Bovine Trypsinogen Research Articles

Coevolved Positions Represent Key Functional Properties in the Trypsin-Like Serine Proteases Protein Family

Trypsin-like serine proteases are a group of homologous enzymes which exert multiple roles in both vertebrate and invertebrate organisms. Key properties of these enzymes include their activation from an inactive zymogen form to their active form by cleavage of residues in their N-terminus, the presence of a conserved catalytic triad of residues, and the existence of different patterns of substrate selectivity for residue cleavage between the various members of this protein family. In this article, we apply the decomposition of residue coevolution networks computational method to find sets of residues related to some of these key properties, especially to zymogen activation. Positive selection detection, normal modes analysis, and the calculation of thermal couplings between the bovine trypsinogen and bovine trypsin structures residues yielded further information for understanding the zymogen activation process and highlighted the importance of some of the coevolved set residues during these transitions.

Biochemical characterization of recombinant Penaeus vannamei trypsinogen.

Trypsinogens are the inactive precursors of trypsins (EC 3.4.21.4), which are digestive serine proteases. Despite knowing the properties of trypsins from Pacific white shrimp, Penaeus vannamei, the biochemical properties of shrimp trypsinogens including activation mechanisms and kinetics are unknown, due to difficulties isolating them from natural sources. In the present work, we describe the purification and biochemical characterization of four trypsinogen-like isoforms from recombinant P. vannamei trypsinogen, with a special emphasis on understanding its activation kinetics. The major trypsinogen-like isoform had an apparent molecular mass of 29 kDa. The other three forms of recombinant trypsinogen were: an N-glycosylated form of 32 kDa, a possibly O-glycosylated form of 41 kDa, and a likely double-chain form with a subunit of 23 kDa. The autoactivation profile of three-recombinant trypsinogen-like isoforms showed increased trypsin activity at a rate that was higher than that of bovine trypsinogen. This confirms the hypothesis proposed in the literature of a rapid trypsinogen autoactivation in the absence of aspartates in the activation peptide as it is for P. vannamei trypsinogen.

Cathepsin B-Mediated Activation of Trypsinogen in Endocytosing Macrophages Increases Severity of Pancreatitis in Mice

BACKGROUND & AIMS:Acute pancreatitis is characterized by premature intracellular activation of digestive proteases within pancreatic acini and a consecutive systemic inflammatory response. We investigated how these processes interact during severe pancreatitis in mice.METHODS:Pancreatitis was induced in C57Bl/6 wild-type (control), cathepsin B (CTSB)- knockout, and cathepsin L-knockout mice by partial pancreatic duct ligation with supramaximal caerulein injection, or by repetitive supramaximal caerulein injections alone. Immune cells that infiltrated the pancreas were characterized by immunofluorescence detection of Ly6g, CD206, and CD68. Macrophages were isolated from bone marrow and incubated with bovine trypsinogen or isolated acinar cells; the macrophages were then transferred into pancreatitis control or cathepsin-knockout mice. Activities of proteases and nuclear factor (NF)-κB were determined using fluorogenic substrates and trypsin activity was blocked by nafamostat. Cytokine levels were measured using a cytometric bead array. We performed immunohistochemical analyses to detect trypsinogen, CD206, and CD68 in human chronic pancreatitis (n = 13) and acute necrotizing pancreatitis (n = 15) specimens.RESULTS:Macrophages were the predominant immune cell population that migrated into the pancreas during induction of pancreatitis in control mice. CD68-positive macrophages were found to phagocytose acinar cell components, including zymogen-containing vesicles, in pancreata from mice with pancreatitis, as well as human necrotic pancreatic tissues. Trypsinogen became activated in macrophages cultured with purified trypsinogen or co-cultured with pancreatic acini and in pancreata of mice with pancreatitis; trypsinogen activation required macrophage endocytosis and expression and activity of CTSB, and was sensitive to pH. Activation of trypsinogen in macrophages resulted in translocation of NF-kB and production of inflammatory cytokines; mice without trypsinogen activation (CTSB-knockout mice) in macrophages developed less severe pancreatitis compared with control mice. Transfer of macrophage from control mice to CTSB-knockout mice increased the severity of pancreatitis. Inhibition of trypsin activity in macrophages prevented translocation of NF-κB and production of inflammatory cytokines.CONCLUSIONS:Studying pancreatitis in mice, we found activation of digestive proteases to occur not only in acinar cells but also in macrophages that infiltrate pancreatic tissue. Activation of the proteases in macrophage occurs during endocytosis of zymogen-containing vesicles, and depends on pH and CTSB. This process involves macrophage activation via NF-κB-translocation, and contributes to systemic inflammation and severity of pancreatitis.

