Alkaline protease inhibitor: a novel class of antifungal proteins against phytopathogenic fungi.

A Streptomyces sp., which produces an alkaline protease inhibitor (API) exhibiting antifungal activity has been isolated from soil. The protein has been purified to homogeneity. The molecular characterization has revealed that it is a dimer (M(r) 28 kDa) with five disulphide linkages and has a pI of 3.8. API is a competitive type of inhibitor with a K(i) value of 2.5 x 10(-9) M. The inhibitor is stable over a pH range of 6 to 12 and a temperature range of 40 to 95 degrees C. API exhibits antifungal activity (in vitro) against phytopathogenic fungi such as Fusarium, Alternaria, and Rhizoctonia and also against Trichoderma, a saprophytic fungus. The antifungal activity of API appears to be associated with its ability to inhibit the fungal serine alkaline protease(s), which is indispensable for its growth. Retardation of the rate of fungal spore germination, as well as hyphal extention, was observed in the presence of API. Both the protease inhibitory and the antifungal activity were abolished on treatment of API with DTT (5 mM), suggestive of a common site for both the activities. This is the first report on API as a potential biocontrol agent against phytopathogenic fungi.     

Slow-tight binding inhibition of pepsin by an aspartic protease inhibitor from Streptomyces sp. MBR04

The present article reports a low molecular weight aspartic protease inhibitor from a Streptomyces sp. MBR04 exhibiting a two-step inhibition mechanism against pepsin. The kinetic interactions revealed a reversible, competitive, slow-tight binding inhibition with an IC(50) and K(i) values of 4.5 nM and 4 nM respectively. The conformational changes induced upon inhibitor binding to pepsin was monitored by far and near UV analysis, demonstrated that the inhibitor binds to the active site and causes inactivation. Chemical modification of the inhibitor with WRK and TNBS abolished the antiproteolytic activity of the inhibitor.     

Evidence for tryptophan in proximity to histidine and cysteine as essential to the active site of an alkaline protease

The presence, microenvironment, and proximity of an essential Trp with the essential His and Cys residues in the active site of an alkaline protease have been demonstrated for the first time using chemical modification, chemo-affinity labeling, and fluorescence spectroscopy. Kinetic analysis of the N-bromosuccinimide- (NBS) or p-hydroxymercuribenzoate- (PHMB) modified enzyme from Conidiobolus sp. revealed that a single Trp and Cys are essential for activity in addition to the Asp, His, and Ser residues of the catalytic triad. Full protection by casein against inactivation of the enzyme by NBS and quenching of Trp fluorescence upon binding of the enzyme with NBS, substrate (sAAPF-pNA), or inhibitor (SSI) confirmed participation of the Trp residue at the substrate/inhibitor binding site of the alkaline protease. Comparison of the K(sv) values for the charged quenchers CsCI (1.66) and KI (7.0) suggested that the overall Trp microenvironment in the protease is electropositive. The proximity of Trp with His was demonstrated by the sigmoidal shape of the pH-dependent fluorometric titration curve with a pK(F) of 6.1. The vicinity of Trp with Cys was indicated by resonance energy transfer between the intrinsic fluorophore (Trp) and 5-iodoacetamide-fluorescein labeled Cys (extrinsic fluorophore). Our results on the proximity of Trp with essential His and Cys thus confirm the presence of Trp in the active site of the alkaline protease.     

Chemical modification studies of Rhizomucor miehei protease: evidence for the role of basic amino acids in enzyme catalysis.

