Expression and purification of the mouse mammary tumor virus gag-pro transframe protein p30 and characterization of its dUTPase activity.

The mouse mammary tumor virus gag-pro transframe protein (p30) contains the nucleocapsid protein domain derived from the 3' end of gag, fused to 154 residues encoded by the 5' region of the pro open reading frame. The DNA coding for p30 was cloned into the plasmid pALTER-1, and an additional nucleotide was inserted by site-directed mutagenesis to allow the read-through from the gag into the pro open reading frame. The obtained insert was then cloned into pGEX-2T, a plasmid containing the glutathione S-transferase gene of Schistosoma japonicum and a nucleotide sequence encoding for a thrombin cleavage site. The chimeric protein (GST-p30) was isolated by affinity chromatography on a glutathione-Sepharose 4B column, and after thrombin treatment, the excised p30 was further purified on a single-stranded DNA-agarose column. This protein showed dUTPase activity, with only negligible cleavage of dATP, dGTP, dCTP, dTTP, or UTP. Its apparent Km for dUTP was 28 microM. The enzyme was inhibited by EDTA, but its effect could be reversed by Mg2+ and other divalent cations. dUTPase activity was also detected in purified mouse mammary tumor virus, and p30 was the only protein recognized by antibodies directed towards the carboxyl-terminal sequence of the dUTPase coding region.     

Cleavage specificity of the subtilisin-like protease C1 from soybean

The cleavage specificity of protease C1, isolated from soybean (Glycine max (L.) Merrill) seedling cotyledons, was examined using oligopeptide substrates in an HPLC based assay. A series of peptides based on the sequence Ac-KVEKEESEEGE-NH2 was used, mimicking a natural cleavage site of protease C1 in the alpha subunit of the storage protein beta-conglycinin. A study of substrate peptides truncated from either the N- or C-terminus indicates that the minimal requirements for cleavage by protease C2 are three residues N-terminal to the cleaved bond, and two residues C-terminal (i.e. P3-P2'). The maximal rate of cleavage is reached with substrates containing four to five residues N-terminal to the cleaved bond and four residues C-terminal (i.e. P4 or P5 to P4'). The importance of Glu residues at the P1, P1', and P4 positions was examined using a series of substituted nonapeptides (P5-P4') with a base sequence of Ac-KVEKEESEE-NH2. At the P1 position, the relative ranking, based on kcat/Km, was E>Q>K>A>D>F>S. Substitutions at the P1' position yield the ranking E congruent withQ>A>S>D>K>F, while those at P4' had less effect on kcat/Km, yielding the ranking F congruent with S congruent with E congruent withD>K>A congruent withQ. These data show that protease C1 prefers to cleave at Glu-Glu and Glu-Gln bonds, and that the nature of the P4' position is less important. The fact that there is specificity in the cleavage of the oligopeptides suggests that the more limited specific cleavage of the alpha and alpha' subunits of beta-conglycinin by protease C1 is due to a combination of the sequence cleavage specificity of the protease and the accessibility of appropriate scissile peptide bonds on the surface of the substrate protein.     

New vectors for high level expression of recombinant proteins in bacteria.

A system has been developed for synthesis and rapid purification of recombinant polypeptides expressed in frame with glutathione S-transferase (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). Expressed fusion proteins are purified from bacterial extracts by glutathione-agarose affinity chromatography. A thrombin protease cleavage site allowed for proteolysis of the fusion protein. We reported the construction of the vector pGEX-KG (K. Guan and J. E. Dixon, 1991, Anal. Biochem. 192, 262-267) which has a glycine-rich "kinker" immediately after the thrombin cleavage site. This kinker dramatically improved the thrombin cleavage efficiency of several fusion proteins. One potential drawback of expressing proteins in this vector is that, following proteolytic cleavage, unrelated amino acids from the vector remain at the amino terminus of the released protein. These extensions could affect enzymatic activity or protein structure. We have constructed two new vectors, pGEX-KT and pGEX-KN, which have the glycine kinker placed N-terminal to the thrombin cleavage site in order to minimize the unrelated amino acids associated with the cleaved protein. The change in location of the kinker had no effect on the increased thrombin cleavage efficiency. A strategy combining the kinker in the vector pGEX-KN with polymerase chain reaction has also been developed to express fusion proteins which when cleaved with thrombin released a protein having no amino terminal extensions of any kind.     

Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase

Several systems have been developed to allow for rapid and efficient purification of recombinant proteins expressed in bacteria. The expression of polypeptides in frame with glutathione S-transferase (GST) allows for purification of the fusion proteins from crude bacterial extracts under nondenaturing conditions by affinity chromatography on glutathione agarose (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). This vector expression system has also incorporated specific protease cleavage sites to facilitate proteolysis of the bacterial fusion proteins. In our hands, the cleavage of these fusion proteins at a thrombin cleavage site proceeded slowly. To facilitate the cleavage of fusion proteins, we have introduced a glycine-rich linker (glycine kinker) containing the sequence P.G.I.S.G.G.G.G.G located immediately following the thrombin cleavage site. This glycine kinker greatly increases the thrombin cleavage efficiency of several fusion proteins. The introduction of the glycine kinker into fusion proteins allows for the cleavage of the fusion proteins while they are attached to the affinity resin resulting in a single step purification of the recombinant protein. More than 2 mg of the highly purified protein was obtained from the equivalent of 100 ml of bacterial culture within a few hours when a protein tyrosine phosphatase was employed as a test protein. The vector, pGEX-KG, has also been modified to facilitate cloning of a variety of cDNAs in all reading frames and has been successfully used to express several eukaryotic proteins.     

Functional expression of Arabidopsis thaliana anthranilate synthase subunit I in Escherichia coli.

Anthranilate synthase is involved in tryptophan (Trp) biosynthesis. Functional expression of subunit I from Arabidopsis (ASA1) was achieved in bacteria as a protein fused with glutathione S-transferase (GST). The active product was purified in a single step on a glutathione-Sepharose column. The Vmax (45 nmol min-1mg-1), the apparent K(M) for chorismate (180 microM), and the feedback inhibition by Trp (complete inhibition by 10 microM Trp) of the purified fusion product (GST-ASA1) were comparable to anthranilate synthase purified from plants. Polyclonal antibodies raised against the fusion project and purified by affinity chromatography on a GST-ASA1-Sepharose column cross-reacted with a 61.5-kD protein in a partially purified anthranilate synthase preparation from corn seedlings. GST-ASA1 cleavage by thrombin, as well as site-directed mutagenesis modifications of the Trp allosteric site, inactivated the recombinant protein.     

Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate

SARS main protease is essential for life cycle of SARS coronavirus and may be a key target for developing anti-SARS drugs. Recently, the enzyme expressed in Escherichia coli was characterized using a HPLC assay to monitor the formation of products from 11 peptide substrates covering the cleavage sites found in the SARS viral genome. This protease easily dissociated into inactive monomer and the deduced Kd of the dimer was 100 microM. In order to detect enzyme activity, the assay needed to be performed at micromolar enzyme concentration. This makes finding the tight inhibitor (nanomolar range IC50) impossible. In this study, we prepared a peptide with fluorescence quenching pair (Dabcyl and Edans) at both ends of a peptide substrate and used this fluorogenic peptide substrate to characterize SARS main protease and screen inhibitors. The fluorogenic peptide gave extremely sensitive signal upon cleavage catalyzed by the protease. Using this substrate, the protease exhibits a significantly higher activity (kcat = 1.9 s(-1) and Km = 17 microM) compared to the previously reported parameters. Under our assay condition, the enzyme stays as an active dimer without dissociating into monomer and reveals a small Kd value (15 nM). This enzyme in conjunction with fluorogenic peptide substrate provides us a suitable tool for identifying potent inhibitors of SARS protease.     

Expression, purification, and properties of recombinant encephalomyocarditis virus RNA-dependent RNA polymerase.

Encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST), which allowed easy purification of the fusion protein by affinity chromatography on immobilized glutathione. Inclusion of a thrombin cleavage site between the GST carrier and the viral enzyme facilitated the release of purified mature EMC virus RNA polymerase from the GST carrier by proteolysis with thrombin. The purified recombinant enzyme has a molecular mass of about 52 kDa and is recognized by polyclonal immune serum raised against a peptide sequence corresponding to the C-terminal region of the protein. The recombinant enzyme comigrates with immunoprecipitated EMC virus RNA polymerase from infected mouse L929 cell extracts when run in parallel lanes on a sodium dodecyl sulfate-polyacrylamide gel. The enzyme exhibits rifampin-resistant, poly(A)-dependent poly(U) polymerase activity and RNA polymerase activity, which are both oligo(U) dependent. Template-size products are synthesized in in vitro reactions with EMC virus genomic RNA or globin mRNA. The availability of recombinant EMC virus RNA polymerase in a purified form will allow biochemical analysis of its role in the replication of the virus as well as structure-function studies of this unique class of enzyme.     

Mutagenesis of Ala290, which modulates substrate subsite affinity at the catalytic interface of dimeric ThMA

The goal of this study was to develop a maltose-producing enzyme using protein engineering and to clarify the relation between the substrate specificity and the structure of the substrate-binding site of dimeric maltogenic amylase isolated from Thermus (ThMA). Ala290 at the interface of ThMA dimer in the vicinity of the substrate-binding site was substituted with isoleucine, which may cause a structural change due to its bulky side chain. TLC analysis of the action pattern of the mutant ThMA-A290I, using maltooligosaccharides as substrates, revealed that ThMA-A290I used maltotetraose to produce mostly maltose, while wild-type ThMA produced glucose as well as maltose. The wild-type enzyme eventually hydrolyzed the maltose produced from maltotetraose into glucose, but the mutant enzyme did not. For both enzymes, the cleavage frequency of the glycosidic bond of maltooligosaccharides was the highest at the second bond from the reducing end. The mutant ThMA had a much higher Km value for maltose than the wild-type ThMA. The kinetic parameter, kcat/Km) of ThMA-A290I for maltose was 48 times less than that of wild-type ThMA, suggesting that the subsite affinity and hydrolysis mode of ThMA were modulated by the residue located at the interface of ThMA dimer near the active site. The conformational rearrangement in the catalytic interface probably led to the change in the substrate binding affinity of the mutant ThMA. Our results provide basic information for the enzymatic preparation of high-maltose syrup.     

Identification of a high affinity leukotriene C4-binding protein in rat liver cytosol as glutathione S-transferase

A soluble high affinity binding unit for leukotriene (LT) C4 in the high speed supernatant of rat liver homogenate was characterized at 4 degrees C as having a single type of saturable affinity site with a dissociation constant of 0.77 +/- 0.27 nM (mean +/- S.E., n = 5). The binding activity was identified as the liver cytosolic subunit 1 (Ya) of glutathione S-transferase, commonly known as ligandin, by co-purification with the catalytic activity during DEAE-cellulose column chromatography and 11,12,14,15-tetrahydro-LTC4 (LTC2)-affinity gel column chromatography; resolution into two major bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Mr 23,000 and 25,000, of which only the smaller protein was labeled with [3H]LTC4 coupled via a photoaffinity cross-linking reagent; and immunodiffusion analysis with rabbit antiserum to glutathione S-transferase which showed a line of identity between the purified LTC4-binding protein and rat liver glutathione S-transferase. The affinity-purified binding protein bound 800 pmol of [3H] LTC4/mg of protein and possessed 12 mumol/min/mg of glutathione transferase activity as assayed with 1-chloro-2,4-dinitrobenzene as substrate. The enzyme activity of the cytosolic LTC4-binding protein was inhibited by submicromolar quantities of unlabeled LTC4, and the binding activity for [3H]LTC4 was blocked by the ligandin substrates, hematin and bilirubin. The high affinity interaction between LTC4 and glutathione S-transferase suggests that glutathione S-transferase may have a role in LTC4 disposition and that previous studies of LTC4 binding to putative receptors in nonresponsive tissues may require redefinition of the binding unit.     