Improved Production of Active Streptomyces griseus Trypsin with a Novel Auto-Catalyzed Strategy.

N-terminal sequences play crucial roles in regulating expression, translation, activation and enzymatic properties of proteins. To reduce cell toxicity of intracellular trypsin and increase secretory expression, we developed a novel auto-catalyzed strategy to produce recombinant trypsin by engineering the N-terminus of mature Streptomyces griseus trypsin (SGT). The engineered N-terminal peptide of SGT was composed of the thioredoxin, glycine-serine linker, His6-tag and the partial bovine trypsinogen pro-peptide (DDDDK). Furthermore, we constructed a variant TLEI with insertion of the artificial peptide at N-terminus and site-directed mutagenesis of the autolysis residue R145. In fed-batch fermentation, the production of extracellular trypsin activity was significantly improved to 47.4 ± 1.2 U·ml−1 (amidase activity, 8532 ± 142.2 U·ml−1 BAEE activity) with a productivity of 0.49 U·ml−1·h−1, which was 329% greater than that of parent strain Pichia pastoris GS115-SGT. This work has significant potential to be scaled-up for microbial production of SGT. In addition, the N-terminal peptide engineering strategy can be extended to improve heterologous expression of other toxic enzymes.

COST-EFFECTIVE AND EFFICIENT APPARATUS FOR ELECTROELUTION OF MICRO- AND MACROGRAM QUANTITIES OF PROTEINS FROM POLYACRYLAMIDE GELS

Protein recovery from gel electrophoresis plays a significant role in functional genomics and proteomics. To assist in this, a simple, cost-effective, and efficient apparatus for electroelution of proteins has been designed. The performance of the apparatus was demonstrated using the proteins bovine serum albumin (BSA), phosphorylase, ovalbumin, pepsin, and trypsinogen. In all the cases the yield of elution was found to be consistently greater than 85% and the proteins could be eluted without degradation in less than 15 min. The utility of this method can be extended to protein elution from denatured and native polyacrylamide gels, DNA purification from agarose gels, and oligomeric primers purification from polyacrylamide gels. In addition to this, the method offers an effortless purification and characterization of microbial extracellular proteins. The eluted proteins can be directly used in N-terminal amino acid sequencing, and in amino acid and proteomics analyses.

Production of functional recombinant bovine trypsin in transgenic rice cell suspension cultures

A synthetic bovine trypsinogen (sbTrypsinogen) was synthesized on the basis of rice-optimized codon usage via an overlap PCR strategy, prior to being expressed under the control of the sucrose starvation-inducible rice α-amylase 3D (RAmy3D) promoter. Secretion of trypsin into the culture medium was achieved by using the existing signal peptide. The plant expression vector was introduced into rice calli ( Oryza sativa L. cv. Dongjin), mediated by Agrobacterium tumefaciens. The integration of the sbTrypsinogen gene into the chromosome of the transgenic rice callus was verified via genomic DNA PCR amplification, and sbTrypsin expression in transgenic rice suspension cells was confirmed via Northern blot analysis. Western blot analysis detected glycosylated proteins in the culture medium, having masses from 24 to 26 kDa, following induction by sugar starvation. Proteolytic activity of the rice-derived trypsin was confirmed by gelatin zymogram, and was similar to that of the commercial bovine-produced trypsin. The yields of sbTrypsin that accumulated in the transgenic rice cell suspension medium were 15 mg/L at 5 days after sugar starvation.

