Papaya Proteinase IV Research Articles

Phytochemistry and nutraceutical properties of Carica papaya (Linn.): A review

Nutraceuticals derived from a medicinal plant having therapeutic significance are a gift to mankind to acquire healthy life. Carica papaya Linn. (aricaceae) commonly known as papaya. It is a significant fruit tree and is found in tropical and subtropical parts of the world. The extract from various parts of plant especially fruit and leaves contains many phytonutrients viz; vitamin A, B1, and vitamin C, calcium hydrate charcoal, phosphorus, iron, protein as well as some endopeptidases like namely papain, glycyl endopeptidase, chymopapain, and caricain. The extract of this plant is identified to be efficacious against diversified ailments like malaria, inflammation, digestive disorder, Microbes, Fungi, and many infectious disorders. Its prophylactic and therapeutic values enhance immunity and ensure a healthier life. Each part of this plant has its nutraceutical properties.Keywords: Nutraceuticals, Carica papaya, Papain, Caricain, Chymopapain

Papain-like cysteine proteases in Carica papaya: lineage-specific gene duplication and expansion

BackgroundPapain-like cysteine proteases (PLCPs), a large group of cysteine proteases structurally related to papain, play important roles in plant development, senescence, and defense responses. Papain, the first cysteine protease whose structure was determined by X-ray crystallography, plays a crucial role in protecting papaya from herbivorous insects. Except the four major PLCPs purified and characterized in papaya latex, the rest of the PLCPs in papaya genome are largely unknown.ResultsWe identified 33 PLCP genes in papaya genome. Phylogenetic analysis clearly separated plant PLCP genes into nine subfamilies. PLCP genes are not equally distributed among the nine subfamilies and the number of PLCPs in each subfamily does not increase or decrease proportionally among the seven selected plant species. Papaya showed clear lineage-specific gene expansion in the subfamily III. Interestingly, all four major PLCPs purified from papaya latex, including papain, chymopapain, glycyl endopeptidase and caricain, were grouped into the lineage-specific expansion branch in the subfamily III. Mapping PLCP genes on chromosomes of five plant species revealed that lineage-specific expansions of PLCP genes were mostly derived from tandem duplications. We estimated divergence time of papaya PLCP genes of subfamily III. The major duplication events leading to lineage-specific expansion of papaya PLCP genes in subfamily III were estimated at 48 MYA, 34 MYA, and 16 MYA. The gene expression patterns of the papaya PLCP genes in different tissues were assessed by transcriptome sequencing and qRT-PCR. Most of the papaya PLCP genes of subfamily III expressed at high levels in leaf and green fruit tissues.ConclusionsTandem duplications played the dominant role in affecting copy number of PLCPs in plants. Significant variations in size of the PLCP subfamilies among species may reflect genetic adaptation of plant species to different environments. The lineage-specific expansion of papaya PLCPs of subfamily III might have been promoted by the continuous reciprocal selective effects of herbivore attack and plant defense.

Antioxidant, immunomodulatory and antiproliferative effects of gelatin hydrolysate from unicorn leatherjacket skin

The in vitro cellular bioactivities including, antioxidant, immunomodulatory and antiproliferative effects of a gelatin hydrolysate (GH) prepared from unicorn leatherjacket skin, using partially purified glycyl endopeptidase, were investigated in order to optimize the use of fish skin waste products as functional food ingredients. GH under the tested concentrations (750-1500 µg mL(-1) ) protected against H2 O2 -induced DNA damage in U937 cells. GH also protected against the H2 O2 -induced reduction in cellular antioxidant enzyme activities, superoxide dismutase and catalase, in HepG2 cells. GH demonstrated immunomodulatory potential by reducing pro-inflammatory cytokine (interleukin-6 (IL-6) and IL-1β) production and nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 macrophage cells. Cell proliferation in human colon cancer (Caco-2) cells was significantly reduced in a dose-dependent manner following incubation with GH. These results indicate that GH has several bioactivities which support its potential as a promising functional food ingredient with various health benefits. © 2015 Society of Chemical Industry.