The effect of chemical modification on milk clotting and proteolytic activities of aspartyl protease obtained from Rhizomucor miehei NRRL 3500 was examined in the absence and the presence of its specific inhibitor pepstatin A. The effect on the ratio of milk clotting activity (MC) to proteolytic activity (PA), an index of the quality of milk clotting proteases was also determined. Modification of the enzyme with trinitrobenzenesulfonic acid, diethylpyrocarbonate and phenylglyoxal produced an increase in the ratio of MC/PA, while modification with 2- hydroxy-5-nitrobenzyl bromide did not affect the ratio. Modification with N-acetylimidazole resulted in a marginal increase in MC/PA ratio. Protection using pepstatin A during modification with phenylglyoxal, N-acetylimidazole and 2-hydroxy-5-nitrobenzyl bromide, protected both MC and PA. In the case of modification by diethylpyrocarbonate, pepstatin A protected only MC. Pepstatin A did not protect both the activities on the modification of the enzyme by trinitrobenzene sulfonic acid. These observations indicate the presence of arginine, tyrosine and tryptophan at the catalytic site of the enzyme, for eliciting MC and PA of the enzyme. In general, modification of the positively charged residues increases the MC/PA ratio of the enzyme. In addition the modified lysine residues responsible for the inactivation of the enzyme were not involved in the active site of the enzyme. Thus the lysine residues might have a secondary role in enzyme catalysis. Further, histidine at the catalytic site was found to be exclusively involved in milk clotting activity. The enzyme with modified histidine residues were more susceptible to autocatalysis, indicating that histidine residues protect the enzyme against autolysis.     

Relationship between protease activity and a sialoglycopeptide inhibitor isolated from bovine brain

We have recently described the isolation and purification to homogeneity of a new sialoglycopeptide from bovine brain cell surfaces that reversibly inhibits protein synthesis and DNA synthesis of normal but not transformed cells. Active inhibitory preparations, however, were shown to contain a protease activity that was not lost upon purification. Several experiments were performed to establish the relationship between the proteolytic activity of the sialoglycopeptide and the biological inhibitory activity. Both the protease activity and inhibitory activity were stable at pH 6-8 but were reduced or completely destroyed below pH 4 and above pH 9. Acid inactivation was reversible and upon dialysis, both the biological inhibitory and protease activities were regained. Deglycosylation and CNBr cleavage indicated that the polypeptide backbone, rather than carbohydrate moiety, played an important role in the protease and biological inhibitory activities. Furthermore, chemical modification of amino and tyrosine groups indicated that both residues are essential for both activities. Thus, the biological inhibitory activity and protease activity are very closely related and most likely reside with the same polypeptide sequence.     

Chemical modification of 3-HBA-6-hydroxylase by phenylglyoxal: kinetic and physicochemical studies on the modified enzyme.

The inactivation of 3-HBA-6-hydroxylase isolated from Micrococcus species by phenylglyoxal and protection offered by 3-HBA against inactivation indicate the presence of arginine residue at or near the substrate binding site. The loss of enzyme activity was time and concentration dependent and displayed pseudo-first order kinetics. A 'n' value of 0.9 was obtained thus suggesting the modification of a single arginine residue per active site which led to the loss of enzyme activity. The enzyme activity could be restored by extensive dialysis at neutral pH. Quenching of the intrinsic fluorescence and reduction in the ellipticity value at 280 nm in the near-UV CD spectrum of the enzyme was noticed after its treatment with phenylglyoxal. These observations probably imply distinct perturbations in the environment of adjacent aromatic amino acid residues such as tryptophan as a consequence of arginine modification.     

Participation of S-S loops in inhibitory activity of peanut protease inhibitor B-III

A peanut Bowman-Birk (BBI) type protease inhibitors B-III has two regions, 1 and 2, homologous with each other. Each region contains three S-S loops and a reactive site in its outermost loop. The inhibitor was used to investigate the contribution of the S-S loops of BBI-type inhibitors to their inhibitory activity. Two steps of Edman degradation of the native inhibitor cleaved loop III (the innermost S-S loop) of region 1 of B-III, and the antichymotryptic activity of the first reactive site decreased to about 1/4 of that of native B-III. A third step of Edman degradation split loop II and the inhibitory activity at that site became extremely low (about 1/200 of the original value). These results suggest that protease inhibitor B-III maintains its active conformation by means of the three S-S loops and that the conformation is markedly changed by the splitting of loop II.     

Purification and characterization of two leaf polypeptide inhibitors of leaf protease from alfalfa (Medicago sativa)

Two polypeptides with antiproteolytic activities have been isolated from alfalfa leaves. Polypeptide I resembles the previously described plant protease inhibitors in both structural and functional features; it has a molecular weight of 15,000, a random coil secondary structure, and inhibits exogenous protease as well as alfalfa leaf protease. Polypeptide II is a novel type of plant inhibitor with a molecular weight of 6300 and a highly organized structure with a high (40-50%) alpha-helix content. It only inhibits endogenous protease with a molar stoichiometry polypeptide/enzyme protein of 1.     

