Dissecting the Active Site of the Collagenolytic Cathepsin L3 Protease of the Invasive Stage of Fasciola hepatica

Background

A family of secreted cathepsin L proteases with differential activities is essential for host colonization and survival in the parasitic flatworm Fasciola hepatica. While the blood feeding adult secretes predominantly FheCL1, an enzyme with a strong preference for Leu at the S₂ pocket of the active site, the infective stage produces FheCL3, a unique enzyme with collagenolytic activity that favours Pro at P₂.

Methodology/Principal Findings

Using a novel unbiased multiplex substrate profiling and mass spectrometry methodology (MSP-MS), we compared the preferences of FheCL1 and FheCL3 along the complete active site cleft and confirm that while the S₂ imposes the greatest influence on substrate selectivity, preferences can be indicated on other active site subsites. Notably, we discovered that the activity of FheCL1 and FheCL3 enzymes is very different, sharing only 50% of the cleavage sites, supporting the idea of functional specialization. We generated variants of FheCL1 and FheCL3 with S₂ and S₃ residues by mutagenesis and evaluated their substrate specificity using positional scanning synthetic combinatorial libraries (PS-SCL). Besides the rare P₂ Pro preference, FheCL3 showed a distinctive specificity at the S₃ pocket, accommodating preferentially the small Gly residue. Both P₂ Pro and P₃ Gly preferences were strongly reduced when Trp67 of FheCL3 was replaced by Leu, rendering the enzyme incapable of digesting collagen. In contrast, the inverse Leu67Trp substitution in FheCL1 only slightly reduced its Leu preference and improved Pro acceptance in P₂, but greatly increased accommodation of Gly at S₃.

Conclusions/Significance

These data reveal the significance of S₂ and S₃ interactions in substrate binding emphasizing the role for residue 67 in modulating both sites, providing a plausible explanation for the FheCL3 collagenolytic activity essential to host invasion. The unique specificity of FheCL3 could be exploited in the design of specific inhibitors selectively directed to specific infective stage parasite proteinases.     

Selective inhibition of the collagenolytic activity of human cathepsin K by altering its S2 subsite specificity

The primary specificity of papain-like cysteine proteases (family C1, clan CA) is determined by S2-P2 interactions. Despite the high amino acid sequence identities and structural similarities between cathepsins K and L, only cathepsin K is capable of cleaving interstitial collagens in their triple helical domains. To investigate this specificity, we have engineered the S2 pocket of human cathepsin K into a cathepsin L-like subsite. Using combinatorial fluorogenic substrate libraries, the P1-P4 substrate specificity of the cathepsin K variant, Tyr67Leu/Leu205Ala, was determined and compared with those of cathepsins K and L. The introduction of the double mutation into the S2 subsite of cathepsin K rendered the unique S2 binding preference of the protease for proline and leucine residues into a cathepsin L-like preference for bulky aromatic residues. Homology modeling and docking calculations supported the experimental findings. The cathepsin L-like S2 specificity of the mutant protein and the integrity of its catalytic site were confirmed by kinetic analysis of synthetic di- and tripeptide substrates as well as pH stability and pH activity profile studies. The loss of the ability to accept proline in the S2 binding pocket by the mutant protease completely abolished the collagenolytic activity of cathepsin K whereas its overall gelatinolytic activity remained unaffected. These results indicate that Tyr67 and Leu205 play a key role in the binding of proline residues in the S2 pocket of cathepsin K and are required for its unique collagenase activity.     

Fasciola hepatica cathepsin L-like proteases: biology,function, and potential in the development of first generation liver fluke vaccines

Fasciola hepatica secretes cathepsin L proteases that facilitate the penetration of the parasite through the tissues of its host, and also participate in functions such as feeding and immune evasion. The major proteases, cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) are members of a lineage that gave rise to the human cathepsin Ls, Ks and Ss, but while they exhibit similarities in their substrate specificities to these enzymes they differ in having a wider pH range for activity and an enhanced stability at neutral pH. There are presently 13 Fasciola cathepsin L cDNAs deposited in the public databases representing a gene family of at least seven distinct members, although the temporal and spatial expression of each of these members in the developmental stage of F. hepatica remains unclear. Immunolocalisation and in situ hybridisation studies, using antibody and DNA probes, respectively, show that the vast majority of cathepsin L gene expression is carried out in the epithelial cells lining the parasite gut. Within these cells the enzyme is packaged into secretory vesicles that release their contents into the gut lumen for the purpose of degrading ingested host tissue and blood. Liver flukes also express a novel multi-domain cystatin that may be involved in the regulation of cathepsin L activity. Vaccine trials in both sheep and cattle with purified native FheCL1 and FheCL2 have shown that these enzymes can induce protection, ranging from 33 to 79%, to experimental challenge with metacercariae of F. hepatica, and very potent anti-embryonation/hatch rate effects that would block parasite transmission. In this article we review the vaccine trials carried out over the past 8 years, the role of antibody and T cell responses in mediating protection and discuss the prospects of the cathepsin Ls in the development of first generation recombinant liver fluke vaccines.     

