Chemical-enzymatic replacement of Ile64 in the reactive site of soybeen trypsin inhibitor (Kunitz).

All the reactive amino groups in soybean trypsin inhibitor (Kunitz) were protected by guanidination of 9 out of 10 lysyl residues with O-methylisourea and by carbamoylation of the NH2 terminal Asp with potassium cyanate. This derivative was converted to modified inhibitor (Arg63-Ile64 reactive site peptide bond hydrolyzed) by incubation with trypsin at pH 3. The NH2 terminal of Ile64 was allowed to react with phenyl isothiocyanate to produce inactive phenylthiocarbamoyl-modified inhibitor. Treatment with trifluoroacetic acid formed the anilinothiazolinone of Ile64 yielding des-Ile64-modified inhibitor. After renaturation and purification, this material coelectrophoresed with modified inhibitor but did not form a stable complex with trypsin. Incubation with tert-butyloxycarbonyl-(amino acid)-N-hydroxysuccinimide esters yielded [tert-butyloxycarbonyl-(amino acid64)]-modified inhibitor. The tert-butyloxycarbonyl protective group was removed in trifluoroacetic acid. After renaturation, active [amino acid64]-modified inhibitors were obtained for Ile64, Ala64, Leu64, and Gly64 replacements. The resynthesis of the reactive-site peptide bound by kinetic control dissociation of the trypsin-inhibitor complex yielded fully active [Ala64]-virgin inhibitor. Thus, soybean trypsin inhibitor (Kunitz) has been shown to tolerate the replacement of the P1' residue with retention of activity. The importance of P1' residues in the function of protein proteinase inhibitors is discussed.     

Replacement of lysine by arginine, phenylalanine and tryptophan in the reactive site of the bovine trypsin-kallikrein inhibitor (Kunitz) and change of the inhibitory properties.

The reactive-site sequence of a proteinase inhibitor can be written as . . . -P3-P2-P1-P'1-P'2-P'3- . . . , where-P1-P'1-denotes the reactive site. Three semisynthetic homologues have been synthesized of the bovine trypsin-kallikrein inhibitor (Kunitz) with either arginine, phenylalanine or tryptophan in place of the reactive-site residue P1, lysine-15. These homologues correspond to gene products after mutation of the lysine 15 DNA codon to an arginine, phenylalanine or tryptophan DNA codon. Starting from native (virgin) inhibitor, reactive-site hydrolyzed, still active (modified) inhibitor was prepared by chemical and enzymic reactions. Modified inhibitor was then converted into inactive des-Lys15-inhibitor by reaction with carboxypeptidase B. Inactive des-Lys15-inhibitor was reactivated by enzymic replacement of the P1 residue according to Leary and Laskowski, Jr. The introduction of arginine was catalyzed by an inverse reaction with carboxypeptidase B, while phenylalanine or tryptophan were replaced by carboxypeptidase A. The reactivated semisynthetic inhibitors were trapped by complex formation with either trypsin or chymotrypsin. The enzyme - inhibitor complexes were subjected to kinetic-control dissociation, and the semisynthetic virgin inhibitors were isolated. The inhibitory properties of the semisynthetic inhibitors have been investigated against bovine trypsin and chymotrypsin and against porcine pancreatic kallikrein and plasmin. The homologues with either lysine or arginine in the P1 position are equally good inhibitors of trypsin, plasmin and kallikrein. The Arg-15-homologue is a slightly more effective kallikrein inhibitor than the Lys15-inhibitor. The semisynthetic phenylalanine and tryptophan homologues, however, are weak inhibitors of trypsin and still weaker inhibitors of kallikrein, but are excellent inhibitors of chymotrypsin. Their association constant with chymotrypsin is at least ten times higher than that of native Lys-15-inhibitor. A dramatic specificity change is observed with the phenylalanine and tryptophan homologues, which in contrast to the native inhibitor do not at all inhibit porcine plasmin. Thus, the nature of the P1 residue strongly influences the primary inhibitory specificity of the bovine inhibitor (Kunitz).     

Structural consequences of the natural substitution, E9K, on reactive-site-hydrolyzed squash (Cucurbita maxima) trypsin inhibitor (CMTI), as studied by two-dimensional NMR.

