Trypsin Variants Research Articles

New horizons in pancreatic genetics.

Pancreatitis remains an intractable disease because no causative treatment is yet available. Recent studies have uncovered some of its underlying pathophysiology, a requirement for identifying potential treatment targets. These advancements were achieved by human genetic studies and by introducing genetic mechanisms into experimental pancreatitis models. Cationic trypsin mutations are the most prominent genetic risk factor for pancreatitis. Investigators have now introduced genetically modified trypsin variants into transgenic animals. In this manner they characterized the role of cellular defense mechanisms, for example degradation of active trypsin by chymotrypsin-C, but also found that increased autoactivation or decreased degradation, not only boost disease severity but also drive progression to chonic pancreatitis. Other studies found that harmful trypsin effects are not restricted to acinar cells, that other digestive enzymes, notably pancreatic elastase, can also induce cellular injury and that endoplasmic-reticulum-stress is an important mechanism when mutations induce protein misfolding. Identifying genetic subsceptibility factors for a disease never completely uncovers its underlying pathogenesis or potential treatment targets. This requires studying the mechanisms suggested by genetic findings in experimentel disease models. Pancreatitis is a field, in which much progress has now been achieved by adopting this approach.

Investigating proteolytic activity of engineered trypsin variants using naturally occurring macromolecular substrates

Serine proteases constitute numerous physiological roles that include but are not limited to the breakdown of food, the participation in immune responses, and with the triggering of cell apoptosis Engineered serine proteases as therapeutic agents constitute a new class of drugs for potential clinical applications. However, one main complication faced when using serine proteases as therapeutic agents is their relatively short half‐life due to the abundance of tight binding inhibitors in the human body that target serine proteases to regulate proteolytic activity and rapidly remove them from circulation. For a protease to be effective it must display resistance towards inhibitors. Previously engineered trypsin variants K60A, K60V, Y39A, Y39F have shown to diminish prime side interactions, affecting micromolecular inhibition through the disruption of hydrogen bond networks occurring at residue positions 39 and 60. These variants displayed resistance towards certain naturally occurring inhibitors, bovine pancreatic trypsin inhibitor and M84R ecotin. Kinetic data was performed using commercially available tripeptide Z‐GPR‐pNA, also indicating no change of specificity. In order to ascertain the viability of these engineered variants as potential therapeutics we must study the interactions between naturally available macro molecular substrates. Experimental methods required the design and utilization of a novel spectroscopicassay tomonitor the proteolytic activity of trypsin variants using chymotrypsinogen (Cg) as our naturally occurring macromolecular substrate. Within the body, trypsin cleaves triggering the activation of zymogen Cg into its active form. Fluorescence will be measured using chymotrypsin specific 4‐methylumbelliferyl p‐tri‐methylammoniocinnamate chloride (MUTMAC). Preliminary data has shown that a 10:1 concentration ratio (100nM Trypsin to 10μM Cg) proved most efficient for activation. However, only ~22% of chymotrypsinogen has been activated using wild‐type trypsin, suggesting that further optimization of this assay is still required. Future work will aim to elucidate the proteolytic rates of these trypsin variant K60A, K60V, Y39A, Y39F where we hope to obtain a better understanding of the interactions between these variants and naturally occurring substrates.Support or Funding InformationNIH MS/PhD Bridge: R25‐GM048972This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Correlating structure and ligand affinity in drug discovery: a cautionary tale involving second shell residues.

A high-resolution crystallographic structure determination of a protein-ligand complex is generally accepted as the 'gold standard' for structure-based drug design, yet the relationship between structure and affinity is neither obvious nor straightforward. Here we analyze the interactions of a series of serine proteinase inhibitors with trypsin variants onto which the ligand-binding site of factor Xa has been grafted. Despite conservative mutations of only two residues not immediately in contact with ligands (second shell residues), significant differences in the affinity profiles of the variants are observed. Structural analyses demonstrate that these are due to multiple effects, including differences in the structure of the binding site, differences in target flexibility and differences in inhibitor binding modes. The data presented here highlight the myriad competing microscopic processes that contribute to protein-ligand interactions and emphasize the difficulties in predicting affinity from structure.

Thermodynamic signatures in macromolecular interactions involving conformational flexibility.