Analysis of Fasciola cathepsin L5 by S2 subsite substitutions and determination of the P1-P4 specificity reveals an unusual preference

Fasciola parasites (liver flukes) express numerous cathepsin L proteases that are believed to be involved in important functions related to host invasion and parasite survival. These proteases are evolutionarily divided into clades that are proposed to reflect their substrate specificity, most noticeably through the S(2) subsite. Single amino acid substitutions to residues lining this site, including amino acid residue 69 (aa69; mature cathepsin L5 numbering) can have profound influences on subsite architecture and influence enzyme specificity. Variations at aa69 among known Fasciola cathepsin L proteases include leucine, tyrosine, tryptophan, phenylalanine and glycine. Other amino acids (cysteine, serine) might have been expected at this site due to codon usage as cathepsin L isoenzymes evolved, but C69 and S69 have not been observed. The introduction of L69C and L69S substitutions into FhCatL5 resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants in Fasciola. An FhCatL5 L69F variant showed an increase in the ability to cleave substrates with P(2) proline, indicating F69 variants expressed by the fluke would likely have this ability. An FhCatL2 Y69L variant showed a decreased acceptance of P(2) proline, further highlighting the importance of Y69 for FhCatL2 P(2) proline acceptance. Finally, the P(1)-P(4) specificity of Fasciola cathepsin L5 was determined and, unexpectedly, aspartic acid was shown to be well accepted at P(2,) which is unique amongst Fasciola cathepsins examined to date.     

Human cathepsin V functional expression, tissue distribution, electrostatic surface potential, enzymatic characterization, and chromosomal localization

Cathepsin V, a thymus and testis-specific human cysteine protease, was expressed in Pichia pastoris, and its physicokinetic properties were determined. Recombinant procathepsin V is autocatalytically activated at acidic pH and is effectively inhibited by various cysteine protease class-specific inhibitors. The S2P2 subsite specificity of cathepsin V was found to be intermediate between those of cathepsins S and L. The substrate binding pocket, S2, accepted both aromatic and nonaromatic hydrophobic residues, whereas cathepsins L and S preferred either an aromatic or nonaromatic hydrophobic residue, respectively. In contrast to cathepsin L, but similar to cathepsin S, cathepsin V exhibited only a very weak collagenolytic activity. Furthermore, cathepsin V was determined to be significantly more stable at mildly acidic and neutral pH than cathepsin L, but distinctly less stable than cathepsin S. A homology structure model of cathepsin V revealed completely different electrostatic potentials on the molecular surface when compared with human cathepsin L. The model-based electrostatic potential of human cathepsin V was neutral to weakly positive at and in the vicinity of the active site cleft, whereas that of cathepsin L was negative over extended regions of the surface. Surprisingly, the electrostatic potential of the human cathepsin V model structure resembled that of the model structure of mouse cathepsin L. These differences in the electrostatic potential at the molecular surfaces provide a reactivity determinant that may be the source of differences in substrate selectivity and pH stability. Cathepsin V was mapped to the chromosomal region 9q22.2, a site adjacent to the cathepsin L locus. The high sequence identity and the overlapping chromosomal gene loci suggest that both proteases evolved from an ancestral cathepsin L-like precursor by gene duplication.     

Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica: expansion of a repertoire of virulence-associated factors

