human neutrophil elastase, crab collagenase, bovine trypsin and human thrombin. The S1 sites of these enzymes share a common general shape of significant rigidity. Large and branched P1 residues adapt in their complexes similar conformations regardless of the polarity and size differences between their S1 pockets. Conversely, long and flexible residues such as P1 Met are present in the disordered form and display a conformational diversity despite similar inhibitory properties with respect to most enzymes studied. Thus, the S1 specificity profiles of the serine proteases appear to result from the precise complementarity of the P1-S1 interface and minor conformational adjustments occurring upon the inhibitor binding.     

Structure-function relationship of serine protease-protein inhibitor interaction.

We report our progress in understanding the structure-function relationship of the interaction between protein inhibitors and several serine proteases. Recently, we have determined high resolution solution structures of two inhibitors Apis mellifera chymotrypsin inhibitor-1 (AMCI-I) and Linum usitatissimum trypsin inhibitor (LUTI) in the free state and an ultra high resolution X-ray structure of BPTI. All three inhibitors, despite totally different scaffolds, contain a solvent exposed loop of similar conformation which is highly complementary to the enzyme active site. Isothermal calo- rimetry data show that the interaction between wild type BPTI and chymotrypsin is entropy driven and that the enthalpy component opposes complex formation. Our research is focused on extensive mutagenesis of the four positions from the protease binding loop of BPTI: P1, P1', P3, and P4. We mutated these residues to different amino acids and the variants were characterized by determination of the association constants, stability parameters and crystal structures of protease-inhibitor complexes. Accommodation of the P1 residue in the S1 pocket of four proteases: chymotrypsin, trypsin, neutrophil elastase and cathepsin G was probed with 18 P1 variants. High resolution X-ray structures of ten complexes between bovine trypsin and P1 variants of BPTI have been determined and compared with the cognate P1 Lys side chain. Mutations of the wild type Ala16 (P1') to larger side chains always caused a drop of the association constant. According to the crystal structure of the Leu16 BPTI-trypsin complex, introduction of the larger residue at the P1' position leads to steric conflicts in the vicinity of the mutation. Finally, mutations at the P4 site allowed an improvement of the association with several serine proteases involved in blood clotting. Conversely, introduction of Ser, Val, and Phe in place of Gly12 (P4) had invariably a destabilizing effect on the complex with these proteases.     

Structural basis for accelerated cleavage of bovine pancreatic trypsin inhibitor (BPTI) by human mesotrypsin

Human mesotrypsin is an isoform of trypsin that displays unusual resistance to polypeptide trypsin inhibitors and has been observed to cleave several such inhibitors as substrates. Whereas substitution of arginine for the highly conserved glycine 193 in the trypsin active site has been implicated as a critical factor in the inhibitor resistance of mesotrypsin, how this substitution leads to accelerated inhibitor cleavage is not clear. Bovine pancreatic trypsin inhibitor (BPTI) forms an extremely stable and cleavage-resistant complex with trypsin, and thus provides a rigorous challenge of mesotrypsin catalytic activity toward polypeptide inhibitors. Here, we report kinetic constants for mesotrypsin and the highly homologous (but inhibitor sensitive) human cationic trypsin, describing inhibition by, and cleavage of BPTI, as well as crystal structures of the mesotrypsin-BPTI and human cationic trypsin-BPTI complexes. We find that mesotrypsin cleaves BPTI with a rate constant accelerated 350-fold over that of human cationic trypsin and 150,000-fold over that of bovine trypsin. From the crystal structures, we see that small conformational adjustments limited to several side chains enable mesotrypsin-BPTI complex formation, surmounting the predicted steric clash introduced by Arg-193. Our results show that the mesotrypsin-BPTI interface favors catalysis through (a) electrostatic repulsion between the closely spaced mesotrypsin Arg-193 and BPTI Arg-17, and (b) elimination of two hydrogen bonds between the enzyme and the amine leaving group portion of BPTI. Our model predicts that these deleterious interactions accelerate leaving group dissociation and deacylation.     

The pancreatic islet β-cell-enriched transcription factor Pdx-1 regulates Slc30a8 gene transcription through an intronic enhancer

PRSS3/mesotrypsin is an atypical isoform of trypsin, the up-regulation of which has been implicated in promoting tumour progression. Mesotrypsin inhibitors could potentially provide valuable research tools and novel therapeutics, but small-molecule trypsin inhibitors have low affinity and little selectivity, whereas protein trypsin inhibitors bind poorly and are rapidly degraded by mesotrypsin. In the present study, we use mutagenesis of a mesotrypsin substrate, APPI (amyloid precursor protein Kunitz protease inhibitor domain), and of a poor mesotrypsin inhibitor, BPTI (bovine pancreatic trypsin inhibitor), to dissect mesotrypsin specificity at the key P(2)' position. We find that bulky and charged residues strongly disfavour binding, whereas acidic residues facilitate catalysis. Crystal structures of mesotrypsin complexes with BPTI variants provide structural insights into mesotrypsin specificity and inhibition. Through optimization of the P(1) and P(2)' residues of BPTI, we generate a stable high-affinity mesotrypsin inhibitor with an equilibrium binding constant K(i) of 5.9 nM, a >2000-fold improvement in affinity over native BPTI. Using this engineered inhibitor, we demonstrate the efficacy of pharmacological inhibition of mesotrypsin in assays of breast cancer cell malignant growth and pancreatic cancer cell invasion. Although further improvements in inhibitor selectivity will be important before clinical potential can be realized, the results of the present study support the feasibility of engineering protein protease inhibitors of mesotrypsin and highlight their therapeutic potential.     

