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)     

Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214.

Much of the catalytic power of trypsin is derived from the unusual buried, charged side chain of Asp102. A polar cave provides the stabilization for maintaining the buried charge, and it features the conserved amino acid Ser214 adjacent to Asp102. Ser214 has been replaced with Ala, Glu, and Lys in rat anionic trypsin, and the consequences of these changes have been determined. Three-dimensional structures of the Glu and Lys variant trypsins reveal that the new 214 side chains are buried. The 2.2-A crystal structure (R = 0.150) of trypsin S214K shows that Lys214 occupies the position held by Ser214 and a buried water molecule in the buried polar cave. Lys214-N zeta is solvent inaccessible and is less than 5 A from the catalytic Asp102. The side chain of Glu214 (2.8 A, R = 0.168) in trypsin S214E shows two conformations. In the major one, the Glu carboxylate in S214E forms a hydrogen bond with Asp102. Analytical isoelectrofocusing results show that trypsin S214K has a significantly different isoelectric point than trypsin, corresponding to an additional positive charge. The kinetic parameter kcat demonstrates that, compared to trypsin, S214K has 1% of the catalytic activity on a tripeptide amide substrate and S214E is 44% as active. Electrostatic potential calculations provide corroboration of the charge on Lys214 and are consistent with the kinetic results, suggesting that the presence of Lys214 has disturbed the electrostatic potential of Asp102.     

Studies of specificity and catalysis in trypsin by structural analysis of site-directed mutants

We are probing the determinants of catalytic function and substrate specificity in serine proteases by kinetic and crystallographic characterization of genetically engineered site-directed mutants of rat trypsin. The role of the aspartyl residue at position 102, common to all members of the serine protease family, has been tested by substitution with asparagine. In the native enzyme, Asp102 accepts a hydrogen bond from the catalytic base His57, which facilitates the transfer of a proton from the enzyme nucleophile Ser195 to the substrate leaving group. At neutral pH, the mutant is four orders of magnitude less active than the naturally occurring enzyme, but its binding affinity for model substrates is virtually undiminished. Crystallographic analysis reveals that Asn102 donates a hydrogen bond to His57, forcing it to act as donor to Ser195. Below pH 6, His57 becomes statistically disordered. Presumably, the di-protonated population of histidyl side chains are unable to hydrogen bond to Asn102. Steric conflict may cause His57 to rotate away from the catalytic site. These results suggest that Asp102 not only provides inductive and orientation effects, but also stabilizes the productive tautomer of His57. Three experiments were carried out to alter the substrate specificity of trypsin. Glycine residues at positions 216 and 226 in the substrate-binding cavity were replaced by alanine residues in order to differentially affect lysine and arginine substrate binding. While the rate of catalysis by the mutant enzymes was reduced in the mutant enzymes, their substrate specificity was enhanced relative to trypsin. The increased specificity was caused by differential effects on the catalytic activity towards arginine and lysine substrates. The Gly----Ala substitution at 226 resulted in an altered conformation of the enzyme which is converted to an active trypsin-like conformation upon binding of a substrate analog. In a third experiment, Lys189, at the bottom of the specificity pocket, was replaced with an aspartate with the expectation that specificity of the enzyme might shift to aspartate. The mutant enzyme is not capable of cleaving at Arg and Lys or Asp, but shows an enhanced chymotrypsin-like specificity. Structural investigations of these mutants are in progress.     

