Biochemical characterization of the catalytic domains of three different Clostridial collagenases

Clostridial collagenases are used for a broad spectrum of biotechnological applications and represent prime target candidates for both therapy and diagnosis of clostridial infections. In this study, we biochemically characterized the catalytic domains of three clostridial collagenases, collagenase G (ColG) and H (ColH) from Clostridium histolyticum, and collagenase T (ColT) from C. tetani. All protein samples showed activity against a synthetic peptidic substrate (furylacryloyl-Leu-Gly-Pro-Ala, FALGPA) with ColH showing the highest overall activity and highest substrate affinity. Whereas the K(m) values of all three enzymes were within the same order of magnitude, the turnover rate k(cat) of ColG decreased 50- to 150-fold when compared to ColT and ColH. It is noteworthy that the protein N-terminus significantly impacts their substrate affinity and substrate turnover as well as their inhibition profile with 1,10-phenanthroline. These findings were complemented with the discovery of a strictly conserved double-glycine motif, positioned 28 amino acids upstream of the HEXXH zinc binding site, which is critical for enzymatic activity. These observations have consequences with respect to the topology of the N-terminus relative to the active site as well as possible activation mechanisms.     

High-level expression of his-tagged clostridial collagenase in Clostridium perfringens

Clostridium histolyticum collagenase is used to isolate cells from various organs and tissues for tissue engineering, and also to treat destructive fibrosis; thus, the demand for high-grade enzyme preparations is increasing. In this study, we constructed a plasmid encoding C. histolyticum type II collagenase (ColH) with a C-terminal hexahistidine tag (ColH-his) to facilitate the purification of the enzyme through immobilized metal affinity chromatography (IMAC). When ColH-his was expressed in a protease-deficient mutant of Clostridium perfringens, it was produced in the culture supernatant more efficiently than the untagged ColH. ColH-his exhibited the same hydrolytic activity as ColH against 4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D: -Arg (Pz peptide), a synthetic collagenase substrate. From 100 ml of the culture supernatant, approximately 1 mg of ColH-his was purified by ammonium sulfate precipitation, IMAC, and high-performance liquid chromatography on a MonoQ column. When IMAC was performed on chelating Sepharose charged with Zn(2+) instead of Ni(2+), a potential carcinogenic metal, the specific activities against Pz peptide and type I collagen decreased slightly. However, they were comparable to those reported for other recombinant ColHs and a commercial C. histolyticum collagenase preparation, suggesting that this expression system is useful for large-scale preparation of high-grade clostridial collagenases.     

Cysteine-to-Serine Mutants Dramatically Reorder the Active Site of Human ABO(H) Blood Group B Glycosyltransferase without Affecting Activity: Structural Insights into Cooperative Substrate Binding

A common feature in the structures of GT-A-fold-type glycosyltransferases is a mobile polypeptide loop that has been observed to participate in substrate recognition and enclose the active site upon substrate binding. This is the case for the human ABO(H) blood group B glycosyltransferase GTB, where amino acid residues 177-195 display significantly higher levels of disorder in the unliganded state than in the fully liganded state. Structural studies of mutant enzymes GTB/C80S/C196S and GTB/C80S/C196S/C209S at resolutions ranging from 1.93 to 1.40 Å display the opposite trend, where the unliganded structures show nearly complete ordering of the mobile loop residues that is lost upon substrate binding. In the liganded states of the mutant structures, while the UDP moiety of the donor molecule is observed to bind in the expected location, the galactose moiety is observed to bind in a conformation significantly different from that observed for the wild-type chimeric structures. Although this would be expected to impede catalytic turnover, the kinetics of the transfer reaction are largely unaffected. These structures demonstrate that the enzymes bind the donor in a conformation more similar to the dominant solution rotamer and facilitate its gyration into the catalytically competent form. Further, by preventing active-site closure, these structures provide a basis for recently observed cooperativity in substrate binding. Finally, the mutation of C80S introduces a fully occupied UDP binding site at the enzyme dimer interface that is observed to be dependent on the binding of H antigen acceptor analog.     