Sequence-specific hydrogen-1 NMR assignments were made to all of the 29 amino acid residues of reactive-site-hydrolyzed Cucurbita maxima trypsin inhibitor I (CMTI-I*) by the application of two-dimensional NMR (2D NMR) techniques, and its secondary structural elements (two tight turns, a 3(10)-helix, and a triple-stranded beta-sheet) were identified on the basis of short-range NOESY cross peaks and deuterium-exchange kinetics. These secondary structural elements are present in the intact inhibitor [Holak, T. A., Gondol, D., Otlewski, J., & Wilusz, T. (1989) J. Mol. Biol. 210, 635-648] and are unaffected by the hydrolysis of the reactive-site peptide bond between Arg5 and Ile6, in accordance with the earlier conclusion reached for CMTI-III* [Krishnamoorthi, R., Gong, Y.-X., Lin, C. S., & VanderVelde, D. (1992) Biochemistry 31, 898-904]. Chemical shifts of backbone hydrogen atoms, peptide NH's, and C alpha H's, of CMTI-I* were compared with those of the intact inhibitor, CMTI-I, and of the reactive-site-hydrolyzed, natural, E9K variant, CMTI-III*. Cleavage of the Arg5-Ile6 peptide bond resulted in changes of chemical shifts of most of the backbone atoms of CMTI-I, in agreement with the earlier results obtained for CMTI-III. Comparison of chemical shifts of backbone hydrogen atoms of CMTI-I* and CMTI-III* revealed no changes, except for residues Glu9 and His25. However, the intact forms of the same two proteins, CMTI-I and CMTI-III, showed small but significant perturbations of chemical shifts of residues that made up the secondary structural elements of the inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human mesotrypsin defies natural trypsin inhibitors: from passive resistance to active destruction

More than twenty years ago Rinderknecht et al. identified a minor trypsin isoform resistant to natural trypsin inhibitors in the human pancreatic juice. At the same time, Estell and Laskowski found that an inhibitor-resistant trypsin from the pyloric caeca of the starfish, Dermasterias imbricata rapidly hydrolyzed the reactive-site peptide bonds of trypsin inhibitors. A connection between these two seminal discoveries was made recently, when human mesotrypsin was shown to cleave the reactive-site peptide bond of the Kunitz-type soybean trypsin inhibitor, and degrade the Kazal-type pancreatic secretory trypsin inhibitor. These observations indicate that proteases specialized for the degradation of protease inhibitors are ubiquitous in metazoa, and prompt new investigations into their biological significance. Here we review the history and properties of human mesotrypsin, and discuss its function in the digestive degradation of dietary trypsin inhibitors and possible pathophysiological role in pancreatitis.     

Natural abundance 15N NMR assignments delineate structural differences between intact and reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III.

15N NMR assignments were made to the backbone amide nitrogen atoms at natural isotopic abundance of intact and reactive-site (Arg5-Ile6) hydrolyzed Cucurbita maxima trypsin inhibitor III (CMTI-III and CMTI-III*, respectively) by means of 2D proton-detected heteronuclear single bond chemical shift correlation (HSBC) spectroscopy, utilizing the previously made sequence-specific 1H NMR assignments (Krishnamoorthi et al. (1992) Biochemistry 31, 898-904). Comparison of the 15N chemical shifts of the two forms of the inhibitor molecule revealed significant changes not only for residues located near the reactive-site region, but also for those distantly located. Residues Cys3, Arg5, Leu7, Met8, Cys10, Cys16, Glu19, His25, Tyr27, Cys28 and Gly29 showed significant chemical shift changes ranging from 0.3 to 6.1 ppm, thus indicating structural perturbations that were transmitted throughout the molecule. These findings confirm the earlier conclusions based on 1H NMR investigations.     