Phage display selection of P1 mutants of BPTI directed against five different serine proteinases

The P1 position of protein inhibitors and oligopeptide substrates determines, to a large extent, association energy with many serine proteinases. To test the agreement of phage display selection with the existing thermodynamic data, a small library of all 20 P1 mutants of basic pancreatic trypsin inhibitor (BPTI) was created, fused to protein III, and displayed on the surface of M13 phage. The wild type of displayed inhibitor monovalently and strongly inhibited trypsin with an association constant of Ka = 3 x 10(11) M(-1). The library was applied to select BPTI variants active against five serine proteinases of different specificity (bovine trypsin and chymotrypsin, human leukocyte and porcine pancreatic elastases, human azurocidin). The results of enrichment with four proteinases agreed well with the available thermodynamic data. In the case of azurocidin, the phage display selection allowed determination of the P1 specificity of this protein with the following frequencies for selected P1 variants: 43% Lys, 36% Leu, 7% Met, 7% Thr, 7% Gln.     

Inhibition of factor XIII activation by an anti-peptide monoclonal antibody.

As the final enzyme in the coagulation cascade, activated fibrin stabilizing factor or factor XIII catalyzes the intermolecular cross-linking of fibrin chains. To study this enzyme in plasma, we derived a monoclonal antibody (MAb 309) against a peptide sequence (NH2-G-V-N-L-Q-E-F-C-COOH) in the thrombin activation site of factor XIII. Radioimmunoassays indicate that MAb 309 binds specifically to both platelet and plasma factor XIII. Peptide inhibition studies demonstrate that the MAb binds equally well to the factor XIII (FXIII) zymogen and the active form of FXIII (FXIIIa). In immunoblots of whole platelet lysates, MAb 309 binds only to FXIII and does not cross-react with other proteins. In saturation binding studies, the antibody shows a binding avidity of (1.75 +/- 0.35) x 10(9) M-1. MAb 309 also inhibited 99% of apparent FXIIIa activity in a standard transglutaminase assay. SDS-PAGE analysis of fibrin clots showed that MAb 309 inhibited fibrin gamma-gamma cross-linking. Moreover, MAb 309 accelerated the lysis of plasma clots, consistent with inhibition of fibrin-fibrin and fibrin-alpha 2-antiplasmin cross-linking. Immunoblotting experiments revealed that MAb 309 affected apparent FXIIIa activity by inhibiting the thrombin activation of the FXIII zymogen. In addition to its utility as a specific probe for the FXIII a-subunit, the strategy used to obtain MAb 309 may be used to generate MAbs that inhibit the activation of other coagulation factor zymogens.     

Synthesis and characterization of a pancreatic trypsin inhibitor homologue and a model inhibitor.

The synthesis and characterization of protein proteinase inhibitor homologues with variations in the amino acid composition in the vicinity of the reactive site should aid the understanding of the mechanism by which inhibition of enzymatic activity occurs. A homologue inhibitor in which the reactive-site residue Ala-16 of basic pancreatic trypsin inhibitor (Kunitz) (BPTI) is replaced by Phe has been synthesized to study the effect of this replacement on the dissociation constants of the enzyme-inhibitor complexes. The replacement of Ala-16 by Phe causes a dramatic increase in the K1 value of the trypsin-BPTI complex while that of the chymotrypsin-BPTI complex remains essentially the same. This cannot be explained simply in terms of increased steric crowding. The Phe replacement probably causes a small change in the local conformation of the reactive site of the inhibitor which leads to a large decrease in the stability of the very tight trypsin-BPTI complex. This conformation change apparently can be tolerated in the less tightly bound chymotrypsin-BPTI complex. On the basis of the known structure of BPTI, a cyclic heptadecapeptide containing one disulfide bond was synthesized as a model inhibitor in order to determine if a smaller peptide can be designed to act as a highly efficient inhibitor for trypsin. This heptadecapeptide which contains all of the amino acid residues of BPTI taking part in the interaction of the proteinase inhibitor with trypsin binds 3 X 10(7) time more weakly to the enzyme than native BPTI does. It thus appears that even though only a small part of the inhibitor molecule enters directly into interaction with the enzyme, the remaining portions of the molecule which hold the structure of the inhibitor rigid are essential for the strong interaction.     