Identification of metal ligands in the Clostridium histolyticum ColH collagenase

A Clostridium histolyticum 116-kDa collagenase has an H415EXXH motif but not the third zinc ligand, as found in already characterized zinc metalloproteinases. To identify its catalytic site, we mutated the codons corresponding to the three conserved residues in the motif to other amino acid residues. The mutation affecting His415 or His419 abolished catalytic activity and zinc binding, while that affecting Glu416 did the former but not the latter. These results suggest that the motif forms the catalytic site. We also mutated the codons corresponding to other amino acid residues that are likely zinc ligands. The mutation affecting Glu447 decreased markedly both the enzymatic activity and the zinc content, while that affecting Glu446 or Glu451 had smaller effects on activity and zinc binding. These mutations caused a decrease in kcat but no significant change in Km. These results are consistent with the hypothesis that Glu447 is the third zinc ligand. The spacing of the three zinc ligands is the same in all known clostridial collagenases but not in other known gluzincins, indicating that they form a new gluzincin subfamily. The effects of mutations affecting Glu446 and Glu451 suggest that the two residues are also involved in catalysis, possibly through an interaction with the two zinc-binding histidine residues.     

Molecular dynamics simulations of matrix metalloproteinase 2: role of the structural metal ions

Herein we investigate the role played by the so-called "structural metal ions" in the catalytic domain of the matrix metalloproteinase 2 enzyme (MMP-2 or gelatinase A). We performed seven molecular dynamics simulations that differ in the number and position of the noncatalytic zinc and calcium ions bound to the MMP-2 catalytic domain. An additional simulation including the three fibronectin-type modules inserted into the catalytic domain was also carried out. The analysis of the trajectories confirms that the binding/removal of the structural ions does not perturb the secondary structure elements but influences the position of several solvent-exposed loop regions that are placed near the active site cleft. The position of these loops modulates the accessibility of important anchorage points for substrate binding that have been identified in the active site groove. On the basis of semiempirical quantum chemical calculations, we estimated the relative free energies of the MMP-2 models, obtaining thus that the binding of two zinc and two calcium ions to the MMP-2 catalytic domain is energetically favored. In this MMP-2 model, which shows the most compact structure, all of the substrate binding sites are readily accessible. Globally, our results help to rationalize at the atomic level the calcium and zinc dependence of the hydrolytic activity catalyzed by the MMPs.     

Structural differences of matrix metalloproteinases. Homology modeling and energy minimization of enzyme-substrate complexes

Matrix metalloproteinases are extracellular enzymes taking part in the remodeling of extracellular matrix. The structures of the catalytic domain of MMP1, MMP3, MMP7 and MMP8 are known, but structures of enzymes belonging to this family still remain to be determined. A general approach to the homology modeling of matrix metalloproteinases, exemplified by the modeling of MMP2, MMP9, MMP12 and MMP14 is described. The models were refined using an energy minimization procedure developed for matrix metalloproteinases. This procedure includes incorporation of parameters for zinc and calcium ions in the AMBER 4.1 force field, applying a non-bonded approach and a full ion charge representation. Energy minimization of the apoenzymes yielded structures with distorted active sites, while reliable three-dimensional structures of the enzymes containing a substrate in active site were obtained. The structural differences between the eight enzyme-substrate complexes were studied with particular emphasis on the active site, and possible sites for obtaining selectivity among the MMP's are discussed. Differences in the P1' pocket are well-documented and have been extensively exploited in inhibitor design. The present work indicates that selectivity could be further improved by considering the P2 pocket as well.     

Purification and properties of individual collagenases fromStreptomyces sp. strain 3B

Streptomyces strain 3B constitutively secreted collagenolytic enzymes during the post-exponential growth phase. Purification is described here leading to two collagenases (I and II) with specific activity of 3350 and 3600 U/mg, respectively, the highest activity obtained as yet for any streptomycete collagenase. Analysis of the purified enzymes by the method of zymography revealed that both I and II were homogeneous, with molar mass 116 and 97 kDa, respectively. Both collagenases were identical in their pH (7.5) and temperature optimum (37 degrees C). The inhibition profile of the enzymes by EDTA and 1,10-phenanthroline confirmed these enzymes to be metalloproteinases. By testing the activity with insoluble collagen, acid soluble collagen, gelatin, casein, elastin and Pz-PLGPR it was established that I and II are very specific for insoluble collagen and gelatin, showing a high activity toward acid soluble collagen and Pz-PLGPR. However, collagenases I and II failed to hydrolyze casein and elastin; they belong to true collagenases and resemble the clostridial enzymes.     

