Increasing Enzyme Concentration Research Articles

Purification and characterization of a cold active alkaline protease from Stenotrophomonas sp., isolated from Kashmir, India

A Psychrotolerant alkaline protease producing bacterium IIIM-ST045 was isolated from a soil sample collected from the Thajiwas glacier of Kashmir, India and identified as Stenotrophomonas sp. on the basis of its biochemical properties and 16S ribosomal gene sequencing. The strain could grow well within a temperature range of 4-37°C however, showed optimum growth at 15°C. The strain was found to over-produce proteases when it was grown in media containing lactose as carbon source (157.50 U mg(-1)). The maximum specific enzyme activity (398 U mg(-1)) was obtained using soya oil as nitrogen source, however, the inorganic nitrogen sources urea, ammonium chloride and ammonium sulphate showed the lowest production of 38.9, 62.2 and 57.9 U mg(-1). The enzyme was purified to 18.45 folds and the molecular weight of the partially purified protease was estimated to be ~55 kDa by SDS-PAGE analysis. The protease activity increased as the increase in enzyme concentration while as the optimum enzyme activity was found when casein (1% w/v) was used as substrate. The enzyme was highly active over a wide range of pH from 6.5 to 12.0 showing optimum activity at pH 10.0. The optimum temperature for the enzyme was 15°C. Proteolytic activity reduced gradually with higher temperatures with a decrease of 56% at 40°C. The purified enzyme was checked for the removal of protein containing tea stains using a silk cloth within a temperature range of 10-60°C. The best washing efficiency results obtained at low temperatures indicate that the enzyme may be used for cold washing purposes of delicate fabrics that otherwise are vulnerable to high temperatures.

Production and Characterization of Serratiopeptidase Enzyme from Serratia Marcescens

Production characterization of serratiopeptidase (STP) enzyme by Serratia marcescens was the aim of this study. Serratia marcescens was allowed to grow in Tryptone Yeast Extract Glucose broth culture for the purpose of inducing STP (serrapeptase or serratiopeptidase) enzyme. The optimal conditions for STP production by Serratia marcescens were; 0.5 % substrate(Gelatin) concentration; 24 h incubation period; 32ºC incubation temperature and 6.0 pH ; the best buffer for production of STP enzyme was phosphate buffer. The best broth ingredient was tryptone; An optimum carbon sources was glucose; an optimum nitrogen source for STP enzyme production was tryptone; Valine was the best amino acids for the production of STP enzyme; the utilization of organic acids, acetic, citric, lactic acid decreased STP enzyme production at different concentrations above 3.0%. The STP enzyme was partially purified by ammonium sulfate precipitation and dialysis. The enzyme was found to have 52 KDa molecular weight by SDS – PAGE analysis. The STP enzyme activity increased as the increase in enzyme concentration. The data obtained emphasizes the possibility of production and purification of the microbial STP enzyme for application under industrial scale.

Supramolecular Structures of Enzyme Clusters.

The structural characterization of subtilisin mesoscale clusters, which were previously shown to induce supramolecular order in biocatalytic self-assembly of Fmoc–dipeptides, was carried out by synchrotron small-angle X-ray, dynamic, and static light scattering measurements. Subtilisin molecules self-assemble to form supramolecular structures in phosphate buffer solutions. Structural arrangement of subtilisin clusters at 55 °C was found to vary systematically with increasing enzyme concentration. Static light scattering measurements showed the cluster structure to be consistent with a fractal-like arrangement, with fractal dimension varying from 1.8 to 2.6 with increasing concentration for low to moderate enzyme concentrations. This was followed by a structural transition around the enzyme concentration of 0.5 mg mL–1 to more compact structures with significantly slower relaxation dynamics, as evidenced by dynamic light scattering measurements. These concentration-dependent supramolecular enzyme clusters provide tunable templates for biocatalytic self-assembly.