Reconstitution of new inhibitors through mutual exchange of Ile(64)-Met(84) peptides of soybean trypsin inhibitors, Tia and Tib

Singly modified soybean trypsin inhibitors (STIs), Tia* [Tia cleaved at Arg(63)-Ile(64)] and Tib* [Tib cleaved at Arg(63)-Ile(64)], were prepared by limited proteolysis with trypsin at pH 3.0. These singly modified inhibitors were further modified to yield doubly modified inhibitors, Tia** and Tib**, by limited proteolysis with subtilisin BPN', which cleaved the Met(84)-Leu(85) bonds of Tia* and Tib*, respectively. The doubly modified inhibitors could be separated into two parts: protein moiety A and peptide moiety a (IRFIAEGHPLSLKFDS-FAVIM) for Tia**, and protein moiety B and peptide moiety b (IRFIAEGNPLRLKFDS-FAVIM) for Tib**. These protein and peptide moieties showed no trypsin inhibitory activities alone. However, the inhibitors can be reconstituted through the mutual exchange of the protein and peptide moieties isolated from STIs. The reconstituted inhibitor which has tyrosine at position 62 and histidine at position 71 shows the highest inhibitory activity. Its Ki value for bovine trypsin is around 10(-10) M, which is almost the same as that of Tia for bovine trypsin. The inhibitor possessing either tyrosine at position 62 or histidine at position 71 exhibits a Ki value of around 10(-9) M, which is between those of Tia and Tib. The inhibitor having phenylalanine and asparagine at positions 62 and 71, respectively, shows the weakest inhibitory activity of around 10(-8) M similar to that of Tib for bovine trypsin.     

NMR studies of internal dynamics of serine proteinase protein inhibitors: Binding region mobilities of intact and reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor (CMTI)-III of the squash family and comparison with those of counterparts of CMTI-V of the potato I family.

Serine proteinase protein inhibitors follow the standard mechanism of inhibition (Laskowski M Jr, Kato I, 1980, Annu Rev Biochem 49:593-626), whereby an enzyme-catalyzed equilibrium between intact (I) and reactive-site hydrolyzed inhibitor (I*) is reached. The hydrolysis constant, Khyd, is defined as [I*]/[I]. Here, we explore the role of internal dynamics in the resynthesis of the scissile bond by comparing the internal mobility data of intact and cleaved inhibitors belonging to two different families. The inhibitors studied are recombinant Cucurbita maxima trypsin inhibitor III (rCMTI-III; Mr 3 kDa) of the squash family and rCMTI-V (Mr approximately 7 kDa) of the potato I family. These two inhibitors have different binding loop-scaffold interactions and different Khyd values--2.4 (CMTI-III) and 9 (CMTI-V)--at 25 degrees C. The reactive-site peptide bond (P1-P1') is that between Arg5 and Ile6 in CMTI-III, and that between Lys44 and Asp45 in CMTI-V. The order parameters (S2) of backbone NHs of uniformly 15N-labeled rCMTI-III and rCMTI-III* were determined from measurements of 15N spin-lattice and spin-spin relaxation rates, and [1H]-15N steady-state heteronuclear Overhauser effects, using the model-free formalism, and compared with the data reported previously for rCMTI-V and rCMTI-V*. The backbones of rCMTI-III [(S2) = 0.71] and rCMTI-III* [(S2) = 0.63] are more flexible than those of rCMTI-V [(S2) = 0.83] and rCMTI-V* [(S2) = 0.85]. The binding loop residues, P4-P1, in the two proteins show the following average order parameters: 0.57 (rCMTI-III) and 0.44 (rCMTI-III*); 0.70 (rCMTI-V) and 0.40 (rCMTI-V*). The P1'-P4' residues, on the other hand, are associated with (S2) values of 0.56 (rCMTI-III) and 0.47 (rCMTI-III*); and 0.73 (rCMTI-V) and 0.83 (rCMTI-V*). The newly formed C-terminal (Pn residues) gains a smaller magnitude of flexibility in rCMTI-III* due to the Cys3-Cys20 crosslink. In contrast, the newly formed N-terminal (Pn' residues) becomes more flexible only in rCMTI-III*, most likely due to lack of an interaction between the P1' residue and the scaffold in rCMTI-III. Thus, diminished flexibility gain of the Pn residues and, surprisingly, increased flexibility of the Pn' residues seem to facilitate the resynthesis of the P1-P1' bond, leading to a lower Khyd value.     

Two-dimensional NMR studies of squash family inhibitors. Sequence-specific proton assignments and secondary structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III.