Enzymatic and molecular characteristics of the efficiency and specificity of exfoliative toxin cleavage of desmoglein 1

Exfoliative toxins (ETs) from Staphylococcus aureus blister the superficial epidermis by hydrolyzing a single peptide bond, Glu381-Gly382, located between extracellular domains 3 and 4 of desmoglein 1 (Dsg1). Enzyme activity is dependent on the calcium-stabilized structure of Dsg1. Here we further define the characteristics of this cleavage. Kinetic studies monitoring the cleavage of Dsg1 by ETA, ETB, and ETD demonstrated kcat/Km values of 2-6 x 10(4) m(-1) s(-1), suggesting very efficient proteolysis. Proteolysis by ETA was not efficiently inhibited by broad spectrum serine protease inhibitors, suggesting that the enzyme cleavage site may be inactive or inaccessible before specific binding to its substrate. Using truncated mutants of human Dsg1 and chimeric molecules between human Dsg1 and either human Dsg3 or canine Dsg1, we show that for cleavage, human-specific amino acids from Dsg1 are necessary in extracellular domain 3 upstream of the scissile bond. If these residues are canine rather than human, ETA binds, but does not cleave, canine Dsg1. These data suggest that the exquisite specificity and efficiency of ETA may depend on the enzyme's binding upstream of the cleavage site with a very specific fit, like a key in a lock.     

Interaction of thrombin with PAR1 and PAR4 at the thrombin cleavage site

Investigations determined the critical amino acids for alpha-thrombin's interaction with protease-activated receptors 1 and 4 (PAR1 and PAR4, respectively) at the thrombin cleavage site. Recombinant PAR1 wild-type (wt) exodomain was cleaved by alpha-thrombin with a Km of 28 microM, a kcat of 340 s-1, and a kcat/Km of 1.2 x 10(7). When the P4 or P2 position was mutated to alanine, PAR1-L38A or PAR1-P40A, respectively, the Km was unchanged, 29 or 23 microM, respectively; however, the kcat and kcat/Km were reduced in each case. In contrast, when Asp39 at P3 was mutated to alanine, PAR1-D39A, Km and kcat were both reduced approximately 3-fold, making the kcat/Km the same as that of PAR1-wt exodomain. Recombinant PAR4-wt exodomain was cleaved by alpha-thrombin with a Km of 61 microM, a kcat of 17 s-1, and a kcat/Km of 2.8 x 10(5). When the P5 or P4 position was mutated to alanine, PAR4-L43A or PAR4-P44A, respectively, there was no change in the Km (69 or 56 microM, respectively); however, the kcat was lowered in each case (9.7 or 7.7 s-1, respectively). Mutation of the P2 position (PAR4-P46A) also had no effect on the Km but markedly lowered the kcat and kcat/Km approximately 35-fold. PAR1-wt exodomain and P4 and P3 mutants were noncompetitive inhibitors of alpha-thrombin hydrolyzing Sar-Pro-Arg-pNA. However, PAR1-P40A displayed a mixed type of inhibition. Mutation of P4, P3, or P2 had no effect on the Ki. All PAR4 exodomains were competitive inhibitors of alpha-thrombin. Mutation of P5, P4, or P2 had no effect on the Ki. These investigations show that Leu at P4 in PAR1 or P5 in PAR4 critically influences the kinetics of alpha-thrombin binding and cleavage of PAR1 and PAR4 exodomains. It also implies that factors other than the hirudin-like binding region on PAR1 exodomain predominate in influencing PAR1 cleavage on cells.     

Characterization of bovine kappa-casein fractions and the kinetics of chymosin-induced macropeptide release from carbohydrate-free and carbohydrate-containing fractions determined by high-performance gel-permeation chromatography.