Human rhinovirus 3C protease: a cysteine protease showing the trypsin(ogen)-like fold

Viral-encoded proteases cleave precursor polyprotein(s) leading to maturation of infectious virions. Strikingly, human rhinovirus 3C protease shows the trypsin(ogen)-like serine protease fold based on two topologically equivalent six-stranded β-barrels, but displays residue Cys147 as the active site nucleophile. By contrast, papain, which is representative of most cysteine proteases, does not display the trypsin(ogen)-like fold. Remarkably, in human rhinovirus 3C cysteine protease, the catalytic residues Cys147, His40 and Glu71 are positioned as Ser195, His57 and Asp102, respectively, building up the catalytic triad of serine proteases in the chymotrypsin–trypsin–elastase family. However, as compared to trypsin-like serine proteases and their zymogens, residue His40 and the oxyanion hole of human rhinovirus 3C cysteine protease, both key structural components of the active site, are located closer to the protein core. Human rhinovirus 3C cysteine protease cleaves preferentially GlnGly peptide bonds or, less commonly, the GlnSer, GlnAla, GluSer or GluGly pairs. Finally, human rhinovirus 3C cysteine protease and the 3CD cysteine protease–polymerase covalent complex bind the 5′ non-coding region of rhinovirus genomic RNA, an essential function for replication of the viral genome.     

Resistance of cathepsin L compared to elastase to proteolysis when complexed with the serpin endopin 2C, and recovery of cathepsin L activity

This study demonstrates unique differences in the conformational nature of cathepsin L compared to elastase when complexed with the serpin endopin 2C, assessed by susceptibilities of protease/endopin 2C complexes to proteolysis by trypsin. Complexed and uncomplexed cathepsin L were resistant to degradation by trypsin, which indicated that trypsin cleavage sites within cathepsin L remain inaccessible when this cysteine protease is complexed with the endopin 2C serpin. In contrast, elastase in complexes with endopin 2C was degraded by trypsin, but uncomplexed elastase was not degraded. These results demonstrate a change in the conformational properties of trypsin cleavage sites within elastase when it is complexed with endopin 2C, compared to uncomplexed elastase. Cathepsin L complexes with endopin 2C were short-lived, but elastase complexes were stable. Furthermore, cathepsin L dissociated from complexes demonstrated recovery of cathepsin L activity, and reducing conditions provided optimum recovery of cathepsin L activity. These findings suggest that cathepsin L, when complexed with endopin 2C, maintains its general conformation in a manner that allows recovery of cathepsin L activity upon dissociation from endopin 2C. These results demonstrate differences in the relative conformational properties of the cysteine protease cathepsin L, compared to the serine protease elastase, in complexes with the serpin endopin 2C.     

Separation of the two domains of human mucus proteinase inhibitor: inhibitory activity is only located in the carboxy-terminal domain

Human mucus proteinase inhibitor is a two-domain protein which inactivates bovine trypsin and chymotrypsin, leukocyte elastase and cathepsin G. In order to localize the site(s) responsible for these inhibitory activities, the two domains were isolated after specific cleavage of the Asp49-Pro50 bond following mild acid treatment of the bronchial inhibitor. The carboxy-terminal domain was active against leukocyte elastase, trypsin and chymotrypsin whereas the amino-terminal domain, which contained a putative antitryptic active site, was devoid of activity. This implicates that, in the whole molecule, the inhibitory activity region is localized only in the carboxy-terminal domain.     

Introduction of alpha-hydroxymethyamino acid residues in substrate specificity P1 position of trypsin inhibitor SFTI-1 from sunflower seeds retains its activity

In many complexes formed by serine proteinases and their inhibitors, the hydroxyl group provided by water molecule or by the inhibitor Ser residue is located close to the inhibitor P1-P1' reactive site. In order to investigate the role of this group, we synthesized analogues of trypsin inhibitor SFTI-1 isolated from the seeds of sunflower modified in P1 by alpha-hydroxymethylserine (HmSer) and both enantiomers of alpha-hydroxymethylvaline (HmVal). All the synthesized analogues inhibited bovine beta-trypsin and human leukocyte elastase. SFTI-1 analogues with HmVal and HmSer appear to be potent inhibitors of bovine beta-trypsin, whereas [Val5]SFTI-1 is practically inactive. Also trypsin inhibitory activity of [Ser5]SFTI-1 is significantly lower. Since the electrostatic interaction between protonated epsilon-NH2 group of the inhibitor P1 position and beta-carboxylate of trypsin Asp189 is the main driving force for interaction of both molecules, the results obtained are very interesting. We believe that these SFTI-1 analogues belong to a novel class of serine proteinase inhibitors.     