The solution structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III (CMTI-III*) was investigated by two-dimensional proton nuclear magnetic resonance (2D NMR) spectroscopy. CMTI-III*, prepared by reacting CMTI-III with trypsin which cleaved the Arg5-Ile6 peptide bond, had the two fragments held together by a disulfide linkage. Sequence-specific 1H NMR resonance assignments were made for all the 29 amino acid residues of the protein. The secondary structure of CMTI-III*, as deduced from NOESY cross peaks and identification of slowly exchanging hydrogens, contains two turns (residues 8-12 and 24-27), a 3(10)-helix (residues 13-16), and a triple-stranded beta-sheet (residues 8-10, 29-27, and 21-25). This secondary structure is similar to that of CMTI-I [Holak, T. A., Gondol, D., Otlewski, J., & Wilusz, T. (1989) J. Mol. Biol. 210, 635-648], which has a Glu instead of a Lys at position 9. Sequential proton assignments were also made for the virgin inhibitor, CMTI-III, at pH 4.71, 30 degrees C. Comparison of backbone hydrogen chemical shifts of CMTI-III and CMTI-III* revealed significant changes for residues located far away from the reactive-site region as well as for those located near it, indicating tertiary structural changes that are transmitted through most of the 29 residues of the inhibitor protein. Many of these residues are functionally important in that they make contact with atoms of the enzyme in the trypsin-inhibitor complex, as revealed by X-ray crystallography [Bode, W., Greyling, H. J., Huber, R., Otlewski, J., & Wilusz, T. (1989) FEBS Lett. 242, 285-292].(ABSTRACT TRUNCATED AT 250 WORDS)     

The amino acid sequence of a Bowman-Birk type proteinase inhibitor from faba beans (Vicia faba L.).

The amino acid sequence of a Bowman-Birk type proteinase inhibitor (FBI) from seeds of faba bean (Vicia faba L.) was determined by analysis of peptide fragments generated by reduction and S-carboxymethylation of enzymatically modified inhibitors, which were obtained from native FBI by limited proteolysis with TPCK-trypsin or TLCK-chymotrypsin at pH 3.5. The established sequence showed that FBI is highly homologous with Vicia angustifolia inhibitor (VAI0 but lacks the portion corresponding to the C-terminal 9 amino acids of VAI. The trypsin reactive-site peptide bond in FBI was also indicated to be Lys(16)-Ser(17) and the chymotrypsin reactive-site peptide bond to be Tyr(42)-Ser(43) by limited proteolysis with TPCK-trypsin or TLCK-chymotrypsin and by sequence comparison with other Bowman-Birk type inhibitors.     

Evidence that Highly Conserved Residues of <Emphasis Type="Italic">Delonix regia</Emphasis> Trypsin Inhibitor Are Important for Activity

Delonix regia trypsin inhibitor (DrTI) consists of a single-polypeptide chain with a molecular mass of 22 kDa and containing two disulfide bonds (Cys44–Cys89 and Cys139–Cys149). Sequence comparison with other plant trypsin inhibitors of the Kunitz family reveals that DrTI contains a negatively charged residue (Glu68) at the reactive site rather than the conserved Arg or Lys found in other Kunitz-type trypsin inhibitors. Site-directed mutagenesis yielded five mutants containing substitutions at the reactive site and at one of the disulfide bonds. Assay of the recombinant proteins showed mutant Glu68Leu and Glu68Lys to have only 4–5% of the wild-type activity. These provide evidence that the Glu68 residue is the reactive site for DrTI and various other Kunitz-type trypsin inhibitors. The Cys139Gly mutant lost its inhibitory activity, whereas the Cys44Gly mutant did not, indicating that the second disulfide bond (Cys139–Cys149) is critical to DrTI inhibitory activity, while the first disulfide bond (Cys44–Cys89) is not required.     