Role of polymer–protein interaction on partitioning pattern of bovine pancreatic trypsinogen and alpha-chymotrypsinogen in polyethyleneglycol/sodium tartrate aqueous two-phase systems

The partitioning pattern of bovine trypsinogen (TRPz) and alpha-chymotrypsinogen (ChTRPz) was investigated in a low impact aqueous two-phase system formed by polyethyleneglycol (PEG) and sodium tartrate (NaTart) pH 5.00. ChTRPz exhibited higher partition coefficients than TRPz did in all the assayed systems. The decrease in PEG molecular weight and the increase in tie line length were observed to displace the partitioning equilibrium of both proteins to the top phase, while phase volume ratios in the range 0.5–1.5 showed not to affect protein partitioning behaviour. Systems formed by PEG of molecular weight 600 with composition corresponding to a high tie line length (PEG 12.93%, w/w and NaTart 21.20%, w/w) are able to recover most of both zymogens in the polymer-enriched phase. A crucial role of PEG–protein interaction in the partitioning mechanism was evidenced by isothermal calorimetric titrations. The major content of highly exposed tryptophan rests, present in ChTRPz molecule, could be considered to be determinant of its higher partition coefficient due to a selective charge transfer interaction with PEG molecule. A satisfactory correlation between partition coefficient and protein surface hydrophobicity was observed in systems formed with PEGs of molecular weight above 4000, this finding being relevant in the design of an extraction process employing aqueous two-phase systems.

Features of partitioning pattern of two pancreatic enzymatic precursors: Trypsinogen and chymotrypsinogen in polyethyleneglycol–sodium citrate aqueous biphasic systems

The partitioning behaviour of bovine trypsinogen and alpha-chymotrypsinogen, enzymatic precursors with similar physicochemical properties, was investigated in different polyethyleneglycol/sodium citrate aqueous two-phase systems. The effect of different factors such as polyethyleneglycol molecular weight, pH, tie line length and temperature was also examined. The increase of pH and the decrease of polyethyleneglycol molecular weight displaced the partitioning equilibrium of both proteins to the top phase. An enthalpy–entropy compensation pattern was observed, indicating the participation of water molecules in the partitioning mechanism. TRPz phase equilibrium showed to be more displaced to the citrate-rich phase than ChTRPz for most of the assayed systems. From a practical view, the aqueous two-phase system formed by polyethylenglycol of molecular weight 1450 and sodium citrate pH 8.20 showed the best capability for separating both proteins. When a mixture formed by equal quantities of both zymogens was partitioned in this system, significant recoveries (about 60%) were obtained. Purity values were improved significantly (84–89%) by either developing a second extractive step or increasing the top–bottom volume ratio.

Systematic Analysis of Aggregates from 38 Kinds of Non Disease-Related Proteins: Identifying the Intrinsic Propensity of Polypeptides to Form Amyloid Fibrils

The ability to form amyloid fibrils from a wide range of proteins would open up the opportunity to augment studies of the molecular basis of amyloid fibril formation. We investigated 36 different conditions with respect to four model proteins to evaluate their ability to form amyloid fibrils. In a 5% ethanol solution at pH 2 at 57 degrees C, hen egg white lysozyme, bovine beta-lactoglobulin, and bovine trypsinogen formed mature-type fibrils, while only histone H2A formed immature-type fibrils. Under these conditions, 25 of the 38 proteins formed amyloid fibrils. In addition, three additional proteins formed fibrils in a solution containing 5% trifluoroethanol instead of 5% ethanol. In summary, a total 28 proteins formed amyloid fibrils. Under these extreme conditions, chemical fragmentation was observed. Destabilization of the native structure, strengthening of hydrogen bonds, and chemical fragmentation are thought to play important roles in the formation of amyloid fibrils.

