Binding Site Of Trypsin Research Articles

Covalent and non-covalent interaction of myofibrillar protein and cyanidin-3-O-glucoside: focus on structure, binding sites and in vitro digestion properties.

The aim of this study was to investigate the effects of covalent and non-covalent interactions between myofibrillar protein (MP) and cyanidin-3-O-glucoside (C3G) on protein structure, binding sites, and digestion properties. Four methods of inducing covalent cross-linking were used in the preparation of MP-C3G conjugates, including tyrosinase-catalyzed oxidation, alkaline pH shift treatment, free radical grafting, and ultrasonic treatment. A comparison was made between MP-C3G conjugates and complexes, and the analysis included sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), C3G binding ratio, liquid chromatography-tandem mass spectrometry (LC-MS/MS), protein side-chain amino acids, circular dichroism spectroscopy, three-dimensional fluorescence, particle size, and in vitro simulated digestion. Covalent bonding between C3G and amino acid side chains in MP was confirmed by LC-MS/MS. In covalent bonding, tryptophan residues, free amino groups and sulfhydryl groups were all implicated. Among the 22 peptides covalently modified by C3G, 30 modification sites were identified, located in lysine, histidine, tryptophan, arginine and cysteine. In vitro simulated digestion experiments showed that the addition of C3G significantly reduced the digestibility of MP, with the covalent conjugate showing lower digestibility than the non-covalent conjugate. Moreover, the digestibility of protein decreased more during intestinal digestion, possibly because covalent cross-linking of C3G and MP further inhibited trypsin targeting sites (lysine and arginine). Covalent cross-linking of C3G with myofibrillar proteins significantly affected protein structure and reduced protein digestibility by occupying more trypsin binding sites. © 2023 Society of Chemical Industry.

Insight into the structural basis of the dual inhibitory mode of Lima bean (Phaseolus lunatus) serine protease inhibitor.

Bovine pancreatic trypsin was crystallized, in-complex with Lima bean trypsin inhibitor (LBTI) (Phaseolus lunatus L.), in the form of a ternary complex. LBTI is a Bowman-Birk-type bifunctional serine protease inhibitor, which has two independent inhibitory loops. Both of the loops can inhibit trypsin, however, only the hydrophobic loop is specific for inhibiting chymotrypsin. The structure of trypsin incomplex with the LBTI has been solved and refined at 2.25 Å resolution, in the space group P41, with Rwork /Rfree values of 18.1/23.3. The two binding sites of LBTI differ in only two amino acids. Lysine and leucine are the key residues of the two different binding loops positioned at the P1, and involved in binding the S1 binding site of trypsin. The asymmetric unit cell contains two molecules of trypsin and one molecule of LBTI. The key interactions include hydrogen bonds between LBTI and active site residues of trypsin. The 3D structure of the enzyme-inhibitor complex provided details insight into the trypsin inhibition by LBTI. To the best of our knowledge, this is the first report on the structure of trypsin incomplex with LBTI.

Pathogenic cellular role of the p.L104P human cationic trypsinogen variant in chronic pancreatitis.

Mutations in the PRSS1 gene encoding human cationic trypsinogen are associated with hereditary and sporadic chronic pancreatitis. High-penetrance PRSS1 mutations found in hereditary pancreatitis alter activation and/or degradation of cationic trypsinogen, thereby promoting intrapancreatic trypsinogen activation. In contrast, a number of rare PRSS1 variants identified in subjects with sporadic chronic pancreatitis cause misfolding and endoplasmic reticulum (ER) stress. Mutation p.L104P is unique among natural PRSS1 variants, since it affects the substrate binding site of trypsin. The aim of the present study was to establish the clinical significance of variant p.L104P through functional analysis. We found that p.L104P trypsin exhibited decreased activity on peptide and protein substrates; however, autoactivation was slightly accelerated. Remarkably, binding of the physiological trypsin inhibitor serine protease inhibitor Kazal type 1 (SPINK1) was decreased by 70-fold. In the presence of the trypsinogen-degrading enzyme chymotrypsin C, mutant p.L104P autoactivated to higher trypsin levels than wild-type trypsinogen. This apparent resistance to degradation was due to slower cleavage at Arg(122) rather than Leu(81) Finally, secretion of mutant p.L104P from transfected cells was markedly reduced due to intracellular retention and aggregation with concomitant elevation of ER stress markers. We conclude that PRSS1 variant p.L104P exhibits a variety of phenotypic changes that can increase risk for chronic pancreatitis. Mutation-induced misfolding and associated ER stress are the dominant effects that support a direct pathogenic role in chronic pancreatitis.