Human inter-alpha-trypsin inhibitor: localization of the Kunitz-type domains in the N-terminal part of the molecule and their release by a trypsin-like proteinase

The N-terminal amino-acid sequence of human ITI has been found to be identical with that of the acid-stable human 30-kDa inhibitors (HI-30) from urine, serum, and those released from inter-alpha-trypsin inhibitor by trypsin or chymotrypsin. Serum HI-30 and HI-30 released by trypsin differ from the urinary inhibitor by an additional C-terminal arginine residue. Compared to these two inhibitors the inhibitor released by chymotryptic proteolysis is elongated C-terminally by an additional phenylalanine residue. These results strongly favour HI-30 as the N-terminus of the inter-alpha-trypsin inhibitor and its release from this inhibitor in vivo by cleavage of the Arg123-Phe124 peptide bond by trypsin-like proteinases.     

Assignment of the 13C-NMR spectra of virgin and reactive-site modified turkey ovomucoid third domain

The virgin (reactive-site Leu18-Glu19 peptide bond intact) and modified (reactive-site Leu18-Glu19 peptide bond hydrolyzed) forms of turkey ovomucoid third domain (OMTKY3 and OMTKY3*, respectively) have been analyzed by proton-detected 1H(13C) two-dimensional single-bond correlation (1H[13C]SBC) spectroscopy. Previous 1H-nmr assignments of these proteins [A.D. Robertson, W.M. Westler, and J.L Markley (1988) Biochemistry, 27, 2519-2529; G. I. Rhyu and J. L. Markley (1988) Biochemistry, 27, 2529-2539] have been extended to directly bonded 13C atoms. Assignments have been made to 52 of the 56 backbone 13C alpha-1H units and numerous side-chain 13C-1H groups in both OMTKY3 and OMTKY3*. The largest changes in the 13C chemical shift upon conversion of OMTKY3 to OMTKY3* occur at or near the reactive site, and tend toward values observed in small peptides. Moreover, the side-chain prochiral methylene protons attached to the C gamma of Glu19 and C delta of Arg21 show nonequivalent chemical shifts in OMTKY3 but more equivalent chemical shifts in OMTKY3*. These results suggest that the reactive site region becomes less ordered upon hydrolysis of the Leu18-Glu19 peptide bond. Comparison of 13C alpha chemical shifts of OMTKY3 and bovine pancreatic trypsin inhibitor [D. Brühuiler and G. Wagner (1986) Biochemistry 25, 5839-5843; N. R. Nirmala and G. Wagner (1988) Journal of the American Chemical Society, 110, 7557-7558] with small peptide values [R. Richarz and K. Wüthrich (1978) Biopolymers, 17, 2133-2141] suggests that 13C alpha chemical shifts of residues residing in helices are generally about 2 ppm downfield of resonances from nonhelical residues.     

The complete amino acid sequence of barley trypsin inhibitor

The amino acid sequence of barley trypsin inhibitor has been determined. The protein is a single polypeptide consisting of 121 amino acid residues and has Mr = 13,305. No free sulfhydryl groups were detected by Ellman's reagent, which indicates the presence of five disulfide bridges in the molecule. The primary site of interaction with trypsin was tentatively assigned to the arginyl-leucyl residues at positions 33 and 34. On comparison of the sequence of this inhibitor with those of other proteinase inhibitors, we found that the barley trypsin inhibitor could not be classified into any of the established families of proteinase inhibitors (Laskowski, M., Jr., and Kato, I. (1980) Annu. Rev. Biochem. 49, 593-626) and that this inhibitor should represent a new inhibitor family. On the other hand, this trypsin inhibitor showed a considerable similarity to wheat alpha-amylase inhibitor (Kashlan, N., and Richardson, M. (1981) Phytochemistry (Oxf.) 20, 1781-1784) throughout the whole sequence, suggesting a common ancestry for both proteins. This is the first case of a possible evolutionary relationship between two inhibitors directed to totally different enzymes, a proteinase and a glycosidase.     

Primary structure of ascidian trypsin inhibitors in the hemolymph of a solitary ascidian, Halocynthia roretzi

The amino acid sequences of trypsin inhibitors I and II from the hemolymph of a solitary ascidian, Halocynthia roretzi, were determined after reduction and S-pyridylethylation. The results indicated that inhibitor I consists of a single polypeptide chain with 55 amino acid residues and four intramolecular disulfide bridges, whereas inhibitor II is composed of two polypeptide chains corresponding to a form derived from inhibitor I by cleavage at the Lys16-Met17 bond. Lys16 may be the reactive-site residue of these inhibitors, because carboxypeptidase B treatment destroys most of the inhibitory activity of inhibitor II but not that of inhibitor I.     

