Monomeric Enzyme Research Articles

Role of connecting loop I in catalysis and allosteric regulation of human glucokinase.

Glucokinase (GCK, hexokinase IV) is a monomeric enzyme with a single glucose binding site that displays steady-state kinetic cooperativity, a functional characteristic that affords allosteric regulation of GCK activity. Structural evidence suggests that connecting loop I, comprised of residues 47-71, facilitates cooperativity by dictating the rate and scope of motions between the large and small domains of GCK. Here we investigate the impact of varying the length and amino acid sequence of connecting loop I upon GCK cooperativity. We find that sequential, single amino acid deletions from the C-terminus of connecting loop I cause systematic decreases in cooperativity. Deleting up to two loop residues leaves the kcat value unchanged; however, removing three or more residues reduces kcat by 1000-fold. In contrast, the glucose K0.5 and KD values are unaffected by shortening the connecting loop by up to six residues. Substituting alanine or glycine for proline-66, which adopts a cis conformation in some GCK crystal structures, does not alter cooperativity, indicating that cis/trans isomerization of this loop residue does not govern slow conformational reorganizations linked to hysteresis. Replacing connecting loop I with the corresponding loop sequence from the catalytic domain of the noncooperative isozyme human hexokinase I (HK-I) eliminates cooperativity without impacting the kcat and glucose K0.5 values. Our results indicate that catalytic turnover requires a minimal length of connecting loop I, whereas the loop has little impact upon the binding affinity of GCK for glucose. We propose a model in which the primary structure of connecting loop I affects cooperativity by influencing conformational dynamics, without altering the equilibrium distribution of GCK conformations.

Dry entrapment of enzymes by epoxy or polyester resins hardened on different solid supports

Embedding of enzymes was performed with epoxy or polyester resin by mixing in a dried enzyme preparation before polymerization was started. This fast and low-cost immobilization method produced enzymatically active layers on different solid supports.As model enzymes the well-characterized Thermomyces lanuginosus lipase and a new threonine aldolase from Ashbya gossypii were used. It was shown that T. lanuginosus lipase recombinantly expressed in Aspergillus oryzae is a monomeric enzyme with a molecular mass of 34kDa, while A. gossypii threonine aldolase expressed in Escherichia coli is a pyridoxal-5′-phosphate binding homotetramer with a mass of 180kDa.The enzymes were used freeze dried, in four different preparations: freely diffusing, adsorbed on octyl sepharose, as well as cross-linked enzyme aggregates or as suspensions in organic solvent. They were mixed with standard two-component resins and prepared as layers on solid supports made of different materials e.g. metal, glass, polyester. Polymerization led to encapsulated enzyme preparations showing activities comparable to literature values.

Biochemical characterization of a functional recombinant aryl‐alcohol dehydrogenase from Taiwanofungus camphorata (583.4)

A TcAAD cDNA encoding a putative aryl‐alcohol dehydrogenase (AAD) was cloned from Taiwanofungus camphorata (Tc). The deduced amino acid sequence is conserved among the reported AADs. A 3‐D structural model of the TcAAD has been created based on the known structure of voltage‐dependent potassium channels subunit beta‐2 (PDB code: 3EAU). To characterize the TcAAD, the coding region was subcloned into an expression vector and transformed into Saccharomyces cerevisiae. The recombinant His6‐tagged TcAAD was overexpressed and purified by Ni affinity chromatography. The purified enzyme showed a band of approximately 39 kDa on a 12 % SDS‐PAGE. The molecular mass determined by MALDI‐TOF is 40.58 kDa which suggests that the purified enzyme is a monomeric enzyme. Using veratraldehyde as a substrate, the KM, Vmax of TcADD was determined at pH 6.0. Using benzyl alcohol derivatives as substrates, the oxidizing power of TcADD via NAD+ at pH 9.6 was studied.Grant Funding Source: National Science Council of the Republic of China, Taiwan under grant 100‐2313‐B‐019‐003‐MY3

Monoacylglycerol Acyltransferase-2 Is a Tetrameric Enzyme That Selectively Heterodimerizes with Diacylglycerol Acyltransferase-1