Human cationic trypsinogen is sulfated on Tyr154

The crystal structure of human pancreatic cationic trypsin showed the chemical modification of Tyr154, which was originally described as phosphorylation [Gaboriaud C, Serre L, Guy-Crotte O, Forest E & Fontecilla-Camps JC (1996) J Mol Biol259, 995-1010]. Here we report that Tyr154 is sulfated, not phosphorylated. Cationic and anionic trypsinogens were purified from human pancreatic juice and subjected to alkaline hydrolysis. Modified tyrosine amino acids were separated on a Dowex cation-exchange column and analyzed by thin layer chromatography. Both human cationic and anionic trypsinogens contained tyrosine sulfate, but no tyrosine phosphate, whereas bovine trypsinogen contained neither. Furthermore, incorporation of [(35)S]SO(4) into human cationic trypsinogen transiently expressed by human embryonic kidney 239T cells was demonstrated. Mutation of Tyr154 to Phe abolished radioactive sulfate incorporation, confirming that Tyr154 is the site of sulfation in cationic trypsinogen. Sulfated pancreatic cationic trypsinogen exhibited faster autoactivation than a nonsulfated recombinant form, suggesting that tyrosine sulfation of trypsinogens might enhance intestinal digestive zymogen activation in humans. Finally, sequence alignment revealed that the sulfation motif is only conserved in primate trypsinogens, suggesting that typsinogen sulfation is absent in other vertebrates.

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

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

Allostery in a Coarse-Grained Model of Protein Dynamics

We propose a criterion for optimal parameter selection in coarse-grained models of proteins and develop a refined elastic network model (ENM) of bovine trypsinogen. The unimodal density-of-states distribution of the trypsinogen ENM disagrees with the bimodal distribution obtained from an all-atom model; however, the bimodal distribution is recovered by strengthening interactions between atoms that are backbone neighbors. We use the backbone-enhanced model to analyze allosteric mechanisms of trypsinogen and find relatively strong communication between the regulatory and active sites.

Binding of bovine pancreatic trypsin inhibitor to trypsinogen: spectroscopic and volumetric studies.

We have investigated the binding of bovine pancreatic trypsin inhibitor (BPTI) to bovine trypsinogen by combining ultrasonic velocimetry, high precision densimetry, and fluorescence spectroscopy. We report the changes in volume, adiabatic compressibility, van't Hoff enthalpy, entropy, and free energy that accompany the association of the two proteins at 25 degrees C and pH 8.0. We have used the measured changes in volume and compressibility in conjunction with available structural data to characterize the binding-induced changes in the hydration properties and intrinsic packing of the two proteins. Our estimate reveals that 110 +/- 40 water molecules become released to the bulk from the hydration shells of BPTI and trypsinogen. Furthermore, we find that the intrinsic coefficient of adiabatic compressibility of the two proteins decreases by 14 +/- 2%, which is suggestive of the binding-induced rigidification of the proteins' interior. BPTI-trypsinogen association is an entropy-driven event which proceeds with an unfavorable change in enthalpy. The favorable change in entropy results from partial compensation between two predominant terms. Namely, a large favorable change in hydrational entropy slightly prevails over a close in magnitude but opposite in sign change in configurational entropy. The reduction in configurational entropy and, consequently, protein dynamics is consistent with the observed decrease in intrinsic compressibility. In general, results of this work emphasize the vital role that water plays in modulating protein recognition events.

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

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

High-level bacterial expression and 15N-alanine-labeling of bovine trypsin. Application to the study of trypsin-inhibitor complexes and trypsinogen activation by NMR spectroscopy.

We describe here the high-level expression of bovine trypsinogen in E. coli, its refolding and activation to beta-trypsin, and the selective incorporation of (15)N-labeled alanine through supplementation of the growth medium. Using this procedure, we expressed (15)N-labeled S195A trypsinogens, both on a wild-type and on a D189S background, in amounts suitable for NMR spectroscopy. 2D [(1)H-(15)N]-HSQC NMR was used to follow conformational changes upon activation of trypsinogen and formation of noncovalent complexes between S195A or S195A/D189S trypsin and protein proteinase inhibitors of different structural families and different sizes, as well as to examine the effects of introduction of the D189S mutation. Spectra of good quality were obtained for both trypsins alone and in complexes of increasing size with the proteinase inhibitors BPTI (total molecular mass 31 kDa), SBTI (total molecular mass 44 kDa), and the serpin alpha(1)-proteinase inhibitor Pittsburgh (alpha(1)PI Pittsburgh) (total molecular mass 69 kDa). Assignments of alanines 55 and 56, close to the active site histidine, and of alanine 195, present in the S195A variant used for most of the studies, were made by mutagenesis. These three alanines, together with two others, probably close to the S1 specificity pocket, were very sensitive to complex formation. In contrast, the remaining 10 alanines were invariant in chemical shift in all 3 of the noncovalent complexes formed, reflecting the conservation of structure in complexes with BPTI and SBTI known from X-ray crystal structures, but also indicating that there is no change in backbone conformation for the noncovalent complex with alpha(1)PI, for which there is no crystal structure. This was true both for S195A and for S195A/D189S trypsins. This high-level expression and labeling approach will be of great use for solution NMR studies on trypsin-serpin complexes, as well as for structural and mechanistic studies on trypsin variants.