The energetics of macromolecular interactions are complex, particularly where protein flexibility is involved. Exploiting serendipitous differences in the plasticity of a series of closely related trypsin variants, we analyzed the enthalpic and entropic contributions accompanying interaction with L45K-eglin C. Binding of the four variants show significant differences in released heat, although the affinities vary little, in accordance with the principle of enthalpy-entropy compensation. Binding of the most disordered variant is almost entirely enthalpically driven, with practically no entropy change. As structures of the complexes reveal negligible differences in protein-inhibitor contacts, we conclude that solvent effects contribute significantly to binding affinities.

Determining the role(s) of prime‐side residues in macromolecular inhibition of trypsin‐fold serine proteases (933.2)

Trypsin‐fold serine proteases are among the most abundant of proteases. By identifying residues that are important for enzyme‐inhibitor interactions in trypsin, we may advance the development of protease‐based therapies in general. Lysine‐60 (K60) is a highly conserved prime‐side residue in trypsin‐fold serine proteases. In trypsin‐inhibitor co‐crystals, K60 hydrogen bonds to tyrosine‐39 (Y39) and is positioned to restrict conformational mobility of phenylalanine‐41 (F41), possibly optimizing the observed hydrogen bond interactions between inhibitors, Y39 and F41. Substitution of K60 with other residues may disrupt these interactions and provide insight to their significance with respect to inhibitor binding. To test this hypothesis, we created trypsin variants K60G, K60A, K60V, K60I, K60M, K60R, Y39S, and Y39F and characterized them with respect to their activities and sensitivities to macromolecular inhibitors soybean trypsin inhibitor (SBTI), bovine pancreatic trypsin inhibitor (BPTI) and ecotin, an inhibitor expressed in E. coli. Our initial results show that, compared to wild‐type trypsin, all variants maintain similar catalytic activity, but‐K60A, K60V, K60I, K60M, and Y39F are more sensitive to inhibition while variants K60R and Y39S are less sensitive to inhibition. Collectively, these results suggest that K60 and Y39 do indeed play significant roles in inhibitor binding.Grant Funding Source: This work was supported by NSF

Engineering the oxyanion hole of trypsin for promoting the reverse of proteolysis

Although proteases are capable of synthesizing peptide bonds via the reverse of proteolysis, they are not proficient at peptide fragment ligation. Further manipulations are needed to shift the native enzyme activity from the cleavage to the synthesis of peptides especially when longer peptides or even proteins are the target molecules of the reaction. This account reports on the synthetic potential of trypsin variants with engineered oxyanion holes mutated by proline mutations, which were designed to minimize proteolytic side reactions during peptide bond synthesis. From the six single and double proline-mutated trypsins, in particular, trypsinQ192P came out as the most promising biocatalyst enabling not only the ligation of cleavage-sensitive peptide fragments but also the selective N-terminal modification of a real protein substrate.

Determining the role(s) of prime‐side residues in macromolecular inhibition of trypsin‐fold serine proteases

Trypsin‐fold serine proteases are among the most abundant of all proteases. By identifying residues that are important for enzyme‐inhibitor interaction in trypsin, we may advance the development of protease‐based therapies in general. Lysine‐60 (K60) is a highly conserved prime‐side residue that may play many roles in trypsin. In co‐crystals, K60 hydrogen bonds to tyrosine‐39 (Y39) and is positioned to restrict conformational mobility of phenylalanine‐41 (F41), possibly limiting the observed hydrogen bond interactions between those residues and macromolecular inhibitors. Substitution of K60 with other amino acid residues may disrupt these interactions and provide insight to their significance with respect to inhibitor binding. To test this hypothesis, we created trypsin variants K60G, K60A, K60V, K60I, and K60R and characterized them with respect to their activities and sensitivities to the macromolecular inhibitors soybean trypsin inhibitor (SBTI) and bovine pancreatic trypsin inhibitor (BPTI). Our initial results show that, compared to wild‐type trypsin, K60A and K60V are catalytically indistinguishable, more resistant to autolysis and more sensitive to inhibition by SBTI and BPTI. Further experiments with ecotin and other trypsin variants can provide additional insight on specific interactions. This work was supported by NSF CAREER Award MCB‐0643988–02 and NIH MARC T34‐GM008574.