Acyl-CoA:monoacylglycerol acyltransferases (MGATs) and diacylglycerol acyltransferases (DGATs) catalyze the two consecutive steps in the synthesis of triacylglycerol, a key process required for dietary fat absorption into the enterocytes of the small intestine. In this report, we investigated the tendency of MGAT2 to form an enzyme complex with DGAT1 and DGAT2 in intact cells. We demonstrated that in addition to the 38-kDa monomer of the MGAT2 enzyme predicted by its peptide sequence, a 76-kDa moiety was detected in SDS-PAGE without reducing agent and heat inactivation. The 76-kDa MGAT2 moiety was greatly enhanced by treatment with a cross-linking reagent in intact cells. Additionally, the cross-linking reagent dose-dependently yielded a band corresponding to the tetramer (152 kDa) in SDS-PAGE, suggesting that the MGAT2 enzyme primarily functions as a homotetrameric protein and as a tetrameric protein. Likewise, DGAT1 also forms a homodimer under nondenaturing conditions. When co-expressed in COS-7 cells, MGAT2 heterodimerized with DGAT1 without treatment with a cross-linking reagent. MGAT2 also co-eluted with DGAT1 on a gel filtration column, suggesting that the two enzymes form a complex in intact cells. In contrast, MGAT2 did not heterodimerize with DGAT2 when co-expressed in COS-7 cells, despite high sequence homology between the two enzymes. Furthermore, systematic deletion analysis demonstrates that N-terminal amino acids 35-80 of DGAT1, but not a signal peptide at the N terminus of MGAT2, is required for the heterodimerization. Finally, co-expression of MGAT2 with DGAT1 significantly increased lipogenesis in COS-7 cells, indicating the functional importance of the dimerization.

Recent and future grand challenges in protein folding, misfolding, and degradation.

Recent and future grand challenges in protein folding, misfolding, and degradation.

Probing Kinase Activation and Substrate Specificity with an Engineered Monomeric IKK2

Catalytic subunits of the IκB kinase (IKK), IKK1/IKKα, and IKK2/IKKβ function in vivo as dimers in association with the necessary scaffolding subunit NEMO/IKKγ. Recent X-ray crystal structures of IKK2 suggested that dimerization might be mediated by a smaller protein–protein interaction than previously thought. Here, we report that removal of a portion of the scaffold dimerization domain (SDD) of human IKK2 yields a kinase subunit that remains monomeric in solution. Expression in baculovirus-infected Sf9 insect cells and purification of this engineered monomeric human IKK2 enzyme allows for in vitro analysis of its substrate specificity and mechanism of activation. We find that the monomeric enzyme, which contains all of the amino-terminal kinase and ubiquitin-like domains as well as the more proximal portions of the SDD, functions in vitro to direct phosphorylation exclusively to residues S32 and S36 of its IκBα substrate. Thus, the NF-κB-inducing potential of IKK2 is preserved in the engineered monomer. Furthermore, we observe that our engineered IKK2 monomer readily autophosphorylates activation loop serines 177 and 181 in trans. However, when residues that were previously observed to interfere with IKK2 trans autophosphorylation in transfected cells are mutated within the context of the monomer, the resulting Sf9 cell expressed and purified proteins were significantly impaired in their trans autophosphorylation activity in vitro. This study further defines the determinants of substrate specificity and provides additional evidence in support of a model in which activation via trans autophosphorylation of activation loop serines in IKK2 requires transient assembly of higher-order oligomers.

Purification and characterization of detergent-compatible protease from Aspergillus terreus gr.

The possibility of using Aspergillus terreus protease in detergent formulations was investigated. Sodium dodecyl sulfate (SDS) and native polyacrylamide gel electrophoresis indicated that the purified alkaline protease (148.9 U/mg) is a monomeric enzyme with a molecular mass of 16 ± 1 kDa. This was confirmed by liquid chromatography–mass spectrometry. The active enzyme degraded the co-polymerized gelatin. The protease demonstrated excellent stability at pH range 8.0–12.0 with optimum at pH 11.0. It was almost 100 % stable at 50 °C for 24 h, enhanced by Ca2+ and Mg2+, but inhibited by Hg2+, and strongly inhibited by phenylmethyl sulfonyl fluoride. It showed maximum activity against casein followed by gelatin; its Vmax was 12.8 U/ml with its corresponding KM of 5.4 mg/ml. The proteolytic activity was activated by Tween-80, Triton-100 and SDS, and remained unaltered in the presence of H2O2 and NaClO. The enzyme exhibited higher storage stability at 4, 28 and −20 °C. It was stable and compatible to the desired level in the local detergents. The addition of the protease to the Super wheel improved its blood stain removal. The isolated protease can thus be a choice option in detergent industry.