Hepatocyte growth factor activator inhibitor type 1 inhibits protease activity and proteolytic activation of human airway trypsin-like protease

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type transmembrane serine protease inhibitor initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA), a serine protease that converts pro-HGF to the active form. HAI-1 also has inhibitory activity against serine proteases such as matriptase, hepsin and prostasin. In this study, we examined effects of HAI-1 on the protease activity and proteolytic activation of human airway trypsin-like protease (HAT), a transmembrane serine protease that is expressed mainly in bronchial epithelial cells. A soluble form of HAI-1 inhibited the protease activity of HAT in vitro. HAT was proteolytically activated in cultured mammalian cells transfected with its expression vector, and a soluble form of active HAT was released into the conditioned medium. The proteolytic activation of HAT required its own serine protease activity. Co-expression of the transmembrane full-length HAI-1 inhibited the proteolytic activation of HAT. In addition, full-length HAI-1 associated with the transmembrane full-length HAT in co-expressing cells. Like other target proteases of HAI-1, HAT converted pro-HGF to the active form in vitro. These results suggest that HAI-1 functions as a physiological regulator of HAT by inhibiting its protease activity and proteolytic activation in airway epithelium.     

A continuous assay for foot-and-mouth disease virus 3C protease activity

Foot-and-mouth disease virus is a highly contagious pathogen that spreads rapidly among livestock and is capable of causing widespread agricultural and economic devastation. The virus genome is translated to produce a single polypeptide chain that subsequently is cleaved by viral proteases into mature protein products, with one protease, 3C(pro), carrying out the majority of the cleavages. The highly conserved nature of this protease across different viral strains and its crucial role in viral maturation and replication make it a very desirable target for inhibitor design. However, the lack of a convenient and high-throughput assay has been a hindrance in the characterization of potential inhibitors. In this article, we report the development of a continuous assay with potential for high throughput using fluorescence resonance energy transfer-based peptide substrates. Several peptide substrates containing the 3C-specific cleavage site were synthesized, varying both the positions and separation of the fluorescent donor and quencher groups. The best substrate, with a specificity constant k(cat)/K(M) of 57.6+/-2.0M(-1) s(-1), was used in inhibition assays to further characterize the protease's activity against a range of commercially available inhibitors. The inhibition profile of the enzyme showed characteristics of both cysteine and serine proteases, with the chymotrypsin inhibitor TPCK giving stoichiometric inhibition of the enzyme and allowing active site titration of the 3C(pro).     

Sensitive genetic screen for protease activity based on a cyclic AMP signaling cascade in Escherichia coli

We describe a genetic system that allows in vivo screening or selection of site-specific proteases and of their cognate-specific inhibitors in Escherichia coli. This genetic test is based on the specific proteolysis of a signaling enzyme, the adenylate cyclase (AC) of Bordetella pertussis. As a model system we used the human immunodeficiency virus (HIV) protease. When an HIV protease processing site, p5, was inserted in frame into the AC polypeptide, the resulting ACp5 protein retained enzymatic activity and, when expressed in an E. coli cya strain, restored the Cya(+) phenotype. The HIV protease coexpressed in the same cells resulted in cleavage and inactivation of ACp5; the cells became Cya(-). When the entire HIV protease, including its adjacent processing sites, was inserted into the AC polypeptide, the resulting AC-HIV-Pr fusion protein, expressed in E. coli cya, was autoproteolysed and inactivated: the cells displayed Cya(-) phenotype. In the presence of the protease inhibitor indinavir or saquinavir, AC-HIV-Pr autoproteolysis was inhibited and the AC activity of the fusion protein was preserved; the cells were Cya(+). Protease variants resistant to particular inhibitors could be easily distinguished from the wild type, as the cells displayed a Cya(-) phenotype in the presence of these inhibitors. This genetic test could represent a powerful approach to screen for new proteolytic activities and for novel protease inhibitors. It could also be used to detect in patients undergoing highly active antiretroviral therapy the emergence of HIV variants harboring antiprotease-resistant proteases.     