Properties of transglutaminase‐treated red bean protein films

AbstractThe effects of microbial transglutaminase (MTGase) treatment at various enzyme concentrations (4‐40 U g−1) on the properties of cast films from red bean protein isolate were investigated. The evaluated properties included mechanical properties (tensile strength, TS and elongation at break, EB), surface hydrophobicity (Hs), moisture content (MC), total soluble matter (TSM), water vapor transmission rate (WVTR) and permeability (WVP), as well as transparency. The results indicated that the TS and EB, and TSM were greatly improved by the MTGase treatment at low enzyme concentrations of 4‐10 U g−1, but considerably impaired at higher concentrations 20‐40 U g−1. The MTGase treatment also significantly increased the Hs, but decreased MC and WVTR, but the changes were independent of the applied enzyme concentration. The MTGase treatment progressively decreased the moisture loss during the drying of film‐forming solutions, upon the increase in enzyme concentration. The protein solubility analyses in various solvents indicated that the contribution of covalent linkages to film network formation progressively increased upon the increase in enzyme concentration. The results suggest that the modifications of the properties of cast films are closely associated with the extent of MTGase‐induced covalent polymerization and/or aggregation. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

O-GlcNac Modification of Human Cardiac α-Actinin

We measured O-GlcNAcylation of myofibrillar proteins from human hearts by two assays: (1) Western blotting with an antibody (CTD110.6) specific to O-GlcNAc modified proteins and (2) specific enzymatic labelling of O-GlcNAc with N-acetylgalactosamine (UDP-GalNAz) by mutant enzyme Y289L beta-1,4-galactosyltransferase (Y289L GalT), which allows coupling of tetramethylrhodamine (TAMRA) fluorescent tag for direct imaging following SDS-PAGE. In every sample, the predominant modified protein was alpha-actinin, which made up 65±4.48% of the enzymatically labelled myofibrillar proteins. We found that alpha-actinin exists as two bands on 12% SDS-PAGE. EA-53 antibody to alpha-actinin revealed that the faster migrating alpha-actinin band is on average <1.4±0.31% of the slower migrating band. Interestingly, CTD110.6 showed signals from both bands with similar intensities (mean ratio=1.02±0.10), indicating that O-GlcNAc modification is more abundant in the minor band (>80x more concentrated). This was confirmed by the enzymatic labelling method; O-GlcNAc was often not detectable in the slower migrating band, with an average ratio of faster/slower migrating band of 2.45±0.49. Further tests showed that the higher mobility band was not a degradation product of the lower mobility band. O-GlcNAc can be removed from the myofibril proteins by beta-N-acetylglucosaminidase. Using CTD110.6 detection, increasing enzyme concentration decreased the faster migrating band to near zero, but the slower migrating band did not show the same trend, suggesting that the antibody may cross react slightly with unmodified alpha-actinin. We conclude that O-GlcNAcylation is highly concentrated in the low abundance faster migrating species of alpha-actinin. The possibility that the two bands represent two isoforms of alpha-actinin differentially modified by the potentially regulatory O-GlcNAc modification is being investigated.

Biocatalytic induction of supramolecular order

Supramolecular gels, which demonstrate tunable functionalities, have attracted much interest in a range of areas, including healthcare, environmental protection and energy-related technologies. Preparing these materials in a reliable manner is challenging, with an increased level of kinetic defects observed at higher self-assembly rates. Here, by combining biocatalysis and molecular self-assembly, we have shown the ability to more quickly access higher-ordered structures. By simply increasing enzyme concentration, supramolecular order expressed at molecular, nano- and micro-levels is dramatically enhanced, and, importantly, the gelator concentrations remain identical. Amphiphile molecules were prepared by attaching an aromatic moiety to a dipeptide backbone capped with a methyl ester. Their self-assembly was induced by an enzyme that hydrolysed the ester. Different enzyme concentrations altered the catalytic activity and size of the enzyme clusters, affecting their mobility. This allowed structurally diverse materials that represent local minima in the free energy landscape to be accessed based on a single gelator structure.

Effect of the degree of enzymatic hydrolysis on the physicochemical properties and in vitro digestibility of rice starch

Rice starch was subjected to enzymatic treatment with pullulanase and the physicochemical properties of the modified rice starch were investigated as a function of the degree of hydrolysis which ranged from 0.9 to 10.2%. The enzymatic hydrolysis of rice starch caused a decrease in the fraction of rapidly digestible starch (RDS) (78.31→44.65%), whereas the levels of slowly digestible starch (SDS) (0.8→22.18%) and resistant starch (RS) (20.79→34.43%) increased, consequently reducing in vitro starch digestibility. In addition, the swelling factor of rice starch decreased with increasing enzyme concentration while an increase in its syneresis was observed. In rapid visco analyser (RVA) measurement, dramatic decreases in the peak viscosity (452.5→12.0 cp), breakdown (307.0→4.5 cp), and setback (207.0→3.0 cp) were observed in the enzyme-treated rice starch of which X-ray diffraction showed that there was the coexistence of A-and V-type crystallinity.