Conformational dynamics of threonine 195 and the S1 subsite in functional trypsin variants

Replacing the catalytic serine in trypsin with threonine (S195T variant) leads to a nearly complete loss of catalytic activity, which can be partially restored by eliminating the C42-C58 disulfide bond. The 0.69 μs of combined explicit solvent molecular dynamics (MD) simulations revealed continuous rearrangement of T195 with different conformational preferences between five trypsin variants tested. Among three conformational families observed for the T195 residue, one showed the T195 hydroxyl in a conformation analogous to that of the serine residue in wild-type trypsin, positioning the hydroxyl oxygen atom for attack on the carbonyl carbon of the peptide substrate. MD simulations demonstrated that this conformation was more populated for the C42A/C58V/S195T and C42A/C58A/S195T triple variants than for the catalytically inactive S195T variant and correlated with restored enzymatic activities for triple variants. In addition, observation of the increased motion of the S214-G219 segment in the S195T substituted variants suggested an existence of open and closed conformations for the substrate binding pocket. The closed conformation precludes access to the S1 binding site and could further reduce enzymatic activities for triple variants. Double variants with intact serine residues (C42A/C58A/S195 and C42A/C58V/S195) also showed interchange between closed and open conformations for the S214-G219 segment, but to a lesser extent than the triple variants. The increased conformational flexibility of the S1 subsite, which was not observed for the wild-type, correlated with reduced enzymatic activities and suggested a possible mode of substrate regulation for the trypsin variants tested.

Presence versus absence of hydrogen bond donor Tyr-39 influences interactions of cationic trypsin and mesotrypsin with protein protease inhibitors.

Mesotrypsin displays unusual resistance to inhibition by polypeptide trypsin inhibitors and cleaves some such inhibitors as substrates, despite a high degree of conservation with other mammalian trypsins. Substitution of Arg for the generally conserved Gly-193 has been implicated as a critical determinant of the unusual behavior of mesotrypsin toward protein protease inhibitors. Another relatively conserved residue near the trypsin active site, Tyr-39, is substituted by Ser-39 in mesotrypsin. Tyr-39, but not Ser-39, forms a hydrogen bond with the main chain amide nitrogen of the P(4) ' residue of a bound protease inhibitor. To investigate the role of the Tyr-39 H-bond in trypsin-inhibitor interactions, we reciprocally mutated position 39 in mesotrypsin and human cationic trypsin to Tyr-39 and Ser-39, respectively. We assessed inhibition constants and cleavage rates of canonical protease inhibitors bovine pancreatic trypsin inhibitor (BPTI) and the amyloid precursor protein Kunitz protease inhibitor domain by mesotrypsin and cationic trypsin variants, finding that the presence of Ser-39 relative to Tyr-39 results in a 4- to 13-fold poorer binding affinity and a 2- to 18-fold increase in cleavage rate. We also report the crystal structure of the mesotrypsin-S39Y•BPTI complex, in which we observe an H-bond between Tyr-39 OH and BPTI Ile-19 N. Our results indicate that the presence of Ser-39 in mesotrypsin, and corresponding absence of a single H-bond to the inhibitor backbone, makes a small but significant functional contribution to the resistance of mesotrypsin to inhibition and the ability of mesotrypsin to proteolyze inhibitors.

Enhancing the catalytic activity of an engineered threonine protease

In this study, the Cys‐42—Cys‐58 disulfide bridge of S195T trypsin was replaced with two valine residues to: 1) increase threonine protease activity and 2) better understand the structual limitations and requirements of this subsite with respect to catalysis. The activities of C42V/C58V and C42V/C58V/S195T trypsins (VV‐Tn and VVT‐Tn, respectively) were evaluated with benzyloxycarbonyl‐Gly‐Pro‐Arg (Z‐GPR) p‐nitroanilide (pNA) and 7‐amino 4‐methyl coumarin (AMC) substrates. VVT‐Tn was 150 and 10 times more active towards the pNA and AMC substrates, respectively, than C42A/C58V/S195T trypsin (AVT‐Tn; kcat/KM values). In a thermostability study, both variants and WT trypsin retained > 80% of their activity suggesting that the substitutions did not destabilize the enzymes. The pH dependence of activity profiles were identical to WT trypsin, suggesting that any structural differences do not perturb the pKas of catalytically relevant residues. Finally, the KI of p‐aminobenzamidine was identical for native and VV‐Tn but that of VVT‐Tn was half the KI for WT. Therefore, Thr‐195 may affect the primary binding pocket structure. Collectively, these results indicate that the activity of Thr‐195 trypsin variants may be increased by appropriate substitutions at positions 42 and 58. However, the structural pertubations induced by these substitutions make achievement of wild‐type levels of activity less likely.(This work was supported by NSF Award MCB‐0643988‐02)