The multiplicity of human pancreatic secretory trypsin inhibitor

Four forms of pancreatic secretory trypsin inhibitor (PSTI; A1, A2, B, and C) were purified from human pancreatic juice. According to sequence results, the primary structure of B was different from that reported earlier (Greene, L.J., et al. (1976) Method Enzymol. 45, 813-825) at two positions, i.e. Asn21----Asp21, Asp29----Asn29. A1 and A2 were deamidated forms of B judging from peptide mappings with Staphylococcus aureus V8 protease. Gln45 in B was replaced by Glu in A1 and Gln51 in B was replaced by Glu in A2. C was an inhibitor lacking five amino acid residues from the amino terminal of B. B and C inhibited human cationic trypsin activity stoichiometrically with similar dissociation constants, but A1 and A2 showed poorer trypsin inhibitory activity than B and C.     

Three dimensional structures of S189D chymotrypsin and D189S trypsin mutants: the effect of polarity at site 189 on a protease-specific stabilization of the substrate-binding site

The crystal structure of S189D rat chymotrypsin have been determined (resolution 2.55A) and compared, together with D189S rat trypsin to wild-type structures to examine why these single mutations resulted in poorly active, non-specific enzymes instead of converting the specificities of trypsin and chymotrypsin into each other. Both mutants have stable structure but suffer from a surprisingly large number of serious deformations. These are restricted to the activation domain, mainly to the substrate-binding region and are larger in S189D chymotrypsin. A wild-type substrate-binding mode in the mutants is disfavored by substantial displacements of the Cys191-Cys220 disulfide and loop segments 185-195 (loop C2/D2) and 217-224 (loop E2/F2) at the specificity site. As a consequence, the substrate-binding clefts become wider and more solvent-accessible in the middle third and occluded in the lower third. Interestingly, while the Ser189 residue in D189S trypsin adopts a chymotrypsin-like conformation, the Asp189 residue in S189D chymotrypsin is turned out toward the solvent. The rearrangements in D189S trypsin are at the same sites where trypsin and trypsinogen differ and, in S189D chymotrypsin, the oxyanion hole as well as the salt-bridge between Asp194 and the N-terminal of Ile16 are missing as in chymotrypsinogen. Despite these similarities, the mutants do not have zymogen conformation. The Ser189Asp and Asp189Ser substitutions are structurally so disruptive probably because the stabilization of such a different specificity site polarities as those after the removal or introduction of a charged residue are beyond the capability of the wild-type conformation of the substrate-binding region.     

Functional organization and evolution of mammalian hexokinases: mutations that caused the loss of catalytic activity in N-terminal halves of type I and type III isozymes.

Mammalian hexokinases are believed to have evolved from a 100-kDa hexokinase which itself is a product of duplication and fusion of an ancestral gene encoding a 50-kDa glucose 6-phosphate-sensitive hexokinase. Type II hexokinase has been shown to possess two distinct functional active sites, one in each half, which functionally resemble the original 100-kDa hexokinase, whereas type I and III isozymes possess only one active site in the C-terminal halves. This study was conducted to identify which mutations caused the loss of catalytic activity in the N-terminal halves of type I and III isozymes. Arg 174 and Ser 447 in type I isozyme and Asp 244 in type III isozyme are speculated to be the cause, because they reside adjacent to the "catalytic" site and corresponding residues, Gly 174, Asp 447, and Gly 231, are conserved in the N-terminal half of type II isozyme as well as all other 50-kDa units that possess catalytic activity. Mutations G174R and D447S in the N-terminal half of type II isozyme reduced specific activity by approximately 79 and 57%, respectively. Therefore, neither mutation alone can account for the inactivation of the N-terminal active site in type I isozyme. Either mutation, G174R or D447S, had moderate effects on Michaelis constants, K(m), for glucose and ATP. Mg(2+). Intriguingly, mutation D447S introduced a novel inhibition by unchelated ATP (K(i) = 68 microM ATP, competitive vs ATP. Mg(2+)) to the N-terminal active site of type II isozyme. Mutation G231D caused instability to type II hexokinase and near complete loss of catalytic activity (95%), suggesting that mutation G231D not only hinders catalysis at the N-terminal active site but also leads to structural instability in type II hexokinase.     