Maturation Mechanism of Severe Acute Respiratory Syndrome (SARS) Coronavirus 3C-like Proteinase

The 3C-like proteinase (3CLpro) of the severe acute respiratory syndrome (SARS) coronavirus plays a vital role in virus maturation and is proposed to be a key target for drug design against SARS. Various in vitro studies revealed that only the dimer of the matured 3CLpro is active. However, as the internally encoded 3CLpro gets matured from the replicase polyprotein by autolytic cleavage at both the N-terminal and the C-terminal flanking sites, it is unclear whether the polyprotein also needs to dimerize first for its autocleavage reaction. We constructed a large protein containing the cyan fluorescent protein (C), the N-terminal flanking substrate peptide of SARS 3CLpro (XX), SARS 3CLpro (3CLP), and the yellow fluorescent protein (Y) to study the autoprocessing of 3CLpro using fluorescence resonance energy transfer. In contrast to the matured 3CLpro, the polyprotein, as well as the one-step digested product, 3CLP-Y-His, were shown to be monomeric in gel filtration and analytic ultracentrifuge analysis. However, dimers can still be induced and detected when incubating these large proteins with a substrate analog compound in both chemical cross-linking experiments and analytic ultracentrifuge analysis. We also measured enzyme activity under different enzyme concentrations and found a clear tendency of substrate-induced dimer formation. Based on these discoveries, we conclude that substrate-induced dimerization is essential for the activity of SARS-3CLpro in the polyprotein, and a modified model for the 3CLpro maturation process was proposed. As many viral proteases undergo a similar maturation process, this model might be generally applicable.

Production and characterization of protease enzyme from Bacillus laterosporus

Production and partial purification of protease enzyme by Bacillus laterosporus was the aim of this study. Bacillus laterosporus was allowed to grow in shake flask broth culture for purpose of inducing protease enzyme. The protease enzyme was purified by ammonium sulfate precipitation followed by dialysis and further concentrated by Amicon tubes. After concentration, the protein was subjected to 12 % Zymogram gel with gelatin and the molecular weight of the protease enzyme was 15 kDa. The protease activity increased as the increase in enzyme concentration; optimum substrate concentration (starch) was 1.0% (w/v); an optimum incubation temperature was 40°C. Purified protease enzyme had a maximum activity at pH 7.0 of phosphate buffer and the optimum incubation time was 24 h. The protease isolated from Bacillus latrosporus is a mesophilic protease. It is stable at pH 7, at 40°C temperatures and this enzyme can be exploited commercially.

Batter rheology and bread texture of sorghum‐based gluten‐free formulations modified with native or pregelatinised cassava starch and α‐amylase

SummaryThe influence of α‐amylase (0–0.3 U g−1) on the crumb properties of gluten‐free sorghum batter and bread, respectively, was investigated. The formulations were modified using native or pregelatinised cassava starch (i.e. batter A – 17% pregelatinised starch, 83% sorghum, 100% water fwb; batter B – 17% native starch, 83% sorghum, 100% water fwb; and batter C – 30% native starch, 70% sorghum, 80% water fwb). The batters had solid viscoelastic character with the storage modulus predominant over the loss modulus. Storage moduli of batter A decreased with increasing angular frequency, whereas the moduli of batters B and C were independent from the angular frequency. Increasing enzyme concentration did not affect the loss factors of the batters. Batters’ resistance to deformation, from highest to lowest, followed the order C &gt; A &gt; B. Increasing enzyme concentration decreased crumb firmness, cohesiveness, springiness, resilience and chewiness but increased adhesiveness. Overall, breads containing native starch had better crumb properties (i.e. springier and less firm, chewy and adhesive) than breads containing pregelatinised starch.