Identification of a novel polyfluorinated compound as a lead to inhibit the human enzymes aldose reductase and AKR1B10: structure determination of both ternary complexes and implications for drug design

Aldo-keto reductases (AKRs) are mostly monomeric enzymes which fold into a highly conserved (α/β)8 barrel, while their substrate specificity and inhibitor selectivity are determined by interaction with residues located in three highly variable external loops. The closely related human enzymes aldose reductase (AR or AKR1B1) and AKR1B10 are of biomedical interest because of their involvement in secondary diabetic complications (AR) and in cancer, e.g. hepatocellular carcinoma and smoking-related lung cancer (AKR1B10). After characterization of the IC50 values of both AKRs with a series of polyhalogenated compounds, 2,2',3,3',5,5',6,6'-octafluoro-4,4'-biphenyldiol (JF0064) was identified as a lead inhibitor of both enzymes with a new scaffold (a 1,1'-biphenyl-4,4'-diol). An ultrahigh-resolution X-ray structure of the AR-NADP(+)-JF0064 complex has been determined at 0.85 Å resolution, allowing it to be observed that JF0064 interacts with the catalytic residue Tyr48 through a negatively charged hydroxyl group (i.e. the acidic phenol). The non-competitive inhibition pattern observed for JF0064 with both enzymes suggests that this acidic hydroxyl group is also present in the case of AKR1B10. Moreover, the combination of surface lysine methylation and the introduction of K125R and V301L mutations enabled the determination of the X-ray crystallographic structure of the corresponding AKR1B10-NADP(+)-JF0064 complex. Comparison of the two structures has unveiled some important hints for subsequent structure-based drug-design efforts.

Biochemical characterization of a functional recombinant aryl-alcohol dehydrogenase from Taiwanofungus camphorata.

BackgroundAryl-alcohol dehydrogenases (AADs) have been known to involve in the metabolism of aromatic compounds.ResultsOne TcAAD cDNA (GenBank HQ453361) encoding a putative aryl-alcohol dehydrogenase (AAD) was cloned from Taiwanofungus camphorata. The deduced amino acid sequence is conserved among the reported AADs. A 3-D structural model of the TcAAD has been created based on the known structure of voltage-dependent potassium channels subunit beta-2 (PDB code: 3EAU). To characterize the TcAAD, the coding region was subcloned into an expression vector and transformed into Saccharomyces cerevisiae. The recombinant His6-tagged TcAAD was overexpressed and purified by Ni affinity chromatography. The purified enzyme showed a band of approximately 39 kDa on a 12% SDS-PAGE. The molecular mass determined by MALDI-TOF is 40.58 kDa which suggests that the purified enzyme is a monomeric enzyme. Using veratraldehyde as a substrate, the KM, Vmax of TcADD was determined at pH 6.0. Using benzyl alcohol derivatives as substrates, the oxidizing power of TcADD via NAD+ at pH 9.6 was studied.ConclusionsThe coding sequence of the TcAAD cDNA was introduced into an S. cerevisiae expression system and the active enzyme purified and characterized. Understanding the properties of this TcAAD will be beneficial for its potential in xenobiotic detoxification or production of natural flavors.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-55-14) contains supplementary material, which is available to authorized users.

Molybdenum reduction to molybdenum blue in Serratia sp. Strain DRY5 is catalyzed by a novel molybdenum-reducing enzyme.

The first purification of the Mo-reducing enzyme from Serratia sp. strain DRY5 that is responsible for molybdenum reduction to molybdenum blue in the bacterium is reported. The monomeric enzyme has an apparent molecular weight of 105 kDalton. The isoelectric point of this enzyme was 7.55. The enzyme has an optimum pH of 6.0 and maximum activity between 25 and 35°C. The Mo-reducing enzyme was extremely sensitive to temperatures above 50°C (between 54 and 70°C). A plot of initial rates against substrate concentrations at 15 mM 12-MP registered a V max for NADH at 12.0 nmole Mo blue/min/mg protein. The apparent K m for NADH was 0.79 mM. At 5 mM NADH, the apparent V max and apparent K m values for 12-MP of 12.05 nmole/min/mg protein and 3.87 mM, respectively, were obtained. The catalytic efficiency (k cat/K m) of the Mo-reducing enzyme was 5.47 M−1 s−1. The purification of this enzyme could probably help to solve the phenomenon of molybdenum reduction to molybdenum blue first reported in 1896 and would be useful for the understanding of the underlying mechanism in molybdenum bioremediation involving bioreduction.