Trypsin reactivity site of the Vicia angustifolia proteinase inhibitor

Trypsin [EC 3.4.21.4] modified (reactive site cleaved) Vicia angustifolia proteinase inhibitor was prepared at pH 3 with a catalytic amount of trypsin and purified using columns of Sephadex G-50 and DEAE-Sephadex A-25. The modified inhibitor, which still retained antitryptic activity, lost its activity upon treatment with carboxypeptidase B or citraconic anhydride. End-group analyses revealed that the carboxyl-terminal Arg and the amino-terminal Ser residues were newly exposed end-groups in the modified inhibitor. It takes a much longer incubation time (about 1 h) to exhibit the maximal inhibitory activity against trypsin. Reduction and carboxymethylation of the modified inhibitor produced two fragments on Sephadex G-50 chromatography. The smaller fragment consisted of about 32 amino acid residues and possessed a new carboxyl-terminal Arg residue. The larger fragment consisted of about 80 residues and possessed a Ser residue at its amino-terminus. These results indicate that the small fragment was derived from the amino-terminal portion of the modified inhibitor and the large fragment from the carboxyl-terminal. It is also concluded that an Arg-Ser bond is the reactive site as well as the inhibitory site of the V. angustifolia inhibitor against trypsin. The sequence around the antitryptic site exhibits high degrees of homology with other double-headed inhibitors of legume origin, such as the Bowman-Birk inhibitor, lima beam inhibitor, and the major inhibitor in chick-peas.     

Changing the inhibitory specificity and function of the proteinase inhibitor eglin c by site-directed mutagenesis: functional and structural investigation.

Amino acids in the serine proteinase inhibitor eglin c important for its inhibitory specificity and activity have been investigated by site-directed mutagenesis. The specificity of eglin c could be changed from elastase to trypsin inhibition by the point mutation Leu45----Arg (L45R) in position P1 [nomenclature according to Schechter and Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162]. Model building studies based on the crystal structure of mutant L45R [Heinz et al. (1991) J. Mol. Biol. 217, 353-371] were used to rationalize this specificity change. Surprisingly, the double mutant L45R/D46S was found to be a substrate of trypsin and various other serine proteinases. Multidimensional NMR studies show that wild-type eglin c and the double mutant have virtually identical conformations. In the double mutant L45R/D46S, however, the N-H bond vector of the scissile peptide bond shows a much higher mobility, indicating that the internal rigidity of the binding loop is significantly weakened due to the loss or destabilization of the internal hydrogen bond of the P1' residue. Mutant T44P was constructed to examine the role of a proline in position P2, which is frequently found in serine proteinase inhibitors [Laskowski and Kato (1980) Annu. Rev. Biochem. 49, 593-626]. The mutant remains a potent elastase inhibitor but no longer inhibits subtilisin, which could be explained by model building. Both Arg51 and Arg53, located in the core of the molecule and participating in the hydrogen bonding network with residues in the binding loop to maintain rigidity around the scissile bond, were individually replaced with the shorter but equally charged amino acid lysine. Both mutants showed a decrease in their inhibitory potential. The crystal structure of mutant R53K revealed the loss of two hydrogen bonds between the core and the binding loop of the inhibitor, which are partially restored by a solvent molecule, leading to a decrease in inhibition of elastase by 2 orders of magnitude.     

Human mesotrypsin is a unique digestive protease specialized for the degradation of trypsin inhibitors