Structural insights regarding an insecticidal Talisia esculenta protein and its biotechnological potential for Diatraea saccharalis larval control

Talisin is a seed-storage protein from Talisia esculenta that presents lectin-like activities, as well as proteinase-inhibitor properties. The present study aims to provide new in vitro and in silico biochemical information about this protein, shedding some light on its mechanistic inhibitory strategies. A theoretical three-dimensional structure of Talisin bound to trypsin was constructed in order to determine the relative interaction mode. Since the structure of non-competitive inhibition has not been elucidated, Talisin-trypsin docking was carried out using Hex v5.1, since the structure of non-competitive inhibition has not been elucidated. The predicted non-coincidence of the trypsin binding site is completely different from that previously proposed for Kunitz-type inhibitors, which demonstrate a substitution of an Arg(64) for the Glu(64) residue. Data, therefore, provide more information regarding the mechanisms of non-competitive plant proteinase inhibitors. Bioassays with Talisin also presented a strong insecticide effect on the larval development of Diatraea saccharalis, demonstrating LD50 and ED50 of ca. 2.0% and 1.5%, respectively.

Inhibition of endogenous α-amylase and protease of Aspergillus flavus by trypsin inhibitor from cultivated and wild-type soybean

The anti-Aspergillus flavus activity of trypsin inhibitor (TI) from cultivated and wild-type soybean (SBTI and WBTI) was investigated in order to confirm its ability to reduce the activity of endogenous α-amylase, protease enzymes and production of aflatoxin B1 secreted by A. flavus. In the current study, it was demonstrated that purified SBTI/WBTI belonged to the family of Bowman-Birk TI, based on evidence from amino acid composition, the presence of two independent binding sites for trypsin and chymotrypsin, and a lysine residue as the active site for trypsin inhibition. Studying the inhibition of A. flavus showed that the effect of SBTI/SBTI on A. flavus α-amylase activity and aflatoxin B1 production depended on TI concentration. However, no inhibitory effect was observed when sufficient exogenous α-amylase (EC 3.2.1.1, from Bacillus subtilis) was added. The resistance to A. flavus infection was partially due to the ability of SBTI/WBTI to inhibit α-amylase activity, thereby limiting the availability of hydrolyzed reducing sugar for fungal growth and further suppressing aflatoxin B1 biosynthesis. In addition, the relationship between SBTI/WBTI levels and fungal protease expression revealed that A. flavus released a certain quantity of endogenous proteases into the culture medium, and the decreased activity of protease and production of aflatoxin B1 suggested that the inhibition activity might also be mediated by SBTI/WBTI as A. flavus protease inhibitor activity.

Phenotypic Variability of the Homozygous IVS3+2T>C Mutation in the Serine Protease Inhibitor Kazal Type 1 (SPINK1) Gene in Patients with Chronic Pancreatitis

Chronic pancreatitis (CP) is a progressive inflammatory disease that eventually results in the impairment of exocrine and endocrine functions of the pancreas. Recent studies have shown an association between mutations in the serine protease inhibitor Kazal type 1 (SPINK1) gene and CP. SPINK1 provides the first line of defense against prematurely activated trypsinogen by physically blocking the active site of trypsin. The IVS3+2T>C (c.194+2T>C) mutation is a loss-of-function splicing mutation; it affects the consensus splicing donor site in intron 3 and may cause the skipping of the entire exon 3, where the trypsin-binding site is located. We report here three CP patients carrying this mutation in a homozygous form, with no noticeable family history of pancreatitis. The first patient is a 25-year-old male with juvenile-onset idiopathic CP. He suffered from repeated attacks of pancreatitis since 5 years old and underwent pancreatico-jejunostomy. He complained of epigastralgia, and was diagnosed as obstructive pancreatitis in the area of the accessory pancreatic duct. The second patient is a 75-year-old male with alcoholic CP. He did not have apparent attacks of pancreatitis, but had numerous calcifications throughout the pancreas and confirmed exocrine failure and diabetes mellitus. The last patient is a 44-year-old female with late-onset idiopathic CP. She suffered from repeated attacks of pancreatitis since 32 years old. She had numerous stones in the main pancreatic duct in the pancreas head and confirmed exocrine failure. The clinical courses of these patients are apparently different, indicating the phenotypic variability of the SPINK1 IVS3+2T>C mutation-associated CP.