Antimicrobial activity studies on a trypsin-chymotrypsin protease inhibitor obtained from potato

A 5.6 kDa trypsin-chymotrypsin protease inhibitor was isolated from the tubers of the potato (Solanum tuberosum L cv. Gogu) by extraction of the water-soluble fraction, dialysis, ultrafiltration, and C18 reversed-phase high performance liquid chromatography. This inhibitor, which we named potamin-1 (PT-1), was thermostable and possessed antimicrobial activity but lacked hemolytic activity. PT-1 strongly inhibited pathogenic microbial strains, including Candida albicans, Rhizoctonia solani, and Clavibacter michiganense subsp. michiganinse. Automated Edman degradation showed that the N-terminal sequence of PT-1 was NH2-DICTCCAGTKGCNTTSANGAFICEGQSDPKKPKACPLNCDPHIAYA-. The sequence had 62% homology with a serine protease inhibitor belonging to the Kunitz family, and the peptide inhibited chymotrypsin, trypsin, and papain. This protease inhibitor, PT-1, was composed of polypeptide chains joined by disulfide bridge(s). Reduced PT-1 almost completely lost its activity against fungi and proteases indicating that disulfide bridge is essential for its protease inhibitory and antifungal activity. These results suggest that PT-1 is an excellent candidate as a lead compound for the development of novel oral or other anti-infective agents.     

Nonenzymatic anticoagulant activity of the mutant serine protease Ser360Ala-activated protein C mediated by factor Va.

The human plasma serine protease, activated protein C (APC), primarily exerts its anticoagulant function by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. A recombinant active site Ser 360 to Ala mutation of protein C was prepared, and the mutant protein was expressed in human 293 kidney cells and purified. The activation peptide of the mutant protein C zymogen was cleaved by a snake venom activator, Protac C, but the "activated" S360A APC did not have amidolytic activity. However, it did exhibit significant anticoagulant activity both in clotting assays and in a purified protein assay system that measured prothrombinase activity. The S360A APC was compared to plasma-derived and wild-type recombinant APC. The anticoagulant activity of the mutant, but not native APC, was resistant to diisopropyl fluorophosphate, whereas all APCs were inhibited by monoclonal antibodies against APC. In contrast to native APC, S360A APC was not inactivated by serine protease inhibitors in plasma and did not bind to the highly reactive mutant protease inhibitor M358R alpha 1 antitrypsin. Since plasma serpins provide the major mechanism for inactivating APC in vivo, this suggests that S360A APC would have a long half-life in vivo, with potential therapeutic advantages. S360A APC rapidly inhibited factor Va in a nonenzymatic manner since it apparently did not proteolyze factor Va. These data suggest that native APC may exhibit rapid nonenzymatic anticoagulant activity followed by enzymatic irreversible proteolysis of factor Va. The results of clotting assays and prothrombinase assays showed that S360A APC could not inhibit the variant Gln 506-FVa compared with normal Arg 506-FVa, suggesting that the active site of S360A APC binds to FVa at or near Arg 506.     

One functional switch mediates reversible and irreversible inactivation of a herpesvirus protease

Distinct mechanisms have evolved to regulate the function of proteolytic enzymes. Viral proteases in particular have developed novel regulatory mechanisms, presumably due to their comparatively rapid life cycles and responses to constant evolutionary pressure. Herpesviruses are a family of human pathogens that require a viral protease with a concentration-dependent zymogen activation involving folding of two alpha-helices and activation of the catalytic machinery, which results in formation of infectious virions. Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) is unique among the herpesvirus proteases in possessing an autolysis site in the dimer interface, which removes the carboxyl-terminal 27 amino acids comprising an alpha-helix adjacent to the active site. Truncation results in the irreversible loss of dimerization and concomitant inactivation. We characterized the conformational and functional differences between the active dimer, inactive monomer, and inactive truncated protease to determine the different protease regulatory mechanisms that control the KSHV lytic cycle. Circular dichroism revealed a loss of 31% alpha-helicity upon dimer dissociation. Comparison of the full-length and truncated monomers by NMR showed differences in 21% of the protein structure, mainly located adjacent to the dimer interface, with little perturbation of the overall protein upon truncation. Fluorescence polarization and active site labeling, with a transition state mimetic, characterized the functional effects of these conformational changes. Substrate turnover is abolished in both the full-length and truncated monomers; however, substrate binding remained intact. Disruption of the helix 6 interaction with the active site oxyanion loop is therefore used in two independent regulatory mechanisms of proteolytic activity.     