Mutual Inhibition of RecQ Molecules in DNA Unwinding

Helicases make conformational changes and mechanical movements through hydrolysis of NTP to unwind duplex DNA (or RNA). Most helicases require a single-stranded overhang for loading onto the duplex DNA substrates. Some helicases have been observed to exhibit an enhanced unwinding efficiency with increasing length of the single-stranded DNA tail both by preventing reannealing of the unwound DNA and by compensating for premature dissociation of the leading monomers. Here we report a previously unknown mutual inhibition of neighboring monomers in DNA unwinding by the monomeric Escherichia coli RecQ helicase. With single molecule fluorescence resonance energy transfer microscopy, we observed that the unwinding initiation of RecQ at saturating concentrations was more delayed for a long rather than a short tailed DNA. In stopped-flow kinetic studies under both single and multiple turnover conditions, the unwinding efficiency decreased with increasing enzyme concentration for long tailed substrates. In addition, preincubation of RecQ and DNA in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate was observed to alleviate the inhibition. We propose that the mutual inhibition effect results from a forced closure of cleft between the two RecA-like domains of a leading monomer by a trailing one, hence the forward movements of both monomers are stalled by prohibition of ATP binding to the leading one. This effect represents direct evidence for the relative movements of the two RecA-like domains of RecQ in DNA unwinding. It may occur for all superfamily I and II helicases possessing two RecA-like domains.

Regulation of De Novo -Initiated RNA Synthesis in Hepatitis C Virus RNA-Dependent RNA Polymerase by Intermolecular Interactions

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has been proposed to change conformations in association with RNA synthesis and to interact with cellular proteins. In vitro, the RdRp can initiate de novo from the ends of single-stranded RNA or extend a primed RNA template. The interactions between the Delta1 loop and thumb domain in NS5B are required for de novo initiation, although it is unclear whether these interactions are within an NS5B monomer or are part of a higher-order NS5B oligomeric complex. This work seeks to address how polymerase conformation and/or oligomerization affects de novo initiation. We have shown that an increasing enzyme concentration increases de novo initiation by the genotype 1b and 2a RdRps while primer extension reactions are not affected or inhibited under similar conditions. Initiation-defective mutants of the HCV polymerase can increase de novo initiation by the wild-type (WT) polymerase. GTP was also found to stimulate de novo initiation. Our results support a model in which the de novo initiation-competent conformation of the RdRp is stimulated by oligomeric contacts between individual subunits. Using electron microscopy and single-molecule reconstruction, we attempted to visualize the low-resolution conformations of a dimer of a de novo initiation-competent HCV RdRp.

Continuous production of polygalacturonases (PGases) using Aspergillus niger in a surface culture bioreactor and modeling the process

The feasibility of continuous production of PGases by a strain of Aspergillus niger was investigated in a surface culture bioreactor. Pectin was used as the substrate. The fermentation started in batch mode until the medium was covered with the mycelia of the microorganism and after that it turned to continuous mode by introducing the fresh feed. The process continued for 34 days. The thickness of the microorganism layer on the surface of the liquid medium became almost constant after about 1 week. The production of PGases, however, continued throughout the experiment. Maximum exo-polygalacturonase (exo-PGase) and endo-polygalacturonase (endo-PGase) activities of 1.5 and 0.014 U/mL were obtained, respectively. An increase in enzyme concentrations was observed when a similar system was used with a line for medium recycle. A Michaelis-Menten type equation was considered for the rate of PGases production in the surface culture bioreactor and the process was modeled. The model parameters were estimated using an optimization method (Particle Swarm Optimization). The model predicted the experimental data reasonably well.

Activity of NADH oxidase from Thermus thermophilus in water/alcohol binary mixtures is limited by the stability of quaternary structure

NADH oxidase from Thermus thermophilus (NOX) is a homodimeric flavoenzyme; it belongs to the nitroreductase and flavin reductase families. In this work we investigate the effect of water miscible alcohols (methanol, ethanol, propanol and isopropanol) on activity and quaternary structure of this thermophilic enzyme. The enzyme activity of NOX was studied in a broad range (0–60%) of alcohol concentrations. In all studied alcohols, NOX displays optimum activity at low alcohol concentration (<20%). Under these conditions, the enzyme catalytic efficiency ( k cat,app/ K M,app) significantly increases mainly due to ∼10-fold increase of the catalytic constant. At high alcohol concentrations (>30%), NOX activity is time dependent and slowly diminished with an apparent time constant of several hours. We found that prolonged incubation in the presence of alcohols results in: (i) dissociation of the homodimeric native structure of the enzyme and (ii) release of catalytically essential flavin cofactor from the intermonomer interface. The inactivation is fully reversible on increasing enzyme concentration and NOX activity is completely recovered even in the presence of high alcohol concentrations. Moreover, exogenous addition of the flavin cofactor dramatically stabilizes dimeric form of the enzyme and results in shift of apparent K d to the lower values. Theoretical simulations showed that flavin dissociation and monomer/dimer equilibria are strongly coupled.