Serine protease inhibitors of North American leeches

Serine protease inhibitors in extracts from three North American leeches, Nephelopsis obscura, Erpobdella punctata and Hemopis marmorata have been separated by anion exchange chromatography and the activity pattern against human granulocyte elastase and porcine chymotrypsin and trypsin determined. All three leech species contained a major peak with anti-trypsin activity, but Hemopis was unique in that the trypsin inhibitor was equally active against chymotrypsin. Nephelopsis was rich in anti-elastase activity of two types, one which was also active against chymotrypsin, and one which was a specific elastase inhibitor. Erpobdella contained inhibitors against elastase and chymotrypsin but with major activity against the latter.     

Mutational analysis of Ser14 and Asp157 in the nucleotide-binding site of beta-actin.

This paper compares wild-type and two mutant beta-actins, one in which Ser14 was replaced by a cysteine, and a second in which both Ser14 and Asp157 were exchanged (Ser14-->Cys and Ser14-->Cys, Asp157-->Ala, respectively). Both of these residues are part of invariant sequences in the loops, which bind the ATP phosphates, in the interdomain cleft of actin. The increased nucleotide exchange rate, and the decreased thermal stability and affinity for DNase I seen with the mutant actins indicated that the mutations disturbed the interdomain coupling. Despite this, the two mutant actins retained their ATPase activity. In fact, the mutated actins expressed a significant ATPase activity even in the presence of Ca2+ ions, conditions under which actin normally has a very low ATPase activity. In the presence of Mg2+ ions, the ATPase activity of actin was decreased slightly by the mutations. The mutant actins polymerized as the wild-type protein in the presence of Mg2+ ions, but slower than the wild-type in a K+/Ca2+ milieu. Profilin affected the lag phases and elongation rates during polymerization of the mutant and wild-type actins to the same extent, whereas at steady-state, the concentration of unpolymerized mutant actin appeared to be elevated. Decoration of mutant actin filaments with myosin subfragment 1 appeared to be normal, as did their movement in the low-load motility assay system. Our results show that Ser14 and Asp157 are key residues for interdomain communication, and that hydroxyl and carboxyl groups in positions 14 and 157, respectively, are not necessary for ATP hydrolysis in actin.     

Chymotrypsin C (caldecrin) stimulates autoactivation of human cationic trypsinogen

Trypsin-mediated trypsinogen activation (autoactivation) facilitates digestive zymogen activation in the duodenum but may precipitate pancreatitis if it occurs prematurely in the pancreas. Autoactivation of human cationic trypsinogen is inhibited by a repulsive electrostatic interaction between the unique Asp218 on the surface of cationic trypsin and the conserved tetra-aspartate (Asp19-22) motif in the trypsinogen activation peptide (Nemoda, Z., and Sahin-Tóth, M. (2005) J. Biol. Chem. 280, 29645-29652). Here we describe that this interaction is regulated by chymotrypsin C (caldecrin), which can specifically cleave the Phe18-Asp19 peptide bond in the trypsinogen activation peptide and remove the N-terminal tripeptide. In contrast, chymotrypsin B, elastase 2A, or elastase 3A (proteinase E) are ineffective. Autoactivation of N-terminally truncated cationic trypsinogen is stimulated approximately 3-fold, and this effect is dependent on the presence of Asp218. Because chymotrypsinogen C is activated by trypsin, and chymotrypsin C stimulates trypsinogen activation, these reactions establish a positive feedback mechanism in the digestive enzyme cascade of humans. Furthermore, inappropriate activation of chymotrypsinogen C in the pancreas may contribute to the development of pancreatitis. Consistent with this notion, the pancreatitis-associated mutation A16V in cationic trypsinogen increases the rate of chymotrypsin C-mediated processing of the activation peptide 4-fold and causes accelerated trypsinogen activation in vitro.     