Seeking “Etwas Neues”—From Bioorganic Chemistry to Alzheimer's Disease—

Introduction to bioorganic chemistry by Prof. Kanaoka at the entrance of my research works affects greatly throughout the life afterward. Chemical modification studies of enzyme proteins taught me quality of chemical reactions. For example, triethyloxonium fluoroborate (Et3O+ BF4(-)), a Meerwein reagent, selectively reacted with a particular carboxyl group (Asp-177) in the substrate binding site of trypsin, even though the reaction was performed in aqueous solution. A series of ion channel studies intoxicate me how exciting the science works are. Purification of sodium channel protein from electric eels initiated the collaboration work to reveal total primary structure of the molecule, as an inaugurating work of ion channel molecules. Photoaffinity labeling proved to be an efficient method to elucidate ligand binding sites, such as TTX binding site within the sodium channel and the sites for calcium anatagonists in L-type calcium channels. Encounter with CD36 molecule expands our works to more pathobiochemical field. We revealed CD36, a class B scavenger receptor, is related to development of atherosclerosis by phagocytosis of ox-LDL in macrophages and even matured adipocytes. In microglia, however, CD36 plays clearance role of oligomeric beta-amyloid peptides in IL-4 activated type-2 microglia, suggesting the activation of type-2 microglia may be useful for developing a new method to treat or prevent from Alzheimer's disease.

N34S Mutation in the SPINK1 Gene Is Not Associated With Alternative Splicing

Previous studies have shown an association between chronic pancreatitis (CP) and mutations, especially the N34S mutation, in the serine protease inhibitor Kazal type 1 (SPINK1) gene. But the underlying molecular mechanisms are unknown. The aberrant splicing caused by the cosegregating intronic mutations might play a role, but this hypothesis has not been tested. We here examined the messenger RNA sequences of the SPINK1 gene in patients carrying the mutations. RNA was isolated from the surgically resected pancreas of 2 CP patients carrying the homozygous N34S mutation and from the gastric biopsy specimen of a CP patient carrying the heterozygous [-215G>A; IVS3+2T>C] mutation. The entire coding region of the SPINK1 gene was amplified by reverse transcription-polymerase chain reaction, subcloned, and sequenced. The level of the wild-type SPINK1 transcript was assessed by real-time polymerase chain reaction. Alternative splicing was not associated with the N34S mutation. On the other hand, the [-215G>A; IVS3+2T>C] mutation caused skipping of whole exon 3, where the trypsin binding site is located. This mutated protein was predicted to consist of 63 amino acids: deletion of amino acid sequence from residues 30 to 64 and shifting of reading frame at amino acid 65 with a novel stop codon. The expression of the wild-type SPINK1 transcript was decreased to 62% of the healthy control in the CP patient carrying the heterozygous [-215G>A; IVS3+2T>C] mutation. Splicing mutation might represent a mechanism for SPINK1-associated CP, but the N34S mutation is not associated with alternative splicing.

Crystal structure of the anticarcinogenic Bowman–Birk inhibitor from snail medic ( Medicago scutellata) seeds complexed with bovine trypsin

The structure of the ternary complex of the anticarcinogenic Bowman–Birk protease inhibitor purified from snail medic ( Medicago scutellata) seeds (MSTI) and two molecules of bovine trypsin has been solved by X-ray diffraction analysis of single crystals to a resolution of 2.0 Å. This is the highest resolution model of a ternary complex of this type currently available. The two binding loops of the MSTI differ in only one amino acid and have in both cases an arginine in position P1. The distances between the residues of the inhibitor at the binding interface and the trypsin side chains that recognize them are almost identical in the two sites. When compared to the NMR model of the uncomplexed MSTI, the inhibitor in the functional assembly with trypsin shows the largest differences in the two P2′ residues. Compared with the similar ternary complex of the soybean trypsin inhibitor, this model shows very small differences in the polypeptide chain of the trypsin binding sites and its largest difference in the area between Asp 26 and His 32 of the MSTI which in the soybean inhibitor has an extra Leu inserted in position 29.