Mesotrypsin is an enigmatic minor human trypsin isoform, which has been recognized for its peculiar resistance to natural trypsin inhibitors such as soybean trypsin inhibitor (SBTI) or human pancreatic secretory trypsin inhibitor (SPINK1). In search of a biological function, two conflicting theories proposed that due to its inhibitor-resistant activity mesotrypsin could prematurely activate or degrade pancreatic zymogens and thus play a pathogenic or protective role in human pancreatitis. In the present study we ruled out both theories by demonstrating that mesotrypsin was grossly defective not only in inhibitor binding, but also in the activation or degradation of pancreatic zymogens. We found that the restricted ability of mesotrypsin to bind inhibitors or to hydrolyze protein substrates was solely due to a single evolutionary mutation, which changed the serine-protease signature glycine 198 residue to arginine. Remarkably, the same mutation endowed mesotrypsin with a novel and unique function: mesotrypsin rapidly hydrolyzed the reactive-site peptide bond of the Kunitz-type trypsin inhibitor SBTI, and irreversibly degraded the Kazal-type temporary inhibitor SPINK1. The observations suggest that the biological function of human mesotrypsin is digestive degradation of trypsin inhibitors. This mechanism can facilitate the digestion of foods rich in natural trypsin inhibitors. Furthermore, the findings raise the possibility that inappropriate activation of mesotrypsinogen in the pancreas might lower protective SPINK1 levels and contribute to the development of human pancreatitis. In this regard, it is noteworthy that the well known pathological trypsinogen activator cathepsin B exhibited a preference for the activation of mesotrypsinogen of all three human trypsinogen isoforms, suggesting a biochemical mechanism for mesotrypsinogen activation in pancreatic acinar cells.     

The complete amino acid sequence of a major trypsin inhibitor from seeds of foxtail millet (Setaria italica)

The complete amino acid sequence of a major trypsin inhibitor (FMTI-II) from seeds of foxtail millet (Setaria italica) was determined by analysis of peptides derived from the reduced and S-carboxymethylated protein by digestion with TPCK-trypsin and Staphylococcus aureus V8 protease. FMTI-II consists of 67 amino acid residues, including 10 half-cystine residues which are involved in 5 disulfide bridges in the molecule. The established sequence had a high degree of homology to Bowman-Birk type inhibitors from leguminous and gramineous plants. The trypsin reactive-site peptide bond in FMTI-II also appears to be Lys (16)-Ser (17) by comparison with these sequences.     

Aprotinin derivatives with chromophoric leaving groups can be used as highly selective active-site titrants for serine proteinases and permit the determination of kinetic constants of enzyme-inhibitor complexes

This paper reports a novel and valuable approach to active-site titration. The starting substance for the preparation of the active-site titrants is aprotinin (bovine pancreatic trypsin inhibitor) in which the reactive-site peptide bond, Lys15-Ala16, is split. Two cystine disulfide bonds hold together the two peptide chains. The Lys15 of the reactive site is substituted by arginine-, phenylalanine- and valine-4-nitroanilide or by valine-7-amido-4-methylcoumarin. The different incorporated amino acid residues correspond to different specificities against serine proteinases. Serine proteinases with suitable specificity are able to remove 4-nitroaniline or 7-amino-4-methylcoumarin from these aprotinin derivatives while at the same time resynthesis of the reactive-site peptide bond occurs. The proteinase is then trapped in a stable enzyme-inhibitor complex, which prevents the proteinase from releasing further leaving groups. The quantity of 4-nitroaniline or 7-amino-4-methylcoumarin, which can be assayed spectrophotometrically or fluorometrically is equimolar to the quantity of proteinase used and trapped. The aprotinin derivatives with an incorporated Phe15 or Val15 residue are highly specific for chymotrypsin or for elastase from human leukocytes, respectively. The kinetic constants kon and koff of the enzyme-inhibitor complexes, and hence the equilibrium dissociation constants, can be calculated from the respective titration curves.     

Mapping of the catalytic site of CHO-t-PA and the t-PA variant BM 06.022 by synthetic inhibitors and substrates.

BM 06.022 is a t-PA deletion variant that is produced as inactive inclusion bodies in Escherichia coli and transformed into the native form by an in vitro refolding process. Until now, no X-ray and NMR structures of BM 06.022 were available. Therefore a detailed kinetic analysis of the hydrolysis of peptide substrates and of the inhibition by several benzamidine-derived inhibitors was carried out in order to assess that the active site region of the protease domain of BM 06.022 is correctly structured in comparison with t-PA. Our data reveal that the single-chain as well as the two-chain form of BM 06.022 and native t-PA are similar in catalytic and in inhibitor binding properties. This indicates that the active site and the highly complex rearrangement of t-PA upon cleavage of the Arg275-Ile276 bond are maintained in BM 06.022.