Kinetic analysis of a simplified scheme of autocatalytic zymogen activation

Proteolytic enzymes are usually biosynthesized as somewhat larger inactive precursors known as zymogens. These zymogens must undergo an activation process, usually a limited proteolysis, to attain their catalytic activity. When the activating enzyme and the activated enzyme coincide, the process is an autocatalytic zymogen activation. In the present study, a kinetic analysis of the entire progress curve for the autocatalytic zymogen activation reactions is presented. On the basis of the kinetic equations, a novel procedure is developed to evaluate the kinetic parameters of the reactions. This procedure is particularly useful for the fast zymogen autoactivation reactions. As two examples, the novel procedure is used to analyse the autocatalytic activation of bovine trypsinogen and human blood coagulation factor XII (Hageman factor).

Unique catalytic and molecular properties of hydrolases from Aspergillus used in Japanese bioindustries.

This review covers the unique catalytic and molecular properties of three proteolytic enzymes and a glycosidase from Aspergillus. An aspartic proteinase from A. saitoi, aspergillopepsin I (EC 3.4.23.18), favors hydrophobic amino acids at P1 and P'1 like gastric pepsin. However, aspergillopepsin I accommodates a Lys residue at P1, which leads to activation of trypsinogens like duodenum enteropeptidase. Substitution of Asp76 to Ser or Thr and deletion of Ser78, corresponding to the mammalian aspartic proteinases, cathepsin D and pepsin, caused drastic decreases in the activities towards substrates containing a basic amino acid residue at 1. In addition, the double mutant T77D/G78(S)G79 of porcine pepsin was able to activate bovine trypsinogen to trypsin by the selective cleavage of the K6-I7 bond of trypsinogen. Deuterolysin (EC 3.4.24.39) from A. oryzae, which contains 1g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. It was concluded that His128, His132, and Asp164 provide the Zn2+ ligands of the enzyme according to a 65Zn binding assay. Deuterolysin is a member of a family of metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH + D" motif and an aspartic acid as the third zinc ligand. Acid carboxypeptidase (EC 3.4.16.1) from A. saitoi is a glycoprotein that contains both N- and O-linked sugar chains. Site-directed mutagenesis of the cpdS, cDNA encoding A. saitoi carboxypeptidase, was cloned and expressed. A. saitoi carboxypeptidase indicated that Ser153, Asp357, and His436 residues were essential for the enzymic catalysis. The N-glycanase released high-mannose type oligosaccharides that were separated on HPLC. Two, which had unique structures of Man10 GlcNAc2 and Man11GlcNAc2, were characterized. An acidic 1,2-alpha-mannosidase (EC 3.2.1.113) was isolated from the culture of A. saitoi. A highly efficient overexpression system of 1,2-alpha-mannosidase fusion gene (f-msdS) in A. oryzae was made. A yeast mutant capable of producing Man5GlcNAc2 human-compatible sugar chains on glycoproteins was constructed. An expression vector for 1,2-alpha-mannosidase with the "HDEL" endoplasmic reticulum retention/retrieval tag was designed and expressed in Saccharomyces cerevisiae. The first report of production of human-compatible high mannose-type (Man5GlcNAc2) sugar chains in S. cerevisiae was described.

Partially folded intermediates during trypsinogen denaturation.