Secretion of mono- and diacylglycerol lipase from Penicillium camembertii U-150 by Saccharomyces cerevisiae and site-directed mutagenesis of the putative catalytic sites of the lipase.

Yeast cells carrying intronless mono- and diacylglycerol lipase (MDGL) genes, constructed by recombination of the genomic gene and cDNA, secreted MDGL into the culture supernatant. Most of the yeast MDGL were extensively glycosylated while they had a similar glyceride specificity to that of native MDGL. Site-directed mutagenesis was used to directly confirm the involvements in enzyme activity of the presumptive amino acid residues to form the catalytic center of MDGL. These residues were conserved in the primary structure alignment of a lipase family from filamentous fungi. Mutant lipase proteins in which Ser83, Ser145, or His259 was replaced with glycine were secreted by yeast transformants as inactive proteins. Mutant proteins replacing Asp199 with glycine or asparagine were not detected in the culture supernatant. Replacing other two highly conserved aspartic acids (at positions 232 and 243) with glycine did not render the enzyme inactive. These results indicate that Ser83, Ser145, and His259 in MDGL, are essential to enzyme activity. Asp199 is also likely to be involved.     

Increasing the thermostability of a neutral protease by replacing positively charged amino acids in the N-terminal turn of alpha-helices.

The 247-260 and 289-299 alpha-helices of Bacillus subtilis neutral protease have a lysine in their N-terminal turn. These lysines were replaced by Ser or Asp in order to improve electrostatic interactions with the alpha-helix dipole. After replacing Lys by Ser at positions 249 or 290, the thermostability of the enzyme was increased by 0.3 and 1.0 degrees C, respectively. The Asp249 and Asp290 mutants exhibited a stabilization of 0.6 and 1.2 degrees C, respectively. The results show the feasibility of stabilizing enzymes by introducing favourable residues at the end of alpha-helices.     

Identification of residues in the carboxy-terminal domain of Clostridium perfringens alpha-toxin (phospholipase C) which are required for its biological activities

A panel of random mutants within the DNA encoding the carboxy-terminal domain of Clostridium perfringens alpha-toxin was constructed. Three mutants were identified which encoded alpha-toxin variants (Lys330Glu, Asp305Gly, and Asp293Ser) with reduced hemolytic activity. These variants also had diminished phospholipase C activity toward aggregated egg yolk phospholipid and reduced cytotoxic and myotoxic activities. Asp305Gly showed a significantly increased enzymatic activity toward the monodisperse substrate rhoNPPC, whereas Asp293Ser displayed a reduced activity toward this phospholipid analogue. In addition, Asp293Ser showed an increased dependence on calcium for enzymatic activity toward aggregated phospholipid and appeared calcium-depleted in PAGE band-shift assays. In contrast, neither Lys330Glu nor Asp305Gly showed altered dependence on calcium for enzymatic activity toward aggregated phospholipid. Asp305 is located in the interface between the amino- and carboxy-terminal domains, whereas Asp293 and Lys330 are surface exposed residues which may play a role in the recognition of membrane phospholipids.     

皖南尖吻蝮蛇毒中类去整合蛋白/富含半胱氨酸两结构域cDNA的克隆和表达

为了探讨出血毒金属蛋白酶结构功能关系 ,通过 RT- PCR方法 ,从皖南尖吻蝮蛇( Agkistrodon acutus)毒腺总 RNA中扩增得到编码 P- 型出血毒金属蛋白酶的完整类去整合蛋白和富含半胱氨酸两个结构域 c DNA( AA/DC) .它全长 964bp的 c DNA,开放阅读框架编码 2 1 6个氨基酸残基 ,序列比较分析表明它同来自 Bothrops jararaca的 jararhagin- C、来自 Crotalus atrox的 catrocollastatin- C有很高的同源性 .在类去整合蛋白结构域中 ,Ser- Glu- Cys- Asp( SECD)代替了去整合蛋白中相应部位的 Arg- Gly- Asp( RGD)三肽序列 .将编码区基因克隆入 p GEX- 2 T载体中 ,转化大肠杆菌 TG- 1 ,用 IPTG诱导表达 ,表达产物具有抑制胶原诱导的血小板凝集活性 ,但不抑制ADP诱导的血小板凝集 .该研究为进一步阐述蛇毒金属蛋白酶结构功能关系和药物开发奠定了基础 .     