Antioxidative gelatin hydrolysate from unicorn leatherjacket skin as affected by prior autolysis

Gelatin hydrolysates from autolysed non-swollen and swollen unicorn leatherjacket skin prepared using partially purified glycyl endopeptidase (GE) from papaya latex were examined for their antioxidative activities. Autolysed swollen skin was more hydrolysed by GE as indicated by higher α-amino group content, compared with autolysed non-swollen skin. ABTS radical scavenging activity and ferric reducing antioxidant power (FRAP) of hydrolysates were increased with increasing levels of GE used. Antioxidative gelatin hydrolysate from autolysed skin, both non-swollen and swollen skins, using 8 % GE termed ‘NS-8GE’ and ‘SS-8GE’ exhibited different modes of action. When both hydrolysates were tested in lecithin liposome system, the efficiency in retardation of lipid oxidation was in a dose-dependent manner. Antioxidative activity of hydrolysates at 5.0 g/L was comparable to that of 0.1 g/L Trolox, in which the oxidation was almost completely inhibited. NS-8GE and SS-8GE also showed their antioxidative activities in gastrointestinal tract model system (GIMs). ABTS radical scavenging activity of both hydrolysates increased, but FRAP decreased in a duodenal condition. Based on size exclusion chromatography, major antioxidative peptides in NS-8GE and SS-8GE had molecular weight of 1,170 and 750 Da, respectively. Therefore, GE could enhance antioxidative activity of autolysed skin. Additionally, swelling process directly determined the modes of actions of resulting gelatin hydrolysates. NS-8GE had high ABTS radical scavenging activity, whereas SS-8GE showed high FRAP.

Cryoprotective and antioxidative effects of gelatin hydrolysate from unicorn leatherjacket skin

Cryoprotective properties of gelatin hydrolysates from autolysed non-swollen and swollen unicorn leatherjacket skin using partially purified glycyl endopeptidase (GE) from papaya latex were examined. Gelatin hydrolysates from autolysed non-swollen skin showed higher cryoprotective property in salt solution system than that from autolysed swollen skin as indicated by lower enthalpy for melting of eutectic and ice crystal. The cryoprotective effect of gelatin hydrolysate (0.5 and 1.0%) was in dose-dependent manner. Gelatin hydrolysate retarded physicochemical changes of natural actomyosin from washed mince system, as evidenced by lower changes in Ca2+-ATPase activity, surface hydrophobicity and disulfide bond formation, compared with the control. Based on DSC analysis, the enthalpy of myosin and actin was also higher in the presence of gelatin hydrolysate. Gelatin hydrolysate could prevent lipid oxidation in washed mince system as shown by lower TBARS value and less abundance of hexanal, heptanal and 1-pentene-3-ol.

Glycyl endopeptidase from papaya latex: Partial purification and use for production of fish gelatin hydrolysate

An aqueous two-phase system (ATPS) in combination with ammonium sulphate ((NH4)2SO4) precipitation was applied to fractionate glycyl endopeptidase from the papaya latex of Red Lady and Khack Dum cultivars. ATPS containing polyethylene glycol (PEG 2000 and 6000) and salts ((NH4)2SO4 and MgSO4) at different concentrations were used. Glycyl endopeptidase with high purification fold (PF) and yield was found in the salt-rich bottom phase of ATPS with 10%PEG 6000–10% (NH4)2SO4. When ATPS fraction from Red Lady cultivar was further precipitated with 40–60% saturation of (NH4)2SO4, PF of 2.1-fold with 80.23% yield was obtained. Almost all offensive odorous compounds, particularly benzyl isothiocyanate, were removed from partially purified glycyl endopeptidase (PPGE). The fish gelatin hydrolysates prepared using PPGE showed higher ABTS radical scavenging activity and less odour, compared with those of crude extract (CE). Thus antioxidative gelatin hydrolysate with negligible undesirable odour could be prepared with the aid of PPGE.

X-ray crystal structure of CMS1MS2: a high proteolytic activity cysteine proteinase from Carica candamarcensis

CMS1MS2 (CC-Ib) from Carica candamarcensis (Vasconcellea cundinamarcensis) is a cysteine proteinase found as a single polypeptide containing 213 residues of 22,991 Da. The enzyme was purified by three chromatographic steps, two of them involving cationic exchange. Crystals of CMS1MS2 complexed with E-64 were obtained by the hanging drop vapor-diffusion method at 291 K using ammonium sulfate and polyethylene glycol 4000/8000 as precipitant. The complex CMS1MS2-E-64 crystallized in the tetragonal space group P4(1)2(1)2 with unit-cell parameters; a = b = 73.64, c = 118.79 Å. The structure was determined by Molecular Replacement and refined at 1.87 Å resolution to a final R factor of 16.2 % (R (free) = 19.3 %). Based on the model, the structure of CMS1MS2 (PDB 3IOQ) ranks as one of the least basic cysteine isoforms from C. candamarcensis, is structurally closer to papain, caricain, chymopapain and mexicain than to the other cysteine proteinases, while its activity is twice the activity of papain towards BAPNA substrate. Two differences, one in the S2 subsite and another in the S3 subsite of CMS1MS2 may contribute to the enhanced activity relative to papain. In addition, the model provides a structural basis for the sensitivity of CMS1MS2 to inhibition by cystatin, not shown by other enzymes of the group, e.g., glycyl endopeptidase and CMS2MS2.