Solution Structure of a Novel C2-Symmetrical Bifunctional Bicyclic Inhibitor Based on SFTI-1

A novel bifunctional bicyclic inhibitor has been created that combines features both from the Bowman-Birk inhibitor (BBI) proteins, which have two distinct inhibitory sites, and from sunflower trypsin inhibitor-1 (SFTI-1), which has a compact bicyclic structure. The inhibitor was designed by fusing together a pair of reactive loops based on a sequence derived from SFTI-1 to create a backbone-cyclized disulfide-bridged 16-mer peptide. This peptide has two symmetrically spaced trypsin binding sites. Its synthesis and biological activity have been reported in a previous communication [Jaulent and Leatherbarrow, 2004, PEDS 17, 681]. In the present study we have examined the three-dimensional structure of the molecule. We find that the new inhibitor, which has a symmetrical 8-mer half-cystine CTKSIPP'I' motif repeated through a C2 symmetry axis also shows a complete symmetry in its three-dimensional structure. Each of the two loops adopts the expected canonical conformation common to all BBIs as well as SFTI-1. We also find that the inhibitor displays a strong and unique structural identity, with a notable lack of minor conformational isomers that characterise most reactive site loop mimics examined to date as well as SFTI-1. This suggests that the presence of the additional cyclic loop acts to restrict conformational mobility and that the deliberate introduction of cyclic symmetry may offer a general route to locking the conformation of beta-hairpin structures.

Secondary Binding Site of Trypsin: Revealed by Crystal Structure of Trypsin-Peptide Complex

Designed synthetic heterochiral peptides, when added to porcine trypsin, resulted in reduction of enzyme activity. The crystal structure of a complex formed between porcine trypsin and a heterochiral hepta peptide Boc-Pro-DAsp-Aib-Leu-Aib-Leu-Ala-NHMe has been determined at 1.9 Å resolution. The hepta peptide does not bind at the active site, but is located in the interstitial region, and interacts with the calcium-binding loop (residues 60–80). The bound peptide interacts with the active site residue Ser195 through an acetate ion, and with Lys 60 mediated by water molecules. The structure, when compared with the other trypsin-peptide complexes, suggests that the flexibility of surface loops, concerted movement of the loops towards the active site, and the interaction of the bound peptide with Lys 60, may be responsible for the reduction in enzyme activity. This study provides a structural evidence for the earlier biochemical observation regarding the role of surface loops in the catalysis of the enzyme.

Trypsin Inhibition by a Peptide Hormone: Crystal Structure of Trypsin–Vasopressin Complex

The large variety of serine protease inhibitors, available from various sources such as tissues, microorganisms, plants, etc., play an important role in regulating the proteolytic enzymes. The analysis of protease-inhibitor complexes helps in understanding the mechanism of action, as well as in designing inhibitors. Vasopressin, an anti-diuretic nonapeptide hormone, is found to be an effective inhibitor of trypsin, with a K(i) value of 5 nM. The crystal structure of the trypsin-vasopressin complex revealed that vasopressin fulfils all the important interactions for an inhibitor, without any break in the scissile peptide bond. The cyclic nature due to a disulfide bridge between Cys1 and Cys6 of vasopressin provides structural rigidity to the peptide hormone. The trypsin-binding site is located at the C terminus, while the neurophysin-binding site is at the N terminus of vasopressin. This study will assist in designing new peptide inhibitors. This study suggests that vasopressin inhibition of trypsin may have unexplored biological implications.

Probing the Active Site of Trypsin with Rose Bengal: Insights into the Photodynamic Inactivation of the Enzyme¶

ABSTRACTIn this work the active site of trypsin has been probed with the dye rose bengal. The dye binds competitively to the enzyme, and it can be used as a probe of the active site of the enzyme. On the basis of the emission wavelength, the binding site of trypsin is relatively polar and is similar to that of acetone in its polarity. The triplet state of rose bengal is quenched by trypsin. This quenching may be caused by the tryptophan and tyrosine residues that are in the near vicinity of the trypsin active site. This quenching can compete with the formation of singlet oxygen from the excited triplet state of rose bengal. We demonstrate that the singlet oxygen involved in the photoinactivation of trypsin is produced by the free rose bengal in solution and the bound dye is incapable of producing singlet oxygen. This explains the lack of correlation between photoinactivation efficiency and sensitizer binding capability previously reported by Wade and Spikes.

Probing the active site of trypsin with rose bengal: insights into the photodynamic inactivation of the enzyme.