Effect of Cellulase Enzyme on Cellulose Nano-topography

Abstract In this study, modification effects of cellulose foil by cellulase enzyme are studied in respect to topography changes by means of atomic force microscopy (AFM). The results provide useful information to understand the enzyme action in the amorphous and crystalline regions of cellulose. It was revealed, that the treatment of cellulose with the cellulase enzyme depends on its concentration, which selectively attacks amorphous cellulose regions. At lower concentration, a random surface roughening of the cellulose foil was observed. With increasing enzyme concentration, the cellulose surface became smoother. On the basis of this knowledge, a conceptual model to describe the conditioning effect by cellulase on the cleanability of cotton fabrics was developed. With the help of this model, we are able to estimate changes in the nanoporosity of cotton fibres.

Setaria cervidual specific phosphatase: characterization and its effect on eosinophil degranulation

Setaria cervi, a bovine filarial parasite contains significant acid phosphatase (AcP) activity in its various life stages. Two forms of AcP were separated from somatic extract of adult female parasite using cation exchange, gel filtration and concavalin affinity chromatography. One form having a molecular mass of 79 kDa was characterized as dual specific protein tyrosine phosphatase (ScDSP) based on substrate specificity and inhibition studies. With various substrates tested, it showed significant activity in the order of phospho-L-tyrosine>pNPP>ADP>phospho-L-serine. Inhibition by orthovanadate, fluoride, molybdate, and zinc ions further confirms protein tyrosine phosphatase nature of the enzyme. Km and Vmax determined with various substrates were found to be 16.66 mM, 25.0 microM/ml/min with pNPP; 20.0 mM, 40.0 microM/ml/min with phospho-L-tyrosine and 27.0 mM, 25.0 microM/ml/min with phospho-L-serine. KI with pNPP and sodium orthovanadate (IC50 33.0 microM) was calculated to be 50.0 mM. Inhibition with pHMB, silver nitrate, DEPC and EDAC suggested the presence of cysteine, histidine and carboxylate residues at its active site. Cross-reactivity with W. bancrofti-infected sera was demonstrated by Western blotting. ScDSP showed elevated levels of IgE in chronic filarial sera using ELISA. Under in vitro conditions, ScDSP resulted in increased effector function of human eosinophils when stimulated by IgG, which showed a further decrease with increasing enzyme concentration. Results presented here suggest that S. cervi DSP should be further studied to determine its role in pathogenesis and the persistence of filarial parasite.

Efficient Elimination of Nonstoichiometric Enzyme Inhibitors from HTS Hit Lists

High-throughput screening often identifies not only specific, stoichiometrically binding inhibitors but also undesired compounds that unspecifically interfere with the targeted activity by nonstoichiometrically binding, unfolding, and/or inactivating proteins. In this study, the effect of such unwanted inhibitors on several different enzyme targets was assessed based on screening results for over a million compounds. In particular, the shift in potency on variation of enzyme concentration was used as a means to identify nonstoichiometric inhibitors among the screening hits. These potency shifts depended on both compound structure and target enzyme. The approach was confirmed by statistical analysis of thousands of dose-response curves, which showed that the potency of competitive and therefore clearly stoichiometric inhibitors was not affected by increasing enzyme concentration. Light-scattering measurements of thermal protein unfolding further verified that compounds that stabilize protein structure by stoichiometric binding show the same potency irrespective of enzyme concentration. In summary, measuring inhibitor IC(50) values at different enzyme concentrations is a simple, cost-effective, and reliable method to identify and eliminate compounds that inhibit a specific target enzyme via nonstoichiometric mechanisms.