Local packing density is the main structural determinant of the rate of protein sequence evolution at site level.

Functional and biophysical constraints result in site-dependent patterns of protein sequence variability. It is commonly assumed that the key structural determinant of site-specific rates of evolution is the Relative Solvent Accessibility (RSA). However, a recent study found that amino acid substitution rates correlate better with two Local Packing Density (LPD) measures, the Weighted Contact Number (WCN) and the Contact Number (CN), than with RSA. This work aims at a more thorough assessment. To this end, in addition to substitution rates, we considered four other sequence variability scores, four measures of solvent accessibility (SA), and other CN measures. We compared all properties for each protein of a structurally and functionally diverse representative dataset of monomeric enzymes. We show that the best sequence variability measures take into account phylogenetic tree topology. More importantly, we show that both LPD measures (WCN and CN) correlate better than all of the SA measures, regardless of the sequence variability score used. Moreover, the independent contribution of the best LPD measure is approximately four times larger than that of the best SA measure. This study strongly supports the conclusion that a site's packing density rather than its solvent accessibility is the main structural determinant of its rate of evolution.

Characterization of a Thermostable Xylanase from the Extremely Thermophilic Bacterium Thermotoga hypogea

Thermotoga hypogea is an extremely thermophilic bacterium that can utilize both peptides and carbohydrates including xylan as growth substrates at 90 ºC. It was found that its xylanase activity was increased up to 9-fold in the presence of either xylan or xylose in growth media, which was independent of growth phases. More than 95% of xylanase activities were associated with cells of T. hypogea. Ultracentrifugation of the cell-free extract of the organism showed that most xylanase activities were in the soluble fraction. A xylanase was purified from the cell-free extract to homogeneity using an FPLC system. The purified enzyme had a molecular mass of 38 kDa as revealed by SDS-PAGE. Its native molecular mass was determined using gel-filtration to be 29 kDa, indicating it was a monomeric enzyme. The purified xylanase was stable with a half-life of 270 min at 90°C in the presence of xylan and BSA. Its optimal pH and temperature were determined to be 5.5 and 90°C, respectively. An apparent Km-value was determined to be 0.07% (w/v) of xylan. Hydrolytic products from xylan catalyzed by the purified enzyme were xylobiose and xylooligosaccharides (n>2). No cellulase activity was detected for the purified xylanase.

Isolation and Characterization of a Novel Endoglucanase from a Bursaphelenchus xylophilus Metagenomic Library

A novel gene (designated as cen219) encoding endoglucanase was isolated from a Bursaphelenchus xylophilus metagenomic library by functional screening. Sequence analysis revealed that cen219 encoded a protein of 367 amino acids. SDS-PAGE analysis of purified endoglucanase suggested that Cen219 was a monomeric enzyme with a molecular mass of 40 kDa. The optimum temperature and pH for endoglucanase activity of Cen219 was separately 50°C and 6.0. It was stable from 30 to 50°C, and from pH 4.0 to 7.0. The activity was significantly enhanced by Mn2+ and dramatically reduced by detergent SDS and metals Fe3+, Cu2+ or Hg2+. The enzyme hydrolyzed a wide range of β-1, 3-, and β-1, 4-linked polysaccharides, with varying activities. Activities towards microcrystalline cellulose and filter paper were relatively high, while the highest activity was towards oat gum. The Km and Vmax of Cen219 towards CMC was 17.37 mg/ml and 333.33 U/mg, respectively. The findings have an insight into understanding the molecular basis of host–parasite interactions in B. xylophilus species. The properties also make Cen219 an interesting enzyme for biotechnological application.