Insertion of an elastase-binding loop into interleukin-1 beta

The protease-binding sequence EAIPMSIPPE from alpha 1-antitrypsin has been inserted into the cytokine interleukin-1 beta, replacing residues 50-53. The resulting mutant protein was cleaved specifically at a single site by elastase and chymotrypsin, but not by trypsin. The cleavage by elastase was shown to be between Met and Ser of the inserted loop. In contrast, wild-type interleukin is not susceptible to cleavage by any of these enzymes. The mutant protein acts as an inhibitor of elastase, with a KI of approximately 30 microM. The wild type displays no such inhibitory activity. The overall structure of the mutant, as demonstrated by CD, appears to be indistinguishable from that of the wild type. These results indicate that the protease-binding region of alpha 1-antitrypsin can be recognized and is active even within the context of an entirely different protein structure. Given that interleukin-1 beta binds to, and is internalized by, many types of cells, this hybrid protein also demonstrates the feasibility of using interleukin-1 beta as a delivery system for useful therapeutic agents.     

One step purification procedure of elastase from pancreatic powder by affinity chromatography

Pancreatic elastase was isolated from acetone powder of porcine pancreas by a one step purification procedure on a trialanyl CH-Sepharose 4B affinity column. This column exhibited affinity not only for active elastase but also for trypsin and chymotrypsin which were present in the same pancreatic powder. However, as the extent of affinity toward elastase is considerably higher, the proper conditions were determined with which the adsorbed elastase was isolated in a highly purified form. The yield of elastolytic activity ranged from 60–85% and the purified elastase was shown to be one component by polyacrylamide disc electrophoresis.     

Conversion of the Alzheimer's beta-amyloid precursor protein (APP) Kunitz domain into a potent human neutrophil elastase inhibitor

Site-specific mutagenesis techniques have been used to construct active site variants of the Kunitz-type protease inhibitor domain present in the Alzheimer's beta-amyloid precursor protein (APP-KD). Striking alteration of its protease inhibitory properties were obtained when the putative P1 residue, arginine, was replaced with the small hydrophobic residue valine. The altered protein was no longer inhibitory toward bovine pancreatic trypsin, human Factor XIa, mouse epidermal growth factor-binding protein, or bovine chymotrypsin, all of which are strongly inhibited by the unaltered APP-KD (Sinha, S., Dovey, H. F., Seubert, P., Ward, P. J., Blacher, R. W., Blaber, M., Bradshaw, R. A., Arici, M., Mobley, W. C., and Lieberburg, I. (1990) J. Biol. Chem. 265, 8983-8985). Instead, the P1-Val-APP-KD was a potent inhibitor of human neutrophil elastase, with a Ki = 0.8 nM, as estimated by the inhibition of the activity of human neutrophil elastase measured using a chromogenic substrate. It also inhibited the degradation of insoluble elastin by the enzyme virtually stoichiometrically. Replacement of the P1' (Ala) and P2' (Met) residues of P1-Val-MKD with the corresponding residues (Ser, Ile) from alpha 1-proteinase inhibitor resulted in an inactive protein, underscoring the mechanistic differences between the serpins from the Kunitz-type protease inhibitor family. These results confirm the importance of the P1 arginine residue of APP-KD in determining inhibitory specificity, and are also the first time that a single amino acid replacement has been shown to generate a specific potent human neutrophil elastase inhibitor from a human KD sequence.