In this work the active site of trypsin has been probed with the dye rose bengal. The dye binds competitively to the enzyme, and it can be used as a probe of the active site of the enzyme. On the basis of the emission wavelength, the binding site of trypsin is relatively polar and is similar to that of acetone in its polarity. The triplet state of rose bengal is quenched by trypsin. This quenching may be caused by the tryptophan and tyrosine residues that are in the near vicinity of the trypsin active site. This quenching can compete with the formation of singlet oxygen from the excited triplet state of rose bengal. We demonstrate that the singlet oxygen involved in the photoinactivation of trypsin is produced by the free rose bengal in solution and the bound dye is incapable of producing singlet oxygen. This explains the lack of correlation between photoinactivation efficiency and sensitizer binding capability previously reported by Wade and Spikes.

Identification of a Residue Critical for Maintaining the Functional Conformation of BPTI

The effects of amino acid replacements on the backbone dynamics of bovine pancreatic trypsin inhibitor (BPTI) were examined using 15N NMR relaxation experiments. Previous studies have shown that backbone amide groups within the trypsin-binding region of the wild-type protein undergo conformational exchange processes on the μs time scale, and that replacement of Tyr35 with Gly greatly increases the number of backbone atoms involved in such motions. In order to determine whether these mutational effects are specific to the replacement of this residue with Gly, six additional replacements were examined in the present study. In two of these, Tyr35 was replaced with either Ala or Leu, and the other four were single replacements of Tyr23, Phe33, Asn43 or Asn44, all of which are highly buried in the native structure and conserved in homologous proteins. The Y35A and Y35L mutants displayed dynamic properties very similar to those of the Y35G mutant, with the backbone segments including residues 10–19 and 32–44 undergoing motions revealed by enhanced 15N transverse relaxation rates. On the other hand, the Y23L, N43G and N44A substitutions caused almost no detectable changes in backbone dynamics, on either the ns–ps or ms–μs time scales, even though each of these replacements significantly destabilizes the native conformation. Replacement of Phe33 with Leu caused intermediate effects, with several residues that have previously been implicated in motions in the wild-type protein displaying enhanced transverse relaxation rates. These results demonstrate that destabilizing amino acid replacements can be accommodated in a native protein with dramatically different effects on conformational dynamics and that Tyr35 plays a particularly important role in defining the conformation of the trypsin-binding site of BPTI.

Overlapping binding sites for trypsin and papain on a Kunitz-type proteinase inhibitor from Prosopis juliflora.

Proteinase inhibitors are among the most promising candidates for expression by transgenic plants and consequent protection against insect predation. However, some insects can respond to the threat of the proteinase inhibitor by the production of enzymes insensitive to inhibition. Inhibitors combining more than one favorable activity are therefore strongly favored. Recently, a known small Kunitz trypsin inhibitor from Prosopis juliflora (PTPKI) has been shown to possess unexpected potent cysteine proteinase inhibitory activity. Here we show, by enzyme assay and gel filtration, that, unlike other Kunitz inhibitors with dual activities, this inhibitor is incapable of simultaneous inhibition of trypsin and papain. These data are most readily interpreted by proposing overlapping binding sites for the two enzymes. Molecular modeling and docking experiments favor an interaction mode in which the same inhibitor loop that interacts in a canonical fashion with trypsin can also bind into the papain catalytic site cleft. Unusual residue substitutions at the proposed interface can explain the relative rarity of twin trypsin/papain inhibition. Other changes seem responsible for the relative low affinity of PTPKI for trypsin. The predicted coincidence of trypsin and papain binding sites, once confirmed, would facilitate the search, by phage display for example, for mutants highly active against both proteinases.

Structural determinants of trypsin affinity and specificity for cationic inhibitors.

The binding free energies of four inhibitors to bovine beta-trypsin are calculated. The inhibitors use either ornithine, lysine, or arginine to bind to the S1 specificity site. The electrostatic contribution to binding free energy is calculated by solving the finite difference Poisson-Boltzmann equation, the contribution of nonpolar interactions is calculated using a free energy-surface area relationship and the loss of conformational entropy is estimated both for trypsin and ligand side chains. Binding free energy values are of a reasonable magnitude and the relative affinity of the four inhibitors for trypsin is correctly predicted. Electrostatic interactions are found to oppose binding in all cases. However, in the case of ornithine- and lysine-based inhibitors, the salt bridge formed between their charged group and the partially buried carboxylate of Asp189 is found to stabilize the complex. Our analysis reveals how the molecular architecture of the trypsin binding site results in highly specific recognition of substrates and inhibitors. Specifically, partially burying Asp189 in the inhibitor-free enzyme decreases the penalty for desolvation of this group upon complexation. Water molecules trapped in the binding interface further stabilize the buried ion pair, resulting in a favorable electrostatic contribution of the ion pair formed with ornithine and lysine side chains. Moreover, all side chains that form the trypsin specificity site are partially buried, and hence, relatively immobile in the inhibitor-free state, thus reducing the entropic cost of complexation. The implications of the results for the general problem of recognition and binding are considered. A novel finding in this regard is that like charged molecules can have electrostatic contributions to binding that are more favorable than oppositely charged molecules due to enhanced interactions with the solvent in the highly charged complex that is formed.