Bovine kappa-casein was fractionated at pH 8.0 on DEAE-Sepharose with an NaCl gradient, followed by DEAE-cellulose chromatography using a decreasing pH gradient from pH 6.0 to 4.5. At least ten components could be identified, each differing in N-acetylneuraminic acid (NeuAc) and/or phosphorus content. Two components appeared to be multiply-phosphorylated, but did not contain NeuAc. The possible significance of this finding in relation to the mode of phosphorylation and glycosylation in vivo is discussed. A carbohydrate-free fraction as well as two NeuAc-containing fractions were compared in their substrate behaviour towards the action of the milk-clotting enzyme chymosin at pH 6.6 and 30 degrees C. To this end the trichloroacetic acid-soluble reaction products were analysed by high-performance gel-permeation chromatography. In order of increasing carbohydrate content the kcat. values found ranged from 40 to 25 s-1 and the Km values from 9 to 3 microM; the overall substrate properties of these components as reflected by the kinetic parameter kcat./Km ranged from 5 to 8 microM-1 X S-1. Irreversible polymerization of the carbohydrate-free fraction brought about a more-than-2-fold increase in Km, the kcat. value remaining virtually constant. The kcat./Km found for the cleavage of whole kappa-casein at pH 6.6 was of the same magnitude as the kcat./Km found for the polymerized carbohydrate-free fraction (i.e. about 3 microM-1 X S-1). No indication of substrate inhibition was found for the carbohydrate-free fraction.     

Plasmin- and thrombin-accelerated shedding of syndecan-4 ectodomain generates cleavage sites at Lys(114)-Arg(115) and Lys(129)-Val(130) bonds

Syndecans are transmembranous heparan sulfate proteoglycans abundant in the surface of all adherent mammalian cells and involved in vital cellular functions. In this study, we found syndecan-1, -2, -3, and -4 to be constitutively expressed by human umbilical vein endothelial cells. The exposure of the ectodomains of syndecan-1 and -4 to the cell surface and their constitutive shedding into the extracellular compartment was measured by immunoassays. In the presence of plasmin and thrombin, shedding was accelerated and monitored by detection and identification of (35)S-labeled proteoglycans. To elucidate the cleavage site of the syndecan ectodomains, we used a cell-free in vitro system with enzyme and substrate as the only reactants. For this purpose, we constructed recombinant fusion proteins of the syndecan-1 and -4 ectodomain together with maltose-binding protein and enhanced yellow fluorescent protein as reporter proteins attached to the N and C termini via oligopeptide linkers. After protease treatment of the fusion proteins, the electrophoretically resolved split products were sequenced and cleavage sites of the ectodomain were identified. Plasmin generated cleavage sites at Lys(114) downward arrowArg(115) and Lys(129) downward arrowVal(130) in the ectodomain of syndecan-4. In thrombin proteolysates of the syndecan-4 ectodomain, the cleavage site Lys(114) downward arrowArg(115) was also identified. The cleavage sites for plasmin and thrombin within the syndecan-4 ectodomain were not present in the syndecan-1 ectodomain. Cleavage of the syndecan-1 fusion protein by thrombin occurred only at a control cleavage site (Arg downward arrowGly) introduced into the linker region connecting the ectodomain with the enhanced yellow fluorescent protein. Because both plasmin and thrombin are involved in thrombogenic and thrombolytic processes in the course of the pathogenesis of arteriosclerosis, the detachment of heparan sulfate-bearing ectodomains could be relevant for the development of arteriosclerotic plaques and recruitment of mononuclear blood cells to the plaque.     

Interaction of thrombin des-ETW with antithrombin III, the Kunitz inhibitors, thrombomodulin and protein C. Structural link between the autolysis loop and the Tyr-Pro-Pro-Trp insertion of thrombin.