Using Single-Turnover Kinetics with Osmotic Stress To Characterize the EcoRV Cleavage Reaction

Type II restriction endonucleases require metal ions to specifically cleave DNA at canonical sites. Despite the wealth of structural and biochemical information, the number of Mg(2+) ions used for cleavage by EcoRV, in particular, at physiological divalent ion concentrations has not been established. In this work, we employ a single-turnover technique that uses osmotic stress to probe reaction kinetics between an initial specific EcoRV-DNA complex formed in the absence of Mg(2+) and the final cleavage step. With osmotic stress, complex dissociation before cleavage is minimized and the reaction rates are slowed to a convenient time scale of minutes to hours. We find that cleavage occurs by a two-step mechanism that can be characterized by two rate constants. The dependence of these rate constants on Mg(2+) concentration and osmotic pressure gives the number of Mg(2+) ions and water molecules coupled to each kinetic step of the EcoRV cleavage reaction. Each kinetic step is coupled to the binding 1.5-2.5 Mg(2+) ions, the uptake of ∼30 water molecules, and the cleavage of a DNA single strand. We suggest that each kinetic step reflects an independent, rate-limiting conformational change of each monomer of the dimeric enzyme that allows Mg(2+) ion binding. This modified single-turnover protocol has general applicability for metalloenzymes.

Zn-bis-glutathionate is the best co-substrate of the monomeric phytochelatin synthase from the photosynthetic heavy metal-hyperaccumulator Euglena gracilis

The phytochelatin synthase from photosynthetic Euglena gracilis (EgPCS) was analyzed at the transcriptional, kinetic, functional, and phylogenetic levels. Recombinant EgPCS was a monomeric enzyme able to synthesize, in the presence of Zn(2+) or Cd(2+), phytochelatin2-phytochelatin4 (PC2-PC4) using GSH or S-methyl-GS (S-methyl-glutathione), but not γ-glutamylcysteine or PC2 as a substrate. Kinetic analysis of EgPCS firmly established a two-substrate reaction mechanism for PC2 synthesis with Km values of 14-22 mM for GSH and 1.6-2.5 μM for metal-bis-glutathionate (Me-GS2). EgPCS showed the highest Vmax and catalytic efficiency with Zn-(GS)2, and was inactivated by peroxides. The EgPCS N-terminal domain showed high similarity to that of other PCSases, in which the typical catalytic core (Cys-70, His-179 and Asp-197) was identified. In contrast, the C-terminal domain showed no similarity to other PCSases. An EgPCS mutant comprising only the N-terminal 235 amino acid residues was inactive, suggesting that the C-terminal domain is essential for activity/stability. EgPCS transcription in Euglena cells was not modified by Cd(2+), whereas its heterologous expression in ycf-1 yeast cells provided resistance to Cd(2+) stress. Phylogenetic analysis of the N-terminal domain showed that EgPCS is distant from plants and other photosynthetic organisms, suggesting that it evolved independently. Although EgPCS showed typical features of PCSases (constitutive expression; conserved N-terminal domain; kinetic mechanism), it also exhibited distinct characteristics such as preference for Zn-(GS)2 over Cd-(GS)2 as a co-substrate, a monomeric structure, and ability to solely synthesize short-chain PCs, which may be involved in conferring enhanced heavy-metal resistance.

Characterization of a new oxygen-insensitive azoreductase from Brevibacillus laterosporus TISTR1911: Toward dye decolorization using a packed-bed metal affinity reactor

This study reports the identification of a new bacterial azoreductase from Brevibacillus laterosporus TISTR1911, its heterologous production in Escherichia coli, the biochemical characterization and immobilization for use in dye biodegradation processes. The recombinant azoreductase (BrAzo) is a monomeric FMN oxygen-insensitive enzyme with a molecular mass of 23 kDa showing a broad specificity for the reduction of synthetic azo dyes. Double hexahistidine-tagged BrAzo was immobilized onto a nickel chelating column and methyl orange was used to assess its degradation potential using a packed-bed reactor. The dye degradation is described by an exponential model in a downstream batchwise continuous flow mode operated with recycling. The complete degradation of methyl orange (170 μM at 600 mL/h) was achieved in 3 h and continued over 9 cycles. Coupling the immobilized BrAzo with glucose dehydrogenase for NADH regeneration yielded a shorter 1.5 h-degradation period that was maintained throughout 16 cycles.