Cathepsin L proteases secreted by the helminth pathogen Fasciola hepatica have functions in parasite virulence including tissue invasion and suppression of host immune responses. Using proteomics methods alongside phylogenetic studies we characterized the profile of cathepsin L proteases secreted by adult F. hepatica and hence identified those involved in host-pathogen interaction. Phylogenetic analyses showed that the Fasciola cathepsin L gene family expanded by a series of gene duplications followed by divergence that gave rise to three clades associated with mature adult worms (Clades 1, 2, and 5) and two clades specific to infective juvenile stages (Clades 3 and 4). Consistent with these observations our proteomics studies identified representatives from Clades 1, 2, and 5 but not from Clades 3 and 4 in adult F. hepatica secretory products. Clades 1 and 2 account for 67.39 and 27.63% of total secreted cathepsin Ls, respectively, suggesting that their expansion was positively driven and that these proteases are most critical for parasite survival and adaptation. Sequence comparison studies revealed that the expansion of cathepsin Ls by gene duplication was followed by residue changes in the S2 pocket of the active site. Our biochemical studies showed that these changes result in alterations in substrate binding and suggested that the divergence of the cathepsin L family produced a repertoire of enzymes with overlapping and complementary substrate specificities that could cleave host macromolecules more efficiently. Although the cathepsin Ls are produced as zymogens containing a prosegment and mature domain, all secreted enzymes identified by MS were processed to mature active enzymes. The prosegment region was highly conserved between the clades except at the boundary of prosegment and mature enzyme. Despite the lack of conservation at this section, sites for exogenous cleavage by asparaginyl endopeptidases and a Leu-Ser[downward arrow]His motif for autocatalytic cleavage by cathepsin Ls were preserved.     

Helminth pathogen cathepsin proteases: it's a family affair

Helminth pathogens express papain-like cysteine peptidases, termed cathepsins, which have important roles in virulence, including host entry, tissue migration and the suppression of host immune responses. The liver fluke Fasciola hepatica, an emerging human pathogen, expresses the largest cathepsin L cysteine protease family yet described. Recent phylogenetic, biochemical and structural studies indicate that this family contains five separate clades, which exhibit overlapping but distinct substrate specificities created by a process of gene duplication followed by subtle residue divergence within the protease active site. The developmentally regulated expression of these proteases correlates with the passage of the parasite through host tissues and its encounters with different host macromolecules.     

The S2 subsites of cathepsins K and L and their contribution to collagen degradation

The exchange of residues 67 and 205 of the S2 pocket of human cysteine cathepsins K and L induces a permutation of their substrate specificity toward fluorogenic peptide substrates. While the cathepsin L-like cathepsin K (Tyr67Leu/Leu205Ala) mutant has a marked preference for Phe, the Leu67Tyr/Ala205Leu cathepsin L variant shows an effective cathepsin K-like preference for Leu and Pro. A similar turnaround of inhibition was observed by using specific inhibitors of cathepsin K [1-(N-Benzyloxycarbonyl-leucyl)-5-(N-Boc-phenylalanyl-leucyl)carbohydrazide] and cathepsin L [N-(4-biphenylacetyl)-S-methylcysteine-(D)-Arg-Phe-beta-phenethylamide]. Molecular modeling studies indicated that mutations alter the character of both S2 and S3 subsites, while docking calculations were consistent with kinetics data. The cathepsin K-like cathepsin L was unable to mimic the collagen-degrading activity of cathepsin K against collagens I and II, DQ-collagens I and IV, and elastin-Congo Red. In summary, double mutations of the S2 pocket of cathepsins K (Y67L/L205A) and L (L67Y/A205L) induce a switch of their enzymatic specificity toward small selective inhibitors and peptidyl substrates, confirming the key role of residues 67 and 205. However, mutations in the S2 subsite pocket of cathepsin L alone without engineering of binding sites to chondroitin sulfate are not sufficient to generate a cathepsin K-like collagenase, emphasizing the pivotal role of the complex formation between glycosaminoglycans and cathepsin K for its unique collagenolytic activity.     

A single amino acid substitution affects substrate specificity in cysteine proteinases from Fasciola hepatica

The trematode Fasciola hepatica secretes a number of cathepsin L-like proteases that are proposed to be involved in feeding, migration, and immune evasion by the parasite. To date, six full cDNA sequences encoding cathepsin L preproproteins have been identified. Previous studies have demonstrated that one of these cathepsins (L2) is unusual in that it is able to cleave substrates with a proline in the P2 position, translating into an unusual ability (for a cysteine proteinase) to clot fibrinogen. In this study, we report the sequence of a novel cathepsin (L5) and compare the substrate specificity of a recombinant enzyme with that of recombinant cathepsin L2. Despite sharing 80% sequence identity with cathepsin L2, cathepsin L5 does not exhibit substantial catalytic activity against substrates containing proline in the P2 position. Molecular modeling studies suggested that a single amino acid change (L69Y) in the mature proteinases may account for the difference in specificity at the S2 subsite. Recombinant cathepsin L5/L69Y was expressed in yeast and a substantial increase in the ability of this variant to accommodate substrates with a proline residue in the P2 position was observed. Thus, we have identified a single amino acid substitution that can substantially influence the architecture of the S2 subsite of F. hepatica cathepsin L proteases.     