Introducing extended substrate selectivity into trypsin through redesign

The objective of this research was to evaluate how a residue with hydrogen bond capability may affect substrate selection by a previously engineered threonine protease. C42S/C58V/S195T trypsin (SVT‐Tn) was characterized and compared to trypsin variants C42A/C58V/S195T‐Tn (AVT‐Tn) and C42A/C58A/S195T (AAT‐Tn). p‐Nitroanilide (pNA) and 7‐amino 4‐methyl coumarin (AMC) derivatives of benzyloxycarbonyl‐Gly‐Pro‐Arg (Z‐GPR) were use to kinetically evaluate the activity and specificity of SVT‐Tn. Their respective kcat/KM values show that SVT‐Tn is 8‐ and 40‐fold more efficient in Z‐GPR‐pNA and Z‐GPR‐AMC hydrolysis, respectively, than AVT‐Tn. Additionally, SVT‐Tn was 5‐fold less efficient in Z‐GPR‐pNA hydrolysis and 2‐fold more efficient in Z‐GPR‐AMC hydrolysis than AAT‐Tn. However, the (kcat/KM)pNA/(kcat/KM)AMC ratios show that SVT trypsin is 3.5‐fold more selective for the pNA substrate, less discriminatory than the AVT and AAT variants. The results suggest that a serine at position 42 in the S195T/C58V context significantly affects catalytic efficiency in a substrate dependent manner for the two substrates tested, but selectivity between the two substrates was only moderate. However, longer peptide substrates are needed to thoroughly evaluate the degree of the selectivity of SVT‐Tn.(This work was supported by NSF Award MCB‐0643988‐02)

Conversion of trypsin to a functional threonine protease

The hydroxyl group of a serine residue at position 195 acts as a nucleophile in the catalytic mechanism of the serine proteases. However, the chemically similar residue, threonine, is rarely used in similar functional context. Our structural modeling suggests that the Ser 195 --> Thr trypsin variant is inactive due to negative steric interaction between the methyl group on the beta-carbon of Thr 195 and the disulfide bridge formed by cysteines 42 and 58. By simultaneously truncating residues 42 and 58 and substituting Ser 195 with threonine, we have successfully converted the classic serine protease trypsin to a functional threonine protease. Substitution of residue 42 with alanine and residue 58 with alanine or valine in the presence of threonine 195 results in trypsin variants that are 10(2) -10(4) -fold less active than wild type in kcat/KM but >10(6)-fold more active than the Ser 195 --> Thr single variant. The substitutions do not alter the substrate specificity of the enzyme in the P1'- P4' positions. Removal of the disulfide bridge decreases the overall thermostability of the enzyme, but it is partially rescued by the presence of threonine at position 195.