X-ray diffraction studies of human thrombin revealed that compared with trypsin, two insertions (B and C) potentially limit access to the active site groove. When amino acids Glu146, Thr147, and Trp148, adjacent to the C-insertion (autolysis loop), are deleted the resulting thrombin (des-ETW) has dramatically altered interaction with serine protease inhibitors. Whereas des-ETW resists antithrombin III inactivation with a rate constant (Kon) approximately 350-fold slower than for thrombin, des-ETW is remarkably sensitive to the Kunitz inhibitors, with inhibition constants (Ki) decreased from 2.6 microM to 34 nM for the soybean trypsin inhibitor and from 52 microM to 1.8 microM for the bovine pancreatic trypsin inhibitor. The affinity for hirudin (Ki = 5.6 pM) is weakened at least 30-fold compared with recombinant thrombin. The mutation affects the charge stabilizing system and the primary binding pocket of thrombin as depicted by a decrease in Kon for diisopropylfluorophosphate (9.5-fold) and for N alpha-p-tosyl-L-lysine-chloromethyl ketone (51-fold) and a 39-fold increase in the Ki for benzamidine. With peptidyl p-nitroanilide substrates, the des-ETW deletion results in changes in the Michaelis (Km) and/or catalytic (kcat) constants, worsened as much as 85-fold (Km) or 100-fold (kcat). The specific clotting activity of des-ETW is less than 5% that of thrombin and the kcat/Km for protein C activation in the absence of cofactor less than 2%. Thrombomodulin binds to des-ETW with a dissociation constant of approximately 2.5 nM and partially restores its ability to activate protein C since, in the presence of the cofactor, kcat/Km rises to 6.5% that of thrombin. This study suggests that the ETW motif of thrombin prevents (directly or indirectly) its interaction with the two Kunitz inhibitors and is not essential for the thrombomodulin-mediated enhancement of protein C activation.     

Specific cleavage of DNA at CG sites by Co(III) and Ni(II) desferal complexes.

Hydrolysis of endothelin 1 by rat kidney membranes was investigated using a reverse-phase HPLC and an automated gas-phase protein sequencer. Endothelin 1 was hydrolyzed into four major fragments which were detected by HPLC. Phosphoramidon, an inhibitor of neutral endopeptidase 24,11, almost completely suppressed the production of three fragments, but one fragment was not affected by the inhibitor. Analysis of N-terminal sequences of the degradation products revealed that the phosphoramidon-sensitive fragments were generated by cleavage at the Ser5-Leu6 bond of endothelin 1 that was identical with its cleavage site by purified rat endopeptidase 24,11, reported previously. The phosphoramidon-insensitive fragment was produced by cleavage at Leu17-Asp18, which was distinct from the sites by endopeptidase 24,11, but corresponded to that by a phosphoramidon-insensitive metallo-endopeptidase recently isolated from rat kidney membranes by us [(1992) Eur. J. Biochem. 204, 547-552]. Kinetic determination of endothelin 1 hydrolysis by the isolated enzyme yielded values of Km = 71.5 microM and kcat = 1.49 s-1, giving a ratio of kcat/Km = 2.08 x 10(4) s-1.M-1. The Km value was much higher and the kcat/Km value was much lower than those for rat endopeptidase 24,11 reported previously. Thus, endopeptidase 24,11 appears to hydrolyze endothelin 1 more efficiently than the isolated enzyme does. Both enzymes may play physiological roles in the metabolism of endothelin 1 by rat kidney membranes in vivo.     

Expression of selenocysteine-containing glutathione S-transferase in Escherichia coli