Chemical modification of chalcone isomerase by diethyl pyrocarbonate: histidine residues are not essential for catalysis

Chalcone isomerase form soybean is inactivated by treatment with diethyl pyrocarbonate (DEP). The competitive inhibitor 4',4-dihydroxychalcone provides kinetic protection against inactivation by DEP with a binding constant at the site of protection in agreement with its binding constant at the active site. Very high concentrations of the competitive inhibitors 4',4-dihydroxychalcone or morin hydrate offer a 10- to 40-fold maximal protection, suggesting a second slower mechanism for inactivation which cannot be prevented by blockage of the active site. Blockage of the only cysteine residue in chalcone isomerase with p-mercuribenzoate does not affect the rate constant for DEP-dependent inactivation and indicates that the modification of the cysteine residue is not responsible for the activity loss observed in the presence of DEP. Treatment of inactivated enzyme with hydroxylamine does not restore catalytic activity, indicating that the modification of histidine or tyrosine residues is not responsible for the activity loss. All five histidines of chalcone isomerase are modified by DEP at pH 5.7 and ionic strength 1.0 M. The rate constant for the modification of the histidine residues of chalcone isomerase is close to that for the reaction of N-acetyl histidine with DEP, indicating that the histidine residues are quite accessible to the modifying reagent. The rate of histidine modification is the same in native enzyme, in urea-denatured enzyme, and in the presence of a competitive inhibitor. In the presence of the competitive inhibitor morin hydrate, all of the histidine residues of chalcone isomerase can be modified without significant loss in catalytic activity. These results demonstrate that the histidine residues of chalcone isomerase are not essential for catalysis and therefore cannot function as nucleophilic catalysts as previously proposed.     

Purification and characterization of a salinity induced alkaline protease from isolated spinach chloroplasts

In cynobacteria and higher plants, salinity is known to inhibit the activity of several enzymes involved in photosynthesis and hence decreases the overall photosynthetic rate. This gave us an impetus to search for a protease, which may be involved in the turnover of non-functional enzymes produced under salinity stress. Taking the possible changes in pH gradient of the chloroplast under consideration, we have tried to identify a protease, which is induced under salinity and characterized it as an alkaline protease using spinach (Spinacia oleracea) leaves as a model system. The HIC-HPLC purified homogeneous alkaline serine protease from the isolated spinach chloroplasts had two subunits of molecular weight 63 and 32 kDa. The enzyme was maximally active at pH 8.5 and 50°C. The enzyme showed the property to hydrolyze the synthetic substrate like azocaesin and had sufficient proteolytic activity in gelatin bound native PAGE. The enzyme activity was also dependent upon the presence of divalent cations and reduced environment. The active site residues were identified and the homogeneous alkaline serine protease had cysteine, lysine and tryptophan residues at its active site.     

Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates

Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus–GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus–GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader.     

Purification and characterization of a milk clotting protease from Rhizomucor miehei

Benzamidine, an inhibitor of serine proteases, was used as an affinity ligand for the purification of aspartyl protease from culture filtrate of Rhizomucor miehei. The two step purification protocol (ion-exchange and affinity chromatography) resulted in a homogenous enzyme preparation with seven-fold purification and a final recovery of 22%. The purified enzyme was free of brown pigmentation, a factor inherently associated with the enzyme; it was stable and active at acidic pH (optimum pH 4.1 for proteolytic activity and 5.6 for milk clotting activity). The significant positive characteristic of the enzyme is its comparatively lower thermostability; the enzyme was comparable to calf rennet in its properties of thermostability, milk-clotting to proteolytic activity ratio and sensitivity to CaCl2. Limited protease digestion of the purified enzyme with proteinase K yielded a 20kDa fragment as shown by SDS–PAGE. Native gel electrophoresis of the digest showed an additional peak of activity corresponding to the 20kDa fragment on SDS–PAGE, this fragment retained both milk-clotting and proteolytic activities. It was also inhibited by pepstatin A and hence it is presumed that this fragment contained the active site of the enzyme.