Understanding Protein–Ligand Interactions: The Price of Protein Flexibility

In order to design selective, high-affinity ligands to a target protein, it is advantageous to understand the structural determinants for protein–ligand complex formation at the atomic level. In a model system, we have successively mapped the factor Xa binding site onto trypsin, showing that certain mutations influence both protein structure and inhibitor specificity. Our previous studies have shown that introduction of the 172SSFI 175 sequence of factor Xa into rat or bovine trypsin results in the destabilisation of the intermediate helix with burial of Phe174 (the down conformation). Surface exposure of the latter residue (the up conformation) is critical for the correct formation of the aromatic box found in factor Xa–ligand complexes. In the present study, we investigate the influence of aromatic residues in position 174. Replacement with the bulky tryptophan (SSWI) shows reduced affinity for benzamidine-based inhibitors (1) and (4), whereas removal of the side-chain (alanine, SSAI) or exchange with a hydrophilic residue (arginine, SSRI) leads to a significant loss in affinity for all inhibitors studied. The variants could be crystallised in the presence of different inhibitors in multiple crystal forms. Structural characterisation of the variants revealed three different conformations of the intermediate helix and 175 loop in SSAI (down, up and super-up), as well as a complete disorder of this region in one crystal form of SSRI, suggesting that the compromised affinity of these variants is related to conformational flexibility. The influence of Glu217, peripheral to the ligand-binding site in factor Xa, was investigated. Introduction of Glu217 into trypsin variants containing the SSFI sequence exhibited enhanced affinity for the factor Xa ligands (2) and (3). The crystal structures of these variants also exhibited the down and super-up conformations, the latter of which could be converted to up upon soaking and binding of inhibitor (2). The improved affinity of the Glu217-containing variants appears to be due to a shift towards the up conformation. Thus, the reduction in affinity caused by conformational variability of the protein target can be partially or wholly offset by compensatory binding to the up conformation. The insights provided by these studies will be helpful in improving our understanding of ligand binding for the drug design process.

ZZ Made EZ: Influence of Inhibitor Configuration on Enzyme Selectivity

Selectivity of drug targeting is necessary in order to forestall undesired side-effects. Here, we examine the structural grounds for the configuration-dependent selectivity of 2,7-bis(4-amidinobenzylidene)-cycloheptan-1-one ( 1) for factor Xa and trypsin: Previous studies showed that factor Xa is preferentially inhibited by the ( Z, Z) configuration isomer of ( 1), whilst trypsin binds equally well to both ( E, Z) and ( Z, Z) forms. Using engineered trypsin variants, we find similar overall binding modes for the ( E, Z) and ( Z, Z) isomers. Minor changes in van der Waals' contacts to Tyr99 (Leu in trypsin) explain the differential inhibition of factor Xa. We note differences in the experimental electron densities observed from co-crystallisation and soaking experiments: while the co-crystallisation of ( 1) with variants containing Tyr99 (Leu99) reveal the exclusive presence of the ( Z, Z) (( E, Z)) configurations respectively, soaking experiments with either variant result in mixtures of ( E, Z), ( Z, Z) and ( E, E). This discrepancy arises presumably from differences in the spatial (packing considerations) or chemical (crystallisation conditions) microenvironments. The results presented here represent an extreme example of the problems that face structure-based drug design, in particular the dangers inherent in relying on a single crystal structure for interpreting protein–ligand interactions.

The energetic cost of induced fit catalysis: Crystal structures of trypsinogen mutants with enhanced activity and inhibitor affinity.

The contribution of induced fit to enzyme specificity has been much debated, although with little experimental data. Here we probe the effect of induced fit on enzyme specificity using the trypsin(ogen) system. BPTI is known to induce trypsinogen to assume a trypsinlike conformation. Correlations are observed between BPTI affinity and the values of k(cat)/K(m) for the hydrolysis of two substrates by eight trypsin(ogen) variants. The slope of both correlations is -1.8. The crystal structures of the BPTI complexes of four variant trypsinogens were also solved. Three of these enzymes, K15A, DeltaI16V17/D194N, and DeltaI16V17/Q156K trypsinogen, are 10- to 100-fold more active than trypsinogen. The fourth variant, DeltaI16V17 trypsinogen, is the lone outlier in the correlations; its activity is lower than expected based on its affinity for BPTI. The S1 site and oxyanion hole, formed by segments 184A-194 and 216-223, are trypsinlike in all of the enzymes. These structural and kinetic data confirm that BPTI induces an active conformation in the trypsin(ogen) variants. Thus, changes in BPTI affinity monitor changes in the energetic cost of inducing a trypsinlike conformation. Although the S1 site and oxyanion hole are similar in all four variants, the N-terminal and autolysis loop (residues 142-152) segments have different interactions for each variant. These results indicate that zymogen activity is controlled by a simple conformational equilibrium between active and inactive conformations, and that the autolysis loop and N-terminal segments control this equilibrium. Together, these data illustrate that induced fit does not generally contribute to enzyme specificity.

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.

Peptide bond formation mediated by substrate mimetics. Structure-guidedoptimization of trypsin for synthesis.