Peptidoglycan Crosslinking Relaxation Plays an Important Role in Staphylococcus aureus WalKR-Dependent Cell Viability

The WalKR two-component system is essential for viability of Staphylococcus aureus, a major pathogen. We have shown that WalKR acts as the master controller of peptidoglycan metabolism, yet none of the identified regulon genes explain its requirement for cell viability. Transmission electron micrographs revealed cell wall thickening and aberrant division septa in the absence of WalKR, suggesting its requirement may be linked to its role in coordinating cell wall metabolism and cell division. We therefore tested whether uncoupling autolysin gene expression from WalKR-dependent regulation could compensate for its essential nature. Uncoupled expression of genes encoding lytic transglycosylases or amidases did not restore growth to a WalKR-depleted strain. We identified only two WalKR-regulon genes whose expression restored cell viability in the absence of WalKR: lytM and ssaA. Neither of these two genes are essential under our conditions and a ΔlytM ΔssaA mutant does not present any growth defect. LytM is a glycyl–glycyl endopeptidase, hydrolyzing the pentaglycine interpeptide crossbridge, and SsaA belongs to the CHAP amidase family, members of which such as LysK and LytA have been shown to have D-alanyl-glycyl endopeptidase activity, cleaving between the crossbridge and the stem peptide. Taken together, our results strongly suggest that peptidoglycan crosslinking relaxation through crossbridge hydrolysis plays a crucial role in the essential requirement of the WalKR system for cell viability.

Solid-to-solid peptide synthesis by glycyl endopeptidase

Glycyl endopeptidase catalysed solid-to-solid synthesis can be carried out efficiently for the model peptides Z-Gly-Phe-NH 2, Z-Gly-Leu-NH 2, Z-Gly-Tyr-NH 2 and Z-Gly-Tyr-OEt. A small excess of acyl donor Z-Gly improved both the initial rate and final yield, whereas an excess of nucleophile Phe-NH 2 prevented reaction. The highest conversion was achieved when using a substrate molar ratio (Z-Gly:Phe-NH 2) of 2:1. Including solid cysteine in the reaction mixture improved both initial rate and final conversion, probably by protecting the enzyme from oxidation. The reasons why conversion to Z-Gly-Phe-NH 2 stopped at around 83% were investigated. Entrapment of residual solid starting material did not seem significant, while autolysis and inactivation of glycyl endopeptidase in the reaction mixture during catalysis was important. The role of chemical equilibrium position was less clear.

Revisiting the enzymes stored in the laticifers of Carica papaya in the context of their possible participation in the plant defence mechanism.

In the tropical species Carica papaya, the articulated and anastomosing laticifers form a dense network of vessels displayed in all aerial parts of the plant. Damaging the papaya tree inevitably severs its laticifers, eliciting an abrupt release of latex. Besides the well-known cysteine proteinases, papain, chymopapain, caricain and glycyl endopeptidase, papaya latex is also a rich source of other enzymes. Together, these enzymes could provide an important contribution to plant defence mechanisms by sanitising and sealing the wounded areas on the tree.

The 2.1 A structure of a cysteine protease with proline specificity from ginger rhizome, Zingiber officinale.