The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was delta GH2O = 6.99 +/- 1.40 kcal/mol for guanidine hydrochloride and delta GH2O = 6.37 +/- 0.57 kcal/mol for urea. Satisfactory fits of equilibrium unfolding transitions required a three-state model involving an intermediate in addition to the native and unfolded forms. Size exclusion HPLC allowed the detection of an intermediate population of trypsinogen whose Stokes radii varied from 24.1 +/- 0.4 A to 26.0 +/- 0.3 A for 1.5 M and 2.5 M guanidine hydrochloride, respectively. During urea denaturation, the range of Stokes radii varied from 23.9 +/- 0.3 A to 25.7 +/- 0.6 A for 4.0 M and 6.0 M urea, respectively. Maximal intrinsic fluorescence was observed at about 3.8 M urea with 8-aniline-1-naphthalene sulfonate (ANS) binding. These experimental data indicate that the unfolding of bovine trypsinogen is not a simple transition and suggest that the equilibrium intermediate population comprises one intermediate that may be characterized as a molten globule. To obtain further insight by studying intermediates representing different stages of unfolding, we hope to gain a better understanding of the complex interrelations between protein conformation and energetics.

Activating a zymogen without proteolytic processing: mutation of Lys15 and Asn194 activates trypsinogen.

The zymogen and mature enzyme forms of trypsin-like serine proteases exhibit a wide range of activities. The prototypical trypsinogen-trypsin system is an example of a minimally active zymogen and a maximally active mature protease. The present work identifies several features of trypsinogen which govern its activity. Our results indicate that rat trypsin is 10(8)-fold more active than rat trypsinogen. Rat trypsinogen appears to be less active than bovine trypsinogen. His40 is believed to be an important determinant of zymogen activity. We are unable to verify this role for His40 in trypsinogen since the mutation of His40 to Phe appears to change the trypsin-substrate interface. Deletion of the N-terminal Ile16 from trypsin is expected to produce a trypsinogen-like protein since the Ile16-Asp194 salt bridge cannot form. Such mutants have higher activity and BPTI affinity than trypsinogen, which indicates that the activation peptide stabilizes the inactive trypsinogen conformation. The mutation of Lys15 to Ala increases the BPTI affinity and activity of trypsinogen to an even greater extent; thus, removal of Lys15 can account for the effect of the loss of the activation peptide. These results suggest that Lys15 is an important determinant of zymogen activity. The mutation of Asp194 to Asn also increases the BPTI affinity and activity of trypsinogen. This result suggests that in addition to stabilizing the active conformation of trypsin via the Ile16-Asp194 salt bridge, Asp194 also maintains the inactive conformation of trypsinogen. A correlation exists between the values of kcat/Km and BPTI affinity of mutant trypsinogens and trypsins. However, the slope of this correlation is 0.64, which indicates that different "active" conformations are involved in BPTI binding and substrate hydrolysis. DeltaI16V17 trypsinogen is the lone outlier; its BPTI affinity is higher than would be expected based on the value of kcat/Km. We show that the rate of BPTI association is slower for DeltaI16V17 trypsinogen than for a mutant trypsinogen with a similar BPTI affinity. This observation suggests that BPTI binds to an "active" trypsinogen conformation that is not kinetically accessible to substrates.

Engineering of Porcine Pepsin: ALTERATION OF S1 SUBSTRATE SPECIFICITY OF PEPSIN TO THOSE OF FUNGAL ASPARTIC PROTEINASES BY SITE-DIRECTED MUTAGENESIS

The S1 substrate specificity of porcine pepsin has been altered to resemble that of fungal aspartic proteinase with preference for a basic amino acid residue in P1 by site directed mutagenesis. On the basis of primary and tertiary structures of aspartic proteinases, the active site-flap mutants of porcine pepsin were constructed, which involved the replacement of Thr-77 by Asp (T77D), the insertion of Ser between Gly-78 and Ser-79 (G78(S)S79), and the double mutation (T77D/G78(S)S79). The specificities of the mutants were determined using p-nitrophenylalanine-based substrates containing a Phe or Lys residue at the P1 position. The double mutant cleaved the Lys-Phe(4-NO2) bonds, while wild-type enzyme digested other bonds. In addition, the pH dependence of hydrolysis of Lys-containing substrates by the double mutant indicates that the interactions between Asp-77 of the mutant and P1 Lys contribute to the transition state stabilization. The double mutant was also able to activate bovine trypsinogen to trypsin by the selective cleavage of the Lys6-Ile7 bond of trypsinogen. Results of this study suggest that the structure of the active site flap contributes to the S1 substrate specificity for basic amino acid residues in aspartic proteinases.