The Evolution of Enzyme Specificity in <Emphasis Type="Italic">Fasciola spp.</Emphasis>

Fasciola spp., commonly known as liver fluke, are significant trematode parasites of livestock and humans. They secrete several cathepsin L-like cysteine proteases, some of which differ in enzymatic properties and timing of expression in the parasite's life cycle. A detailed sequence and evolutionary analysis is presented, based on 18 cathepsin L-like enzymes isolated from Fasciola spp. (including a novel clone identified in this study). The enzymes form a monophyletic group which has experienced several gene duplication events over the last approximately 135 million years, giving rise to the present-day enzymatic repertoire of the parasite. This timing of these duplications appears to correlate with important points in the evolution of the mammalian hosts. Furthermore, the dates suggest that Fasciola hepatica and Fasciola gigantica diverged around 19 million years ago. A novel analysis, based on the pattern of amino acid diversity, was used to identify sites in the enzyme that are predicted to be subject to positive adaptive evolution. Many of these sites occur within the active site cleft of the enzymes, and hence would be expected to lead to differences in substrate specificity. Using homology modeling, with reference to previously obtained biochemical data, we are able to predict S2 subsite specificity for these enzymes: specifically those that can accommodate bulky hydrophobic residues in the P2 position and those that cannot. A number of other positions subject to evolutionary pressure and potentially significant for enzyme function are also identified, including sites anticipated to diminish cystatin binding affinity.     

Regulation of collagenase activities of human cathepsins by glycosaminoglycans

Cathepsin K, a lysosomal papain-like cysteine protease, forms collagenolytically highly active complexes with chondroitin sulfate and represents the most potent mammalian collagenase. Here we demonstrate that complex formation with glycosaminoglycans (GAGs) is unique for cathepsin K among human papain-like cysteine proteases and that different GAGs compete for the binding to cathepsin K. GAGs predominantly expressed in bone and cartilage, such as chondroitin and keratan sulfates, enhance the collagenolytic activity of cathepsin K, whereas dermatan, heparan sulfate, and heparin selectively inhibit this activity. Moreover, GAGs potently inhibit the collagenase activity of other cysteine proteases such as cathepsins L and S at 37 degrees C. Along this line MMP1-generated collagen fragments in the presence of GAGs are stable against further degradation at 28 degrees C by all cathepsins but cathepsin K, whereas thermal destabilization at 37 degrees C renders the fragments accessible to all cathepsins. These results suggest a novel mechanism for the regulation of matrix protein degradation by GAGs. It further implies that cathepsin K represents the only lysosomal collagenolytic activity under physiologically relevant conditions.     

Squamous cell carcinoma antigen 1 is an inhibitor of parasite-derived cysteine proteases

The physiological significance of the squamous cell carcinoma antigens 1 (SCCA1) and SCCA2, members of the ovalbumin serpin family, remains unresolved. In this study, we examined whether SCCA1 or SCCA2 inhibits protozoa- or helminth-derived cysteine proteases. SCCA1, but not SCCA2, potently inhibited the cysteine protease activities of CPB2.8 from Leishmania mexicana, cruzain from Trypanosoma cruzi, rhodesain from Trypanosoma brucei rhodesience, and cathepsin L2 from Fasciola hepatica. The inhibitory activities of SCCA1 were due to its resistance to cleavage by the cysteine proteases. The findings indicate that induction of cysteine protease inhibitors might be a novel defense mechanism against parasite development.     

Crystal structure of human procathepsin X: a cysteine protease with the proregion covalently linked to the active site cysteine