Substrate mimetics are excellent tools for protease-mediated peptide synthesis that enable the coupling of peptides independently of the primary specificity of the enzyme without undesired cleavages of the newly formed peptide bonds. However, the synthetic utility of this beneficial approach is limited to reactions with nonspecific amino-acid-containing peptides while the coupling of specific ones leads to unwanted cleavages due to the native proteolytic activity of the biocatalyst. This paper reports on the use of site-directed mutagenesis to design trypsin variants with decreased cleavage activity. Starting from the variant D189S, which is known for its low proteolytic potential, Ser189 and Ser190 were exchanged for Ala to further repress the inherent amidase activity of trypsin D189S. The effect of mutations was analysed by model synthesis reactions using specific amino-acid-containing peptides and substrate mimetics as the reactants. Finally, computer-assisted protein-ligand docking studies were performed to get closer insight into the molecular basis of the experimental results.

S′ subsite mapping of serine proteases based on fluorescence resonance energy transfer

A microassay based on fluorescence resonance energy transfer has been developed to determine the S′ specificity of serine proteases. The protease-catalyzed acyl transfer from a fluorescing acyl donor ester to a P ′ 1/P ′ 2 variable hexapeptide library of nucleophiles labeled with a fluorescence quencher leads to an internally quenched peptide product and a fluorescent hydrolysis product. The amount of fluorescence quenching allows one to draw conclusions about the interaction of the nucleophile at the S′ sites of the protease. o-Aminobenzoic acid and 3-nitrotyrosine were used as an efficient donor–acceptor pair for the resonance energy transfer. The P ′ 1/P ′ 2 variable hexapeptide library with the general structure H-Xaa-Ala-Ala-Ala-Tyr(NO 2)-Gly-OH and H-Ala-Xaa-Ala-Ala-Tyr(NO 2)-Gly-OH, where Xaa represents Arg, Lys, Met, Phe, Ala, Gly, Ser, Gln and Glu, was prepared by solid-phase synthesis. Investigations of the S′ specificity of trypsin, chymotrypsin and trypsin variants show that this assay is a fast and sensitive screening method for S′ subsite mapping of serine proteases and is suitable for a high throughput screening. The assay might be useful for the development of restriction proteases and the estimation of yields in enzymatic peptide synthesis.

Introduction of a cysteine protease active site into trypsin.

Active site serine 195 of rat anionic trypsin was replaced with a cysteine by site-specific mutagenesis in order to determine if a thiol group could function as the catalytic nucleophile in serine protease active site environment. Two genetically modified rat thiol trypsins were generated; the first variant contained a single substitution of Ser195 with Cys (trypsin S195C) while the second variant contained the Ser195 to Cys as well as an Asp102 to Asn substitution (trypsin D102N,S195C) that more fully mimics the putative catalytic triad of papain. Both variants were expressed as his J signal peptide-trypsin fusion proteins to high levels under the control of the tac promoter. The mature forms of both variants were secreted into the periplasmic space of Escherichia coli. Trypsin S195C shows a low level of activity toward the activated ester substrate Z-Lys-pNP, while both trypsin S195C and trypsin D102N,S195C were active toward the fluorogenic tripeptide substrate Z-GPR-AMC. Esterase and peptidase activities of both thiol trypsin variants were inhibited by known Cys protease inhibitors as well as by specific trypsin inhibitors. The kcat of trypsin S195C was reduced by a factor of 6.4 x 10(5) relative to that of trypsin while the kcat of trypsin D102N,S195C was lowered by a factor of 3.4 x 10(7) with Z-GPR-AMC as substrate. Km values were unaffected. The loss of activity of trypsin D102N,S195C was partially attributed to an inappropriate Asn102-His57 interaction that precludes the formation of the catalytically competent His57-Cys195 ion pair although loss of the negative charge of D102 at the active site probably contributes to diminished activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of an efficient method for generating and screening active trypsin and trypsin variants.

Des techniques classiques de biochimie et des approches nouvelles de genie genetique (construction d'un vecteur, mutagenese dirigee, expression dans E. coli et screening) sont mises au point et utilisees dans le but d'etablir les bases regissant la specificite d'une serine protease, la trypsine, pour son substrat