A cysteine protease from ginger rhizome (GP-II) cleaves peptides and proteins with proline at the P(2) position. The unusual specificity for proline makes GP-II an attractive tool for protein sequencing and identification of stably folded domains in proteins. The enzyme is a 221 amino acid glycoprotein possessing two N-linked oligosaccharide chains (8% glycosylated by weight) at Asn99 and Asn156. The availability of the sequence of these glycosyl chains afforded the opportunity to observe their structure and impact on protein conformation. The three-dimensional structure of GP-II has been determined by X-ray crystallography to a resolution of 2.1 A (overall R-factor = 0.214, free R = 0.248). The overall structure of GP-II is similar to that of the homologous cysteine proteases papain, actinidin, and glycyl endopeptidase, folding into two distinct domains of roughly equal size which are divided by a cleft. The observed N-linked glycosyl chains (half the total carbohydrate sequence) participate in both crystallographic and noncrystallographic contacts, tethering the proteins together via hydrogen bonds to the carbohydrate residues without intervening ordered water molecules. The putative S(2) binding pocket (the proline recognition site) was identified by superposition of the GP-II structure with structures of four previously determined papain-inhibitor complexes. The particular enzymic amino acids forming the S(2) pocket of GP-II (Trp, Met, and Ala) are similar to those found in the proline binding pockets of the unrelated enzymes alpha-lytic protease and cyclophilin. However, there is no conserved three-dimensional arrangement of these residues between the three enzymes (i.e., no proline binding motif). Thus, the particular amino acids found at S(2) are consistent with a binding pocket for a moiety with the steric characteristics and charge distribution of proline. Size exclusion is also a mechanism for selectivity compared to the S(2) binding pocket of papain. The S(2) binding pocket of GP-II greatly restricts the size of the side chain which could be bound because of the occurrence of a tryptophan in place of the corresponding tyrosine in papain. In light of the nature of the binding pocket, the specificity of GP-II for proline over other small nonpolar amino acids may be attributed to a direct effect of proline on the substrate peptide backbone conformation.

Cathepsin L is capable of truncating cystatin C of 11 N-terminal amino acids

Cystatin C with the 11 N-terminal amino acids truncated shows a much lower affinity for cysteine proteinases than the intact inhibitor. Such truncation of cystatin C is recorded after action of glycyl endopeptidase and cathepsin L. Incubation of cystatin C with papain, cathepsin B or cathepsin H led to no changes in the cystatin C molecule. Isoelectric focusing of the cathepsin L and cystatin C mixture showed the formation of two new bands. One of them appeared whether E-64 or PMSF was added or not, evidently representing a cystatin C/cathepsin L complex. The other band is the truncated cystatin C molecule. N-terminal sequencing after separation by HPLC showed that cystatin C is cleaved by cathepsin L at the Gly11-Gly12 bond. The action of cathepsin L on cystatin C may be explained by the cleavage of the scissile bond in an inappropriate complex.

The proregion of papaya proteinase IV inhibits Colorado potato beetle digestive cysteine proteinases

Three distinct digestive protease systems were induced in larvae of the herbivorous pest, Colorado potato beetle (CPB; Leptinotarsa decemlineata Say), and used as a model to assess the ability of the proregion of papaya proteinase IV (PPIV; glycyl endopeptidase, EC 3.4.22.25) to act as an inhibitor of insect digestive cysteine proteinases. As shown by gelatin/PAGE and complementary inhibition assays, a recombinant form of the proregion produced in Escherichia coli inhibited a fraction of the insect proteases also inhibited by the well-characterized inhibitor of cysteine proteinases, oryzacystatin I (OCI). In contrast with OCI, the inhibitory potency of the proregion was affected by an increase of the temperature, suggesting a certain alteration of its structural integrity by the insect non-target proteases. This apparent susceptibility to proteolysis was confirmed by SDS-PAGE, after challenging the proregion with the different insect extracts. As seen on gel, selective inhibition of the insect aspartate proteinase, cathepsin D, with the inhibitor pepstatin A preserved the activity of the proregion against cysteine proteinases by preventing its hydrolysis. Taken together, these observations suggest the potential of plant protease proregions as regulators of cysteine proteinases in biotechnological systems, and show the ability of protease inhibitors to preserve the integrity of `companion' defense-related proteins from the action of insensitive proteases in target pests.

Structural basis for different inhibitory specificities of human cystatins C and D.

Human cystatins C and D share almost identical primary structures of two out of the three segments proposed to be of importance for enzyme interactions but have markedly different profiles for inhibition of the target cysteine peptidases, cathepsins B, H, L, and S. To investigate if the N-terminal binding regions of the inhibitors are responsible for the different inhibition profiles, and thereby confer biological selectivity, two hybrid cystatins were produced in Escherichia coli expression systems. In one hybrid, the N-terminal segment of cystatin C was placed on the framework of cystatin D, and the second was engineered with the N-terminal segment of cystatin D on the cystatin C scaffold. Truncated cystatin C and D variants, devoid of their N-terminal segments, were obtained by incubation with glycyl endopeptidase and isolated, in a second approach to assess the importance of the N-terminal binding regions for cystatin function and specificity. The affinities of the four cystatin variants for cathepsins B, H, L, and S were measured. By comparison with corresponding results for wild-type cystatins C and D, it was concluded (1) that both the N-terminal and framework part of the molecules significantly contribute to the observed differences in inhibitory activities of cystatins C and D and (2) that the N-terminal segment of cystatin C increases the inhibitory activity of cystatin D against cathepsin S and cathepsin L but results in decreased activity against cathepsin H. These differences in specificity were explained by the residues interacting with the S2 subsite of peptidases (Val- and Ala-10 in cystatin C and D, respectively). Also, removal of the N-terminal segment results in total loss of enzyme affinity for cystatin D but not for cystatin C. Therefore, structural differences in the framework parts, as well as in the N-terminal segments, are critical for both inhibitory specificity and potency. Homology modeling was used to identify residues likely responsible for the generally reduced inhibitory potency of cystatin D.