Serpins of oat (Avena sativa) grain with distinct reactive centres and inhibitory specificity

Most proteinase inhibitors from plant seeds are assumed to contribute to broad-spectrum protection against pests and pathogens. In oat (Avena sativa L.) grain the main serine proteinase inhibitors were found to be serpins, which utilize a unique mechanism of irreversible inhibition. Four distinct inhibitors of the serpin superfamily were detected by native PAGE as major seed albumins and purified by thiophilic adsorption and anion exchange chromatography. The four serpins OSZa-d are the first proteinase inhibitors characterized from this cereal. An amino acid sequence close to the blocked N-terminus, a reactive centre loop sequence, and the second order association rate constant (ka') for irreversible complex formation with pancreas serine proteinases at 24 degrees C were determined for each inhibitor. OSZa and OSZb, both with the reactive centre scissile bond P1-P1' Thr downward arrow Ser, were efficient inhibitors of pancreas elastase (ka' > 105M-1 s-1). Only OSZb was also an inhibitor of chymotrypsin at the same site (ka' = 0.9 x 105M-1 s-1). OSZc was a fast inhibitor of trypsin at P1-P1' Arg downward arrow Ser (ka' = 4 x 106M-1 s-1); however, the OSZc-trypsin complex was short-lived with a first order dissociation rate constant kd = 1.4 x 10-4 s-1. OSZc was also an inhibitor of chymotrypsin (ka' > 106M-1 s-1), presumably at the overlapping site P2-P1 Ala downward arrow Arg, but > 90% of the serpin was cleaved as substrate. OSZd was cleaved by chymotrypsin at the putative reactive centre bond P1-P1' Tyr downward arrow Ser, and no inhibition was detected. Together the oat grain serpins have a broader inhibitory specificity against digestive serine proteinases than represented by the major serpins of wheat, rye or barley grain. Presumably the serpins compensate for the low content of reversible inhibitors of serine proteinases in oats in protection of the grain against pests or pathogens.     

Interaction of trypsin with a trypsin inhibitor from bovine colostrum]

Kinetics of trypsin association with trypsin inhibitor from colostrum (IC) was studied. The association rate constant is 3-10-5 M- minus 1 sec- minus 1 at pH 7,8, 25 degrees C. The rate constant for the complex dissociation was determined from the kinetics of the IC displacement from the complex with trypsin by a specific substrate and was found to be 5-10- minus 6 sec- minus 1 (pH 7,8; 25 degrees C). The equilibrium constant (Ki) was measured in a special experiment and was equal to 4-10- minus 12 M (p H 7,8; 25 degrees C). The similarity of this reaction and the association of trypsin with other protein inhibitors was discussed.     

Protein Disulfide-Isomerase Interacts with a Substrate Protein at All Stages along Its Folding Pathway

In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI) catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding). However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) and showed by NMR that it is partly-folded and highly dynamic. NMR studies show that it binds to PDI at the same site that binds peptide ligands, with rapid binding and dissociation kinetics; surface plasmon resonance shows its interaction with PDI has a K_d of ca. 10⁻⁵ M. For comparison, we characterized the interactions of PDI with native BPTI and fully-unfolded BPTI. Interestingly, PDI does bind native BPTI, but binding is quantitatively weaker than with partly-folded and unfolded BPTI. Hence PDI recognizes and binds substrates via permanently or transiently unfolded regions. This is the first study of PDI''s interaction with a partly-folded protein, and the first to analyze this folding catalyst''s changing interactions with substrates along an oxidative folding pathway. We have identified key features that make PDI an effective catalyst of oxidative protein folding – differential affinity, rapid ligand exchange and conformational flexibility.     

Engineering an unnatural Nalpha-anchored disulfide into BPTI by total chemical synthesis: structural and functional consequences

A disulfide-engineered analogue of bovine pancreatic trypsin inhibitor (BPTI), ((N(alpha)-(CH2)2S-)Gly38)BPTI, has been prepared using a thioester-mediated auxiliary functional group chemical ligation of a N(alpha)-ethanethiol-containing peptide segment with a peptide-alphaCOSR segment. In this study, Nalpha-(ethanethiol)Gly38 replaces the native Cys38, providing the sulfhydryl group required for ligation and folding. Comparisons between ((Nalpha-(CH2)2SH)Gly38)BPTI, synthetic native BPTI and reference BPTI purchased from Sigma were made using mass spectroscopy, enzyme inhibitor association constant determination (K(a)) and 1H-nuclear magnetic resonance total correlated spectroscopy (1H-NMR TOCSY) measurements. The K(a) value for ((Nalpha-(CH2)2SH)Gly38)BPTI was approximately 20-fold lower than synthetic and reference BPTI, which was attributed to perturbations in the binding loop of the protein (near Cys14). This hypothesis was confirmed by two-dimensional (2D) 1H-NMR TOCSY experiments. The data reported here demonstrate that total chemical synthesis by auxiliary functional group chemical ligation is a practical method for the synthesis of a novel class of biologically active protein analogues containing additional functional groups linked to the protein backbone.