Human cathepsin X is one of many proteins discovered in recent years through the mining of sequence databases. Its sequence shows clear homology to cysteine proteases from the papain family, containing the characteristic residue patterns, including the active site. However, the proregion of cathepsin X is only 38 residues long, the shortest among papain-like enzymes, and the cathepsin X sequence has an atypical insertion in the regions proximal to the active site. This protein was recently expressed and partially characterized biochemically. Unlike most other cysteine proteases from the papain family, procathepsin X is incapable of autoprocessing in vitro but can be processed under reducing conditions by exogenous cathepsin L. Atypically, the mature enzyme is primarily a carboxypeptidase and has extremely poor endopeptidase activity. We have determined the three-dimensional structure of the procathepsin X at 1.7 A resolution. The overall structure of the mature enzyme is characteristic for enzymes of the papain superfamily, but contains several novel features. Most interestingly, the short proregion binds to the enzyme with the aid of a covalent bond between the cysteine residue in the proregion (Cys10p) and the active site cysteine residue (Cys31). This is the first example of a zymogen in which the inhibition of enzyme's proteolytic activity by the proregion is achieved through a reversible covalent modification of the active site nucleophile. Such mode of binding requires less contact area between the proregion and the enzyme than observed in other procathepsins, and no auxiliary binding site on the enzyme surface is used. A three-residue insertion in a highly conserved region, just prior to the active site cysteine residue, confers a significantly different shape on the S' subsites, compared to other proteases from papain family. The 3D structure provides an explanation for the rather unusual carboxypeptidase activity of this enzyme and confirms the predictions based on homology modeling. Another long insertion in the cathepsin X amino acid sequence forms a beta-hairpin pointing away from the active site. This insertion, thought to be an equivalent of cathepsin B occluding loop, is located on the side of the protein, distant from the substrate binding site.     

Interdependency of sequence and positional specificities for cysteine proteases of the papain family

The specificity of cysteine proteases is characterized by the nature of the amino acid sequence recognized by the enzymes (sequence specificity) as well as by the position of the scissile peptide bond (positional specificity, i.e., endopeptidase, aminopeptidase, or carboxypeptidase). In this paper, the interdependency of sequence and positional specificities for selected members of this class of enzymes has been investigated using fluorogenic substrates where both the position of the cleavable peptide bond and the nature of the sequence of residues in P2-P1 are varied. The results show that cathepsins K and L and papain, typically considered to act strictly as endopeptidases, can also display dipeptidyl carboxypeptidase activity against the substrate Abz-FRF(4NO2)A and dipeptidyl aminopeptidase activity against FR-MCA. In some cases the activity is even equal to or greater than that observed with cathepsin B and DPP-I (dipeptidyl peptidase I), which have been characterized previously as exopeptidases. In contrast, the exopeptidase activities of cathepsins K and L and papain are extremely low when the P2-P1 residues are A-A, indicating that, as observed for the normal endopeptidase activity, the exopeptidase activities rely heavily on interactions in subsite S2 (and possibly S1). However, cathepsin B and DPP-I are able to hydrolyze substrates through the exopeptidase route even in absence of preferred interactions in subsites S2 and S1. This is attributed to the presence in cathepsin B and DPP-I of specific structural elements which serve as an anchor for the C- or N-terminus of a substrate, thereby allowing favorable enzyme-substrate interaction independently of the P2-P1 sequence. As a consequence, the nature of the residue at position P2 of a substrate, which is usually the main factor determining the specificity for cysteine proteases of the papain family, does not have the same contribution for the exopeptidase activities of cathepsin B and DPP-I.     

Molecular cloning and functional characterization of crustapain: a distinct cysteine proteinase with unique substrate specificity from northern shrimp Pandalus borealis

A cDNA clone encoding a cysteine proteinase of the papain superfamily has been isolated from the hepatopancreas of northern shrimp Pandalus borealis (NsCys). NsCys shares the highest identity of 64% with a cathepsin L-like cysteine proteinase from lobster, and its identity to the well-characterized mammalian cathepsins S, L, and K falls within a narrow range of 54-59%. However, it differs from each of these cathepsins in certain key residues including, for example, the unique occurrence of tryptophan and cysteine residues at the structurally important S2 subsite. Consequently, NsCys produced in Pichia pastoris appears to be distinct in various physicokinetic properties. The recombinant enzyme is active and stable over a wide range of pH values, and its substrate specificity is unusual, as demonstrated by its poor affinity for phenylalanine residues. Instead, it shows the highest specificity for proline residues, a property similar to cathepsin K. Unlike cathepsin K, however, NsCys cleaves valine residues more efficiently than leucine. Similar results were obtained with the natural peptide substrate glucagon. The shrimp proteinase is further distinguished by its potent collagenolytic activity, resulting in a cleavage pattern reminiscent of bacterial collagenase. To distinguish such unique structural and enzymatic properties, we propose the trivial name "crustapain" for the shrimp proteinase, indicating that it is a papain-like cysteine proteinase from a crustacean species.     