The amino acid sequences, structure comparisons and inhibition kinetics of sheep cathepsin L and sheep stefin B

Cathepsin L and stefin B were isolated from sheep liver, the cathepsin L being isolated by a low pH homogenisation method, which increases the proportion of the two-chain form of the enzyme, thus facilitating sequencing. The amino acid sequences of the isolated cathepsin L and stefin B were determined. The two-chain form of cathepsin L contains 217 amino acid residues and has an M r of 23,627. The sequence was obtained by sequencing the native active enzyme, the light and heavy chains and the peptides generated by cyanogen bromide cleavage. These peptides were aligned with peptides obtained by hydrolysis with endoproteinase Lys-C, glycyl endopeptidase and endoproteinase Glu-C. Sheep liver cathepsin L exhibits a high degree of sequence identity to human cathepsin L. Sheep stefin B consists of 98 amino acid residues and its calculated M r is 11,150. The inhibitor has its NH 2-terminal amino acid residue blocked. Its amino acid sequence was determined by sequencing the peptides obtained by cleavage with cyanogen bromide and peptides obtained by hydrolysis with endoproteinase Glu-C and endoproteinase Lys-C. Sheep stefin B shows a high degree of sequence identity with bovine and human stefin B. The kinetics of the interaction between sheep cathepsin L and stefin B were determined, with the interaction of stefin B with papain used as a benchmark to compare with other published results. Despite the considerable homology between bovine and sheep stefin B, the kinetics of their interaction with papain and cathepsin L differed markedly, possibly due to the differences in the so-called “trunk” region of the cystatin molecule.

Kinetic constants for the hydrolysis of aggrecan by the papaya proteinases and their relevance for chemonucleolysis

The four known proteinases from papaya latex, namely papain (EC 3.4.22.2), chymopapain (EC 3.4.22.6), caricain (EC 3.4.22.30), and glycyl endopeptidase (EC 3.4.22.25), were purified to homogeneity and fully characterized by single radial immunodiffusion and active-site titration. A modified HPLC gel permeation assay was used to determine the kinetic constants for aggrecan hydrolysis by the papaya proteinases. The disappearance of intact aggrecan monomer was first-order, indicating that for the four enzymes studied the K m was much larger than 0.5 μM and that k cat/ K m = 1.2 ± 0.1 × 10 6 M −1 s −1 for chymopapain, 1.20 ± 0.08 × 10 6 m −1 s −1 for caricain, 0.90 ± 0.02 × 10 6 m −1 s −1 for papain, and 0.120 ± 0.005 × 10 6 m −1 s −1 for glycyl endopeptidase. Chymodiactin, the chymopapain preparation used for chemonucleolysis, consists of a mixture of chymopapain (70%), caricain (20%), and glycyl endopeptidase (4%). The rate constant for the aggrecan hydrolysis by such a mixture was not significantly different from the rate constant for pure chymopapain. As a result of these observations, we predict that pure chymopapain could replace partially purified chymopapain preparations for chemonucleolysis.