The X-ray crystal structure of the catalytic domain of human neutrophil collagenase inhibited by a substrate analogue reveals the essentials for catalysis and specificity.

Matrix metalloproteinases are a family of zinc endopeptidases involved in tissue remodelling. They have been implicated in various disease processes including tumour invasion and joint destruction. These enzymes consist of several domains, which are responsible for latency, catalysis and substrate recognition. Human neutrophil collagenase (PMNL-CL, MMP-8) represents one of the two 'interstitial' collagenases that cleave triple helical collagens types I, II and III. Its 163 residue catalytic domain (Met80 to Gly242) has been expressed in Escherichia coli and crystallized as a non-covalent complex with the inhibitor Pro-Leu-Gly-hydroxylamine. The 2.0 A crystal structure reveals a spherical molecule with a shallow active-site cleft separating a smaller C-terminal subdomain from a bigger N-terminal domain, composed of a five-stranded beta-sheet, two alpha-helices, and bridging loops. The inhibitor mimics the unprimed (P1-P3) residues of a substrate; primed (P1'-P3') peptide substrate residues should bind in an extended conformation, with the bulky P1' side-chain fitting into the deep hydrophobic S1' subsite. Modelling experiments with collagen show that the scissile strand of triple-helical collagen must be freed to fit the subsites. The catalytic zinc ion is situated at the bottom of the active-site cleft and is penta-coordinated by three histidines and by both hydroxamic acid oxygens of the inhibitor. In addition to the catalytic zinc, the catalytic domain harbours a second, non-exchangeable zinc ion and two calcium ions, which are packed against the top of the beta-sheet and presumably function to stabilize the catalytic domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nereis cuticle collagen. Proteolysis by marine vibrial and clostridial collagenases

Proteolysis of Nereis cuticle collagen by two bacterial collagenases was investigated using viscosimetry, enzyme kinetics, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and ion exchange chromatography of collagenolytic peptides. Collagenase of the marine Vibrio B-30 completely degrades native cuticle collagen at 7 degress C with a turnover number 50 times greater than that of the clostridial collagenase. Although turnover numbers for the two enzymes are comparable when using denatured cuticle collagen as substrate, the vibrial collagenase appears to cleave twice as many peptide bonds per mg of cuticle collagen as does the clostridial enzyme. Sodium dodecyl sulfate gel electrophoresis of collagenase-digested native cuticle collagen reflects the resistance of the collagen to clostridial collagenase; however, the vibrial enzyme completely degrades the cuticle collagen with the formation of one transient intermediate (Mr 400,000). Peptide analysis of fully digested denatured cuticle collagen reveals that the two enzymes have a number of qualitative and quantitative similarities. Despite these, however, only the vibrial collagenase seems capable of extensively degrading native cuticle collagen.     

Structural and computational investigations of VIM-7: insights into the substrate specificity of vim metallo-β-lactamases

The presence of metallo-β-lactamases (MBLs) in many clinically important human bacterial pathogens limits treatment options, as these enzymes efficiently hydrolyze nearly all β-lactam antibiotics. VIM enzymes are among the most widely distributed MBLs, but many of the individual VIM subtypes remain poorly characterized. Pseudomonas aeruginosa VIM-7 is the most divergent among VIM-type MBLs in terms of amino acid sequence. Here we present crystal structures of VIM-7 as the native enzyme, with Cys221 oxidized (VIM-7-Ox), and with a sulfur atom bridging the two active-site zinc ions (VIM-7-S). Comparison with VIM-2 and VIM-4 structures suggests an explanation for the reduced catalytic efficiency of VIM-7 against cephalosporins with a positively charged cyclic substituent at the C3 position (e.g., ceftazidime). Kinetic variations are attributed to substitutions in residues 60-66 (that form a loop adjacent to the active site previously implicated in substrate binding) and to the disruption of two hydrogen-bonding clusters through substitutions at positions 218 and 224. Furthermore, the less negatively charged surface of VIM-7 (compared to VIM-2) may also contribute to the reduced hydrolytic efficiency. Docking of the cephalosporins ceftazidime and cefotaxime into the VIM-2 and VIM-7 structures reveals that amino acid substitutions may cause the mode of substrate binding to differ between the two enzymes. Our structures thus provide new insights into the variation in substrate specificity that is evident across this family of clinically important enzymes.     