Crystal structure of the parasite protease inhibitor chagasin in complex with a host target cysteine protease

Chagasin is a protein produced by Trypanosoma cruzi, the parasite that causes Chagas' disease. This small protein belongs to a recently defined family of cysteine protease inhibitors. Although resembling well-known inhibitors like the cystatins in size (110 amino acid residues) and function (they all inhibit papain-like (C1 family) proteases), it has a unique amino acid sequence and structure. We have crystallized and solved the structure of chagasin in complex with the host cysteine protease, cathepsin L, at 1.75 A resolution. An inhibitory wedge composed of three loops (L2, L4, and L6) forms a number of contacts responsible for high-affinity binding (K(i), 39 pM) to the enzyme. All three loops interact with the catalytic groove, with the central loop L2 inserted directly into the catalytic center. Loops L4 and L6 embrace the enzyme molecule from both sides and exhibit distinctly different patterns of protein-protein recognition. Comparison with a 1.7 A structure of uncomplexed chagasin, also determined in this study, demonstrates that a conformational change of the first binding loop (L4) allows extended binding to the non-primed substrate pockets of the enzyme active site cleft, thereby providing a substantial part of the inhibitory surface. The mode of chagasin binding is generally similar, albeit distinctly different in detail, when compared to those displayed by cystatins and the cysteine protease inhibitory p41 fragment of the invariant chain. The chagasin-cathepsin L complex structure provides details of how the parasite protein inhibits a host enzyme of possible importance in host defense. The high level of structural and functional similarity between cathepsin L and the T. cruzi enzyme cruzipain gives clues to how the cysteine protease activity of the parasite can be targeted. This information will aid in the development of synthetic inhibitors for use as potential drugs for the treatment of Chagas disease.     

Crystal structure of human cathepsin S.

We have determined the 2.5 A structure (Rcryst = 20.5%, Rfree = 28.5%) of a complex between human cathepsin S and the potent, irreversible inhibitor 4-morpholinecarbonyl-Phe-hPhe-vinyl sulfone-phenyl. Noncrystallographic symmetry averaging and other density modification techniques were used to improve electron density maps which were nonoptimal due to systematically incomplete data. Methods that reduce the number of parameters were implemented for refinement. The refined structure shows cathepsin S to be similar to related cysteine proteases such as papain and cathepsins K and L. As expected, the covalently-bound inhibitor is attached to the enzyme at Cys 25, and enzyme binding subsites S3-S1' are occupied by the respective inhibitor substituents. A somewhat larger S2 pocket than what is found in similar enzymes is consistent with the broader specificity of cathepsin S at this site, while Lys 61 in the S3 site may offer opportunities for selective inhibition of this enzyme. The presence of Arg 137 in the S1' pocket, and proximal to Cys 25 may have implications not only for substrate specificity C-terminal to the scissile bond, but also for catalysis.     

Human cathepsin X: A cysteine protease with unique carboxypeptidase activity.

Cathepsin X is a novel cysteine protease which was identified recently from the EST (expressed sequence tags) database. In a homology model of the mature cathepsin X, a unique three residue insertion between the Gln22 of the oxyanion hole and the active site Cys31 was found to be located in the primed region of the binding cleft as part of a surface loop corresponding to residues His23 to Tyr27, which we have termed the "mini-loop". From the model, it became apparent that this distinctive structural feature might confer exopeptidase activity to the enzyme. To verify this hypothesis, human procathepsin X was expressed in Pichia pastoris and converted to mature cathepsin X using small amounts of human cathepsin L. Cathepsin X was found to display excellent carboxypeptidase activity against the substrate Abz-FRF(4NO(2)), with a k(cat)/K(M) value of 1.23 x 10(5) M(-)(1) s(-)(1) at the optimal pH of 5.0. However, the activity of cathepsin X against the substrates Cbz-FR-MCA and Abz-AFRSAAQ-EDDnp was found to be extremely low, with k(cat)/K(M) values lower than 70 M(-)(1) s(-)(1). Therefore, cathepsin X displays a stricter exopeptidase activity than cathepsin B. No inhibition of cathepsin X by cystatin C could be detected up to a concentration of 4 microM of inhibitor. From a model of the protease complexed with Cbz-FRF, the bound carboxypeptidase substrate is predicted to establish a number of favorable contacts within the cathepsin X binding site, in particular with residues His23 and Tyr27 from the mini-loop. The presence of the mini-loop restricts the accessibility of cystatin C as well as of the endopeptidase and MCA substrates in the primed subsites of the protease. The marked structural and functional differences of cathepsin X relative to other members of the papain family of cysteine proteases will be of great value in designing specific inhibitors useful as research tools to investigate the physiological and potential pathological roles of this novel enzyme.