Immunoglobulin E Antibodies to Papaya Proteinases and Their Relevance to Chemonucleolysis

Levels of four papaya cysteine proteinases were determined in Chymodiactin, a pharmaceutical preparation of chymopapain (EC 3.4.22.6) used in chemonucleolysis for the treatment of sciatica. Twelve sera known to contain immunoglobulin E antibodies to Chymodiactin were assayed for immunoglobulin E antibodies to these enzymes. The goal of the study was to determine what contribution each of the four proteinases makes to the allergic response that occasionally occurs during injection of a damaged intervertebral disc with chymopapain preparations. The occurrence of an allergic reaction during chemonucleolysis implies prior sensitization to components of the injected enzyme solution. The latex of the unripe fruit of the papaya plant Carica papaya, from which chymopapain is purified, contains another three immunologically distinct cysteine proteinases: 1) caricain (EC 3.4.22.30), 2) glycyl endopeptidase (EC 3.4.22.25), and 3) papain (EC 3.4.22.2). A dot-blot immunoassay was developed to quantify each enzyme in Chymodiactin. Total serum immunoglobulin E levels and specific immunoglobulin E antibody levels to each of the four papaya cysteine proteinases were assayed by an enzyme-linked immunoassay in 12 sera containing immunoglobulin E antibodies to Chymodiactin. Chymodiactin contained 70% chymopapain, 20% caricain, 4% glycyl endopeptidase, and 0.1% papain. Immunoglobulin E antibodies to all four proteinases were found in most of the 12 sera, but in varying proportions. Antibodies to glycyl endopeptidase were predominant in eight sera, and the mean amounts of immunoglobulin E directed against each protein were: glycyl endopeptidase, 4.21 IU/ml; caricain, 2.9 IU/ml; chymopapain, 1.97 IU/ml; and papain, 1.39 IU/ml. Total serum immunoglobulin E levels showed little correlation with immunoglobulin E responses to Chymodiactin. The results suggested that removal of glycyl endopeptidase and caricain from pharmaceutical preparations of chymopapain may help reduce the incidence of allergic reactions during chemonucleolysis.

Temporary inhibition of papain by hairpin loop mutants of chicken cystatin Distorted binding of the loops results in cleavage of the Gly 9-Ala 10 bond

Temporary inhibition of the cysteine proteinases papain and cathepsin L was observed with several hairpin loop mutants of recombinant chicken cystatin at enzyme concentrations above nanomolar. Kinetic modelling of inhibition data, gel electrophoresis and amino acid sequencing revealed that reappearance of papain activity is due to selective cleavage of the Gly 9-Ala 10 bond in the N-terminal binding area of the chicken cystatin variants, resulting in truncated inhibitors of lower affinity. Cleavage of the same bond by contaminating papaya proteinase IV was ruled out by previous purification of papain and suitable control experiments. According to the proposed kinetic model, cleavage occurs within the enzyme-inhibitor complex with first order rate constants k temp of 2.3 × 10 −3 up to 5 × 10 −1 s −1. A similar k temp K m ratio was found for all mutants (0.7 × 10 6–2.1 × 10 6 s −1·M −1); it is almost identical with the k cat K m ratio of the peptide substrate Z-Phe-Arg-NHMec. These results suggest that distorted contacts of one of the hairpin loops affect binding of the N-terminal contact area in a way that covalent interaction of the Gly 9-Ala 10 bond with the active-site Cys residue of papain can occur and the bond is cleaved in a substrate-like manner.

Expression of the pro-regions of papain and papaya proteinase IV in Escherichia coli and their inhibition of mature cysteine proteinases

Expression of the pro-regions of papain and papaya proteinase IV in <i>Escherichia coli</i> and their inhibition of mature cysteine proteinases

Modification of S1 subsite specificity in the cysteine protease cathepsin B.

Cysteine proteases of the papain family generally exhibit broad P1 specificity. A notable exception is papaya proteinase IV (PPIV), which only accepts Gly at this position. In all other cysteine proteases the S1 subsite residues 23 and 65 (papain numbering) are absolutely conserved as Gly, while in PPIV they are replaced by Glu and Arg, respectively. These differences appear to underlie both PPIV specificity and its resistance to inhibition by cystatins. To test this hypothesis, the equivalent residues (Gly27 and Gly73) in the mammalian cysteine protease cathepsin B were changed to Glu and Arg, respectively. Relative to the wild-type enzyme, the Gly27Glu and Gly73Arg mutants showed a drastic reduction in activity with substrates containing a P1 Arg. In contrast, substrates having a Gly residue in P1 were hydrolyzed effectively. The double mutant (Gly27Glu:Gly73Arg) exhibited no detectable activity against any substrate studied. Inhibition of the Gly73Arg mutant by E-64 [1-(L-trans-epoxysuccinyl-L-leucylamino)-4-guanidinobutane] was found to be similar to that of the wild-type enzyme. In contrast, inhibition by cystatin C exhibited a 20,000-fold reduction. These results demonstrate the dramatic influence of side chains at sequence locations 27 and 73 on the S1 subsite specificity of cysteine proteases.