Unusual commonality in active site structural features of substrate promiscuous and specialist enzymes

Enzyme promiscuity is the ability of (some) enzymes to perform alternate reactions or catalyze non-cognate substrate(s). The latter is referred to as substrate promiscuity, widely studied for its biotechnological applications and understanding enzyme evolution. Insights into the structural basis of substrate promiscuity would greatly benefit the design and engineering of enzymes. Previous studies on some enzymes have suggested that flexibility, hydrophobicity, and active site protonation state could play an important role in enzyme promiscuity. However, it is not known yet whether substrate promiscuous enzymes have distinctive structural characteristics compared to specialist enzymes, which are specific for a substrate. In pursuit to address this, we have systematically compared substrate/catalytic binding site structural features of substrate promiscuous with those of specialist enzymes. For this, we have carefully constructed dataset of substrate promiscuous and specialist enzymes. On careful analysis, surprisingly, we found that substrate promiscuous and specialist enzymes are similar in various binding/catalytic site structural features such as flexibility, surface area, hydrophobicity, depth, and secondary structures. Recent studies have also alluded that promiscuity is widespread among enzymes. Based on these observations, we propose that substrate promiscuity could be defined as a continuum feature that varies from narrow (specialist) to broad range of substrate preferences. Moreover, diversity of conformational states of an enzyme accessible for ligand binding may possibly regulate its substrate preferences.     

Crystal Structure of Serratia fonticola Sfh-I: Activation of the Nucleophile in Mono-Zinc Metallo-β-Lactamases

Metallo-β-lactamases (MBLs) or class B β-lactamases are zinc-dependent enzymes capable of inactivating almost all classes of β-lactam antibiotics. To date, no MBL inhibitors are available for clinical use. Of the three MBL subclasses, B2 enzymes, unlike those from subclasses B1 and B3, are fully active with one zinc ion bound and possess a narrow spectrum of activity, hydrolyzing carbapenem substrates almost exclusively. These remain the least studied MBLs. Sfh-I, originally identified from the aquatic bacterium Serratia fonticola UTAD54, is a divergent member of this group. Previous B2 MBL structures, available only for the CphA enzyme from Aeromonas hydrophila, all contain small molecules bound in their active sites. In consequence, the mechanism by which these enzymes activate the water nucleophile required for β-lactam hydrolysis remains to be unambiguously established. Here we report crystal structures of Sfh-I as a complex with glycerol and in the unliganded form, revealing for the first time the disposition of water molecules in the B2 MBL active site. Our data indicate that the hydrolytic water molecule is activated by His118 rather than by Asp120 and/or zinc. Consistent with this proposal, we show that the environment of His118 in B2 MBLs is distinct from that of the B1 and B3 enzymes, where this residue acts as a zinc ligand, and offer a structure-based mechanism for β-lactam hydrolysis by these enzymes.     

Crystal structure of Toxoplasma gondii porphobilinogen synthase: insights on octameric structure and porphobilinogen formation

Porphobilinogen synthase (PBGS) is essential for heme biosynthesis, but the enzyme of the protozoan parasite Toxoplasma gondii (TgPBGS) differs from that of its human host in several important respects, including subcellular localization, metal ion dependence, and quaternary structural dynamics. We have solved the crystal structure of TgPBGS, which contains an octamer in the crystallographic asymmetric unit. Crystallized in the presence of substrate, each active site contains one molecule of the product porphobilinogen. Unlike prior structures containing a substrate-derived heterocycle directly bound to an active site zinc ion, the product-bound TgPBGS active site contains neither zinc nor magnesium, placing in question the common notion that all PBGS enzymes require an active site metal ion. Unlike human PBGS, the TgPBGS octamer contains magnesium ions at the intersections between pro-octamer dimers, which are presumed to function in allosteric regulation. TgPBGS includes N- and C-terminal regions that differ considerably from previously solved crystal structures. In particular, the C-terminal extension found in all apicomplexan PBGS enzymes forms an intersubunit β-sheet, stabilizing a pro-octamer dimer and preventing formation of hexamers that can form in human PBGS. The TgPBGS structure suggests strategies for the development of parasite-selective PBGS inhibitors.     