Effect of an enzyme preparation on wheat flour and dough color, mixing, and test baking

Abstract Bleaching flours with natural rather than chemical extracts is attractive because it reduces risks upon exposure and appeals to natural food consumers. This paper reports effects of a commercial proprietary blend of ‘natural’ bleaching enzymes on wheat flour and dough color, mixing behavior and test bake performance. Enzyme preparation did not improve whiteness (L*) and yellowness (b*) of flour system, but benzoyl peroxide sharply reduced b*. For whole wheat flour dough, L* increased for enzyme-treated dough after 2-h resting, ending higher than benzoyl peroxide and control dough. Yellowness increased in enzyme-treated dough. When enzyme application was during milling, resulting whole wheat flour dough had much higher L*. Mixograms of flours with increasing enzyme concentration showed severe dough weakness and rapid breakdown. Dialysis of the enzyme preparation in 3500 molecular weight cutoff (MWCO) tubes and freeze-drying prior to flour application recovered dough strength. l -cysteine in the enzyme mix was thought to adversely affect dough strength, and dialysis helped recover dough strength. However, pup loaf baking with dialyzed enzyme showed some loss of baking characteristics (loaf volume, loaf weight, and proof height) over the control. Although the natural enzyme extract addition enhanced whiteness for whole wheat dough, its effects on dough and baking properties were not favorable.

Mechanism of low-molecular-weight heparin reversal by platelet factor 4

Low-molecular-weight heparins (LMWH) exert their anticoagulant effect by accelerating anti-thrombin (AT) inactivation of factor Xa (fXa) and thrombin. To address the hypothesis of a calcium-dependent template mechanism in LMWH activity, we compared the ability of the heparin neutralising agent Platelet Factor 4 (PF4) to inhibit various therapeutic LMWH in a kinetic assay. Neutralization coefficients by PF4 and apparent affinities of PF4 for various LMWH increased in a molecular weight-dependent manner. Protamine sulphate neutralized heparin via a non-specific mechanism. EDTA abolished the calcium-dependent acceleration of the fXa-AT reaction, indicating that the bridging mechanism contributed significantly to LMWH activity. Within a low range of LMWH concentration (< 0.2 U/ml), excess AT over PF4 (4:1) had no effect on PF4 activity, indicating that PF4 and AT binding to heparin were independent of each other. Instead, increasing enzyme concentration reversed the negative effect of heparin- bound AT on PF4-dependent neutralization. Inhibition of thrombin by heparin was also neutralized by PF4, albeit to a higher extent than the fXa-AT reaction. Altogether, these results suggested that an interaction of PF4 with protease mediated the association of PF4 to the heparin chain. We propose that PF4 participates in the anti-fXa dependence of LMWH due to its major effect on the anti-thrombin activity of LMWH and that inhibition of fXa via the template mechanism plays an essential role in LMWH activity and pharmacokinetics, because PF4 specifically targets this mechanism.

NS3 Helicase from the Hepatitis C Virus Can Function as a Monomer or Oligomer Depending on Enzyme and Substrate Concentrations

Hepatitis C virus NS3 helicase can unwind double-stranded DNA and RNA and has been proposed to form oligomeric structures. Here we examine the DNA unwinding activity of monomeric NS3. Oligomerization was measured by preparing a fluorescently labeled form of NS3, which was titrated with unlabeled NS3, resulting in a hyperbolic increase in fluorescence anisotropy and providing an apparent equilibrium dissociation constant of 236 nm. To evaluate the DNA binding activity of individual subunits within NS3 oligomers, two oligonucleotides were labeled with fluorescent donor or acceptor molecules and then titrated with NS3. Upon the addition of increasing concentrations of NS3, fluorescence energy transfer was observed, which reached a plateau at a 1:1 ratio of NS3 to oligonucleotides, indicating that each subunit within the oligomeric form of NS3 binds to DNA. DNA unwinding was measured under multiple turnover conditions with increasing concentrations of NS3; however, no increase in specific activity was observed, even at enzyme concentrations greater than the apparent dissociation constant for oligomerization. An ATPase-deficient form of NS3, NS3(D290A), was prepared to explore the functional consequences of oligomerization. Under single turnover conditions in the presence of excess concentration of NS3 relative to DNA, NS3(D290A) exhibited a dominant negative effect. However, under multiple turnover conditions in which DNA concentration was in excess to enzyme concentration, NS3(D290A) did not exhibit a dominant negative effect. Taken together, these data support a model in which monomeric forms of NS3 are active. Oligomerization of NS3 occurs, but subunits can function independently or cooperatively, dependent upon the relative concentration of the DNA.