RecG and UvsW catalyse robust DNA rewinding critical for stalled DNA replication fork rescue

Helicases that both unwind and rewind DNA have central roles in DNA repair and genetic recombination. In contrast to unwinding, DNA rewinding by helicases has proved difficult to characterize biochemically because of its thermodynamically downhill nature. Here we use single-molecule assays to mechanically destabilize a DNA molecule and follow, in real time, unwinding and rewinding by two DNA repair helicases, bacteriophage T4 UvsW and Escherichia coli RecG. We find that both enzymes are robust rewinding enzymes, which can work against opposing forces as large as 35 pN, revealing their active character. The generation of work during the rewinding reaction allows them to couple rewinding to DNA unwinding and/or protein displacement reactions central to the rescue of stalled DNA replication forks. The overall results support a general mechanism for monomeric rewinding enzymes.

ITPA (inosine triphosphate pyrophosphatase): From surveillance of nucleotide pools to human disease and pharmacogenetics

Cellular nucleotide pools are often contaminated by base analog nucleotides which interfere with a plethora of biological reactions, from DNA and RNA synthesis to cellular signaling. An evolutionarily conserved inosine triphosphate pyrophosphatase (ITPA) removes the non-canonical purine (d)NTPs inosine triphosphate and xanthosine triphosphate by hydrolyzing them into their monophosphate form and pyrophosphate. Mutations in the ITPA orthologs in model organisms lead to genetic instability and, in mice, to severe developmental anomalies. In humans there is genetic polymorphism in ITPA. One allele leads to a proline to threonine substitution at amino acid 32 and causes varying degrees of ITPA deficiency in tissues and plays a role in patients' response to drugs. Structural analysis of this mutant protein reveals that the protein is destabilized by the formation of a cavity in its hydrophobic core. The Pro32Thr allele is thought to cause the observed dominant negative effect because the resulting active enzyme monomer targets both homo- and heterodimers to degradation.

The branched mitochondrial respiratory chain from Debaryomyces hansenii: Components and supramolecular organization

The branched respiratory chain in mitochondria from the halotolerant yeast Debaryomyces hansenii contains the classical complexes I, II, III and IV plus a cyanide-insensitive, AMP-activated, alternative-oxidase (AOX). Two additional alternative oxidoreductases were found in this organism: an alternative NADH dehydrogenase (NDH2e) and a mitochondrial isoform of glycerol-phosphate dehydrogenase (MitGPDH). These monomeric enzymes lack proton pump activity. They are located on the outer face of the inner mitochondrial membrane. NDH2e oxidizes exogenous NADH in a rotenone-insensitive, flavone-sensitive, process. AOX seems to be constitutive; nonetheless, most electrons are transferred to the cytochromic pathway. Respiratory supercomplexes containing complexes I, III and IV in different stoichiometries were detected. Dimeric complex V was also detected. In-gel activity of NADH dehydrogenase, mass spectrometry, and cytochrome c oxidase and ATPase activities led to determine the composition of the putative supercomplexes. Molecular weights were estimated by comparison with those from the yeast Y. lipolytica and they were IV2, I–IV, III2–IV4, V2, I–III2, I–III2–IV, I–III2–IV2, I–III2–IV3 and I–III2–IV4. Binding of the alternative enzymes to supercomplexes was not detected. This is the first report on the structure and organization of the mitochondrial respiratory chain from D. hansenii.

Implantation serine proteinase 2 is a monomeric enzyme with mixed serine proteolytic activity and can silence signalling via proteinase activated receptors

Implantation serine proteinase 2 (ISP2), a S1 family serine proteinase, is known for its role in the critical processes of embryo hatching and implantation in the mouse uterus. Native implantation serine proteinases (ISPs) are co-expressed and co-exist as heterodimers in uterine and blastocyst tissues. The ISP1-ISP2 enzyme complex shows trypsin-like substrate specificity. In contrast, we found that ISP2, isolated as a 34 kDa monomer from a Pichia pastoris expression system, exhibited a mixed serine proteolytic substrate specificity, as determined by a phage display peptide cleavage approach and verified by the in vitro cleavage of synthetic peptides. Based upon the peptide sequence substrate selectivity, a database search identified many potential ISP2 targets of physiological relevance, including the proteinase activated receptor 2 (PAR2). The in vitro cleavage studies with PAR2-derived peptides confirmed the mixed substrate specificity of ISP2. Treatment of cell lines expressing proteinase-activated receptors (PARs) 1, 2, and 4 with ISP2 prevented receptor activation by either thrombin (PARs 1 and 4) or trypsin (PAR2). The disarming and silencing of PARs by ISP2 may play a role in successful embryo implantation.