The isolation and properties of collagenolytic proteases from crab hepatopancreas

A mixture of collagenolytic proteases has been isolated from the Kamchatka crab hepatopancreas. The four individual enzymes were further separated with FPLC and partially characterized. Crab collagenolytic proteases possess a high activity against different types of collagen, especially against calf skin collagen Type III and bovine lens capsule collagen Type IV, which is resistant to the microbial Clostridium sp. collagenases. In contrast with microbial collagenases the crab enzymes are good general proteases, able to cleave standard synthetic and protein substrates and possess a chymotrypsin-, trypsin- and elastase-like specificity. N-Terminal sequence analysis revealed that crab collagenolytic proteases had evolved from a trypsin-like ancestor. Crab proteases, structurally belonging to the trypsin-like enzymes, nevertheless, possess the unique ability, among this class of enzymes, to cleave the native insoluble collagen. It seems that crab collagenolytic proteases and true metalloenzyme vertebrate and microbial collagenases have certain common structural features particularly in the regions of their substrate binding site.     

Crystal structure of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli.

Crystal structures of glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase from Escherichia coli have been determined to 2.0-A resolution in the absence of ligands, and to 2.5-A resolution with the feedback inhibitor AMP bound to the PRPP catalytic site. Glutamine PRPP amidotransferase (GPATase) employs separate catalytic domains to abstract nitrogen from the amide of glutamine and to transfer nitrogen to the acceptor substrate PRPP. The unliganded and AMP-bound structures, which are essentially identical, are interpreted as the inhibited form of the enzyme because the two active sites are disconnected and the PRPP active site is solvent exposed. The structures were compared with a previously reported 3.0-A structure of the homologous Bacillus subtilis enzyme (Smith JL et al., 1994, Science 264:1427-1433). The comparison indicates a pattern of conservation of peptide structures involved with catalysis and variability in enzyme regulatory functions. Control of glutaminase activity, communication between the active sites, and regulation by feedback inhibitors are addressed differently by E. coli and B. subtilis GPATases. The E. coli enzyme is a prototype for the metal-free GPATases, whereas the B. subtilis enzyme represents the metal-containing enzymes. The structure of the E. coli enzyme suggests that a common ancestor of the two enzyme subfamilies may have included an Fe-S cluster.     

Structure- and substrate-based inhibitor design for Clostridium botulinum neurotoxin serotype A

The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins cleave specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex proteins and block the release of neurotransmitters that cause flaccid paralysis and are considered potential bioweapons. Botulinum neurotoxin type A is the most potent among the clostridial neurotoxins, and to date there is no post-exposure therapeutic intervention available. To develop inhibitors leading to drug design, it is imperative that critical interactions between the enzyme and the substrate near the active site are known. Although enzyme-substrate interactions at exosites away from the active site are mapped in detail for botulinum neurotoxin type A, information about the active site interactions is lacking. Here, we present the crystal structures of botulinum neurotoxin type A catalytic domain in complex with four inhibitory substrate analog tetrapeptides, viz. RRGC, RRGL, RRGI, and RRGM at resolutions of 1.6-1.8 A. These structures show for the first time the interactions between the substrate and enzyme at the active site and delineate residues important for substrate stabilization and catalytic activity. We show that OH of Tyr(366) and NH(2) of Arg(363) are hydrogen-bonded to carbonyl oxygens of P1 and P1' of the substrate analog and position it for catalytic activity. Most importantly, the nucleophilic water is replaced by the amino group of the N-terminal residue of the tetrapeptide. Furthermore, the S1' site is formed by Phe(194), Thr(215), Thr(220), Asp(370), and Arg(363). The K(i) of the best inhibitory tetrapeptide is 157 nm.     

Limited proteolysis of type I collagen at hyperreactive sites by class I and II Clostridium histolyticum collagenases: complementary digestion patterns