Evolution of a probable 'glutathione-binding ancestor' resulting in a common thioredoxin-fold for glutathione S-transferases and glutathione peroxidases may possibly suggest that a glutathione S-transferase could be engineered into a selenium-containing glutathione S-transferase (seleno-GST), having glutathione peroxidase (GPX) activity. Here, we addressed this question by production of such protein. In order to obtain a recombinant seleno-GST produced in Escherichia coli, we introduced a variant bacterial-type selenocysteine insertion sequence (SECIS) element which afforded substitution with selenocysteine for the catalytic Tyr residue in the active site of GST from Schistosoma japonica. Utilizing coexpression with the bacterial selA, selB, and selC genes (encoding selenocysteine synthase, SelB, and tRNA(Sec), respectively) the yield of recombinant seleno-GST was about 2.9 mg/L bacterial culture, concomitant with formation of approximately 85% truncation product as a result of termination of translation at the selenocysteine-encoding UGA codon. The mutations inferred as a result of the introduction of a SECIS element did not affect the glutathione-binding capacity (Km = 53 microM for glutathione as compared to 63 microM for the wild-type enzyme) nor the GST activity (kcat = 14.3 s(-1) vs. 16.6 s(-1)), provided that the catalytic Tyr residue was intact. When this residue was changed to selenocysteine, however, the resulting seleno-GST lost the GST activity. It also failed to display any novel GPX activity towards three standard peroxide substrates (hydrogen peroxide, butyl hydroperoxide or cumene hydroperoxide). These results show that recombinant selenoproteins with internal selenocysteine residues may be heterologously produced in E. coli at sufficient amounts for purification. We also conclude that introduction of a selenocysteine residue into the catalytic site of a glutathione S-transferase is not sufficient to induce GPX activity in spite of a maintained glutathione-binding capacity.     

Purification and characterization of a coagulant enzyme from Trimeresurus flavoviridis venom

An enzyme bearing thrombin-like specificity has been purified to homogeneity from the venom of Trimeresurus flavoviridis (the Habu snake). The enzyme is a monomer with a molecular weight of 23,500 as determined by analytical gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein contains approximately 210 amino acid residues and has a relatively high content of aspartic acid and glutamic acid. The isoelectric point was 4.8 and the extinction coefficient at 280 nm for a 1% solution was 11.5. The enzyme acted directly on fibrinogen to form a fibrin clot with 2.0 NIH units. Analysis by high performance liquid chromatography of enzyme-treated fibrinogen revealed the release of a peptide identical in composition to thrombin-induced fibrinopeptide A, but no peptide corresponding to fibrinopeptide B was detected. The enzyme showed esterase and amidase activities on synthetic substrates containing arginine. The enzyme exhibited higher activity toward tosyl-L-arginine methyl ester (TAME) but 6-times lower activity toward benzoyl-L-arginine p-nitroanilide when compared with bovin thrombin. The esterase activity was inhibited by diisopropylfluorophosphate and at a slower rate by phenylmethanesulfonyl fluoride, but was least affected by tosyl-L-lysine chloromethyl ketone, showing that the enzyme is a serine protease like thrombin. The enzyme showed a bell-shaped pH dependence of kcat/Km for hydrolysis of TAME, with a maximum around pH 8.5.     

Auto-inactivation by cleavage within the dimer interface of Kaposi's sarcoma-associated herpesvirus protease.

An autolysis site of functional and structural significance has been mapped within the dimer interface of Kaposi's sarcoma-associated herpesvirus protease. Cleavage 27 residues from the C terminus of the 230 amino acid residue, 25 kDa protein was observed to cause a loss of dimerization and proteolytic activity, even though no active site moieties were lost. Gel-filtration chromatography and analytical ultracentrifugation were used to analyze the changes in oligomerization upon autolysis. The selective auto-disruption of this essential protein-protein interface by proteolytic cleavage resulted in a 60 % loss in mean residue ellipticity by circular dichroism as well as a 20 % weaker, 10 nm red-shifted intrinsic protein fluorescence emission spectrum. These apparent conformational changes induced a strict inhibition of enzymatic activity. An engineered substitution at the P1' position of this cleavage site attenuated autolysis by the enzyme and restored wild-type dimerization. In addition to retaining full proteolytic activity in a continuous fluorescence-based enzyme assay, this protease variant allowed the determination of the enzyme's dimerization dissociation constant of 1.7 (+/-0.9) microM. The structural perturbations observed in this enzyme may play a role in viral maturation, and offer general insight into the allosteric relationship between the dimer interface and active site of herpesviral proteases. The functional coupling between oligomerization and activity presented here may allow for a better understanding of such phenomena, and the design of an enzyme variant stabilized to autolysis should further the structural and mechanistic characterization of this viral protease.