How the Substitution of K188 of Trypsin Binding Site by Aromatic Amino Acids Can Influence the Processing of β-Casein

Aspartyl 189 residue of trypsin is known to be essential for specific lysis of Arg-X and Lys-X bonds. Undertaking to modulate the catalytic properties of this protease, otherwise highly conserved K188 was replaced with aromatic amino acid residues aiming the perturbation of the electrostatics and the amplifying of hydrophobic interactions of the substrate binding site. The catalytic properties of the mutants K188F, K188Y, and K188W were measured at pH 7, 8, 9, and 10 using a pair of synthetic tetrapeptidep-nitroanilide substrates and beta-casein. The kinetic analysis reveals that all the mutants conserve the native trypsin capacity to split peptide bonds containing arginyl and lysyl residues. Surprisingly, however, depending on mutation, the optimum pH of activity changes. As demonstrated only by proteolysis of a natural substrate, all mutants cleave also peptide bonds involving asparagine and glutamine. These stuttered cleavage sites are close to the β-casein fragments in β-sheet according to Hydrophobic Cluster Analysis.

Enhanced protein flexibility caused by a destabilizing amino acid replacement in BPTI

A genetically engineered variant of bovine pancreatic trypsin inhibitor (Y35G BPTI) has been shown previously by X-ray crystallography to have a three-dimensional structure dramatically different from that of the wild-type protein, particularly in the protease-binding region of the molecule. Yet, the Y35G variant is a potent trypsin inhibitor. Described here are 15N NMR relaxation studies to compare the backbone dynamics of Y35G BPTI to those of the wild-type protein. The Tyr35 → Gly substitution increased the transverse relaxation rates of more than one third of all backbone amide groups, but had little effect on the longitudinal relaxation rates, indicating that the substitution facilitates relatively slow backbone motions, estimated to be on the microsecond time-scale. The results indicate that the residues making up the trypsin-binding site undergo large and relatively slow conformational changes in solution, estimated to be on the 5 to 20 μs time-scale. It is thus likely that the crystal structure represents only one of multiple interconverting conformations in solution, only a fraction of which may be competent for binding trypsin. The large thermodynamic destabilization associated with this substitution may arise, in part, from a loss in cooperativity among the multiple stabilizing interactions that are normally favored by the highly ordered structure of the wild-type protein. These results suggest that fully understanding the effects of amino acid replacements on the functional and thermodynamic properties of proteins may often require analysis of the dynamic, as well as the structural, properties of altered proteins.

The role of the Cys191-Cys220 disulfide bond in trypsin: new targets for engineering substrate specificity.

The S1 binding site of trypsin is cross-linked by the conserved Cys191-Cys220 disulfide bond. The substitution of Cys191 and Cys220 with Ala decreases the activity of trypsin by 20-200-fold as measured by kcat/K(m) for the hydrolysis of amide substrates; in contrast, ester hydrolysis is decreased by < 10-fold. Similar decreases are observed in the hydrolysis of oligopeptide and single amino acid substrates. This decrease in activity results from a decrease in the acylation rate. The substrate binding and deacylation rate are not affected by the loss of the disulfide bond. C191A/C220A binds BPTI with the same affinity as trypsin, although the affinity of benzamidine is decreased 10-fold and the affinity of leupeptin is decreased 1000-fold. The CD spectrum of C191A/C220A displays significant differences from that of trypsin; these differences most likely result from the loss of the disulfide chromophore, although perturbation of enzyme structure cannot be discounted. The loss of the Cys191-Cys220 disulfide has no effect on the stability of trypsin as measured by urea denaturation. Single and double substitutions of Ser at positions 191 and 220 have a similar activity to C191A/C220A. These results indicate that the Cys191-Cys220 disulfide bond is not essential for the function, structure or stability of trypsin.