The initial proteolytic events in the hydrolysis of rat tendon type I collagen by the class I and II collagenases from Clostridium histolyticum have been investigated at 15 degrees C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been used to detect the initial cleavage fragments of both the alpha 1(I) and alpha 2 chains, which migrate at different rates in the buffer system employed. Experiments with the class I collagenases indicate that the first cleavage occurs across all three chains of the triple helix close to the C-terminus to produce fragments whose alpha chains have molecular weights of approximately 88,000. The second cleavage occurs near the N-terminus to reduce the molecular weight of the alpha chains to 80,000. Initial proteolysis by the class II collagenases occurs across all three chains at a site in the interior of the collagen triple helix to give N- and C-terminal fragments with alpha-chain molecular weights of 35,000 and 62,000, respectively. The C-terminal fragment is subsequently cleaved to give fragments with alpha-chain molecular weights of 59,000. These results indicate that type I collagen is degraded at several hyperreactive sites by these enzymes. Thus, initial proteolysis by these bacterial collagenases occurs at specific sites, much like the mammalian collagenases. These results with the individual clostridial collagenases provide an explanation for earlier data which indicated that collagen is degraded sequentially from the ends by a crude clostridial collagenase preparation.     

Insights into the anthrax lethal factor–substrate interaction and selectivity using docking and molecular dynamics simulations

The anthrax toxin of the bacterium Bacillus anthracis consists of three distinct proteins, one of which is the anthrax lethal factor (LF). LF is a gluzincin Zn‐dependent, highly specific metalloprotease with a molecular mass of ～90 kDa that cleaves most isoforms of the family of mitogen‐activated protein kinase kinases (MEKs/MKKs) close to their amino termini, resulting in the inhibition of one or more signaling pathways. Previous studies on the crystal structures of uncomplexed LF and LF complexed with the substrate MEK2 or a MKK‐based synthetic peptide provided structure‐activity correlations and the basis for the rational design of efficient inhibitors. However, in the crystallographic structures, the substrate peptide was not properly oriented in the active site because of the absence of the catalytic zinc atom. In the current study, docking and molecular dynamics calculations were employed to examine the LF‐MEK/MKK interaction along the catalytic channel up to a distance of 20 Å from the zinc atom. This residue‐specific view of the enzyme‐substrate interaction provides valuable information about: (i) the substrate selectivity of LF and its inactivation of MEKs/MKKs (an issue highly important not only to anthrax infection but also to the pathogenesis of cancer), and (ii) the discovery of new, previously unexploited, hot‐spots of the LF catalytic channel that are important in the enzyme/substrate binding and interaction.     

Signaling Domain of Sonic Hedgehog as Cannibalistic Calcium-Regulated Zinc-Peptidase

Sonic Hedgehog (Shh) is a representative of the evolutionary closely related class of Hedgehog proteins that have essential signaling functions in animal development. The N-terminal domain (ShhN) is also assigned to the group of LAS proteins (LAS = Lysostaphin type enzymes, D-Ala-D-Ala metalloproteases, Sonic Hedgehog), of which all members harbor a structurally well-defined center; however, it is remarkable that ShhN so far is the only LAS member without proven peptidase activity. Another unique feature of ShhN in the LAS group is a double- center close to the zinc. We have studied the effect of these calcium ions on ShhN structure, dynamics, and interactions. We find that the presence of calcium has a marked impact on ShhN properties, with the two calcium ions having different effects. The more strongly bound calcium ion significantly stabilizes the overall structure. Surprisingly, the binding of the second calcium ion switches the putative catalytic center from a state similar to LAS enzymes to a state that probably is catalytically inactive. We describe in detail the mechanics of the switch, including the effect on substrate co-ordinating residues and on the putative catalytic water molecule. The properties of the putative substrate binding site suggest that ShhN could degrade other ShhN molecules, e.g. by cleavage at highly conserved glycines in ShhN. To test experimentally the stability of ShhN against autodegradation, we compare two ShhN mutants in vitro: (1) a ShhN mutant unable to bind calcium but with putative catalytic center intact, and thus, according to our hypothesis, a constitutively active peptidase, and (2) a mutant carrying additionally mutation E177A, i.e., with the putative catalytically active residue knocked out. The in vitro results are consistent with ShhN being a cannibalistic zinc-peptidase. These experiments also reveal that the peptidase activity depends on .     

Brachyurins, Serine Collagenolytic Enzymes from Crabs

The properties of brachyurins, proteolytic enzymes belonging to a new subfamily of chymotrypsin-like proteases, are considered. These enzymes, found in various species of crustacean, exhibit mixed substrate specificity and a marked collagenolytic activity. The enzymatic and physicochemical properties of brachyurins I and their primary and spatial structures are discussed in detail. A separate chapter is devoted to the preparations of collagenases from the hepatopancreas of king crab: their action on the damaged skin and use in medicine.