Distribution of chymoelastases and trypsin-like enzymes in five species of entomopathogenic deuteromycetes

Nine isolates of the entomopathogenic deuteromycetes Metarhizium anisopliae, Beauveria bassiana, Verticillium lecanii, Nomuraea rileyi, and Aschersonia aleyrodis produced basic (pI greater than 7.0) chymoelastases that possessed extended binding sites, comprising at least four or five subsites, with preference for hydrophobic residues at the primary binding site. Most isolates also produced additional acidic enzymes with similar specificities against ester and amide substrates but which lacked activity against elastin. Both acidic and basic enzymes degraded high protein azure or locust cuticle and, as shown by inhibition studies, possessed essential serine and histidine residues in the active site. In spite of similarities in catalytic properties antibodies generated against a Metarhizium chymoelastase cross-reacted only with enzymes from two (out of four) Metarhizium isolates; enzymes from all other isolates did not cross-react. Two isolates of Metarhizium produced a third class of protease which degraded Bz-AA-AA-Arg-NA substrates (AA, various amino acids) and hide protein azure. Analogous peptidases were produced by other isolates but they were specific for Bz-Phe-Val-Arg-NA and showed less sensitivity to trypsin inhibitors. The possible significance to pathology of the presence of diverse yet similar protease forms in five genera of entomopathogens is discussed.     

球孢白僵菌凝乳弹性蛋白酶(BbPr1)的纯化与特性

以几丁质为底物,加入基本盐培养基中,诱导球孢白僵菌(Beauveria     

球孢白僵菌凝乳弹性蛋白酶(BbPr1)的纯化与特性*

以几丁质为底物,加入基本盐培养基中,诱导球孢白僵菌（Beauveriabassiana）产生分解昆虫表皮的蛋白酶。诱导物中,蝉蜕诱导的蛋白酶总活性、比活较高,经超滤、离子交换层析、亲和层析、制备性IEF电洗脱纯化了一种有凝乳弹性蛋白酶（Pr1）活性的蛋白酶BbPr1。经SDS－PAGE电泳银染后是单带,HPLC凝胶过滤显示单峰。BbPr1为单体酶,分子量为33.6kD左右,pI为7.4。底物专一性测定显示,BbPr1能水解Phe或Leu形成的酸胺键和肽键。BbPrl可被PMSF抑制,表明其活性中心有Ser残基;BbPr1还可被胰凝乳蛋白酶抑制剂TPCK和凝乳弹性蛋白酶抑制剂TEI等所抑制;胰蛋白酶抑制剂beupeptin和Epianstatin,及胃蛋白酶抑制剂Pepstain对BbPr1活性无影响。还研究了BbPr1的最适作用pH和pH稳定性。     

Comparative substrate specificity analysis of recombinant human cathepsin V and cathepsin L

Cathepsins V and L have high identity and few structural differences. In this paper, we reported a comparative study of the hydrolytic activities of recombinant human cathepsins V and L using fluorescence resonance energy transfer peptides derived from Abz-KLRSSKQ-EDDnp (Abz = ortho-aminobenzoic acid and EDDnp = N-(2,4-dinitrophenyl)ethylenediamine). Five series of peptides were synthesized to map the S3 to S2' subsites. The cathepsin V subsites S1 and S3 present a broad specificity while cathepsin L has preference for positively charged residues. The S2 subsites of both enzymes require hydrophobic residues with preference for Phe and Leu. The S1' and S2' subsites of cathepsins V and L are less specific. Based on these data we designed substrates to explore the electrostatic potential differences of them. Finally, the kininogenase activities of these cathepsins were compared using synthetic human kininogen fragments. Cathepsin V preferentially released Lys-bradykinin while cathepsin L released bradykinin. This kininogenase activity by cathepsins V and L was also observed from human high and low molecular weight kininogens.     

Purification and characterization of major extracellular proteinases from Trichophyton rubrum.

Two extracellular proteinases that probably play a central role in the metabolism and pathogenesis of the most common dermatophyte of man, Trichophyton rubrum, were purified to homogeneity. Size-exclusion chromatography and Chromatofocusing were used to purify the major proteinases 42-fold from crude fungal culture filtrate. The major enzyme has pI 7.8 and subunit Mr 44 000, but forms a dimer of Mr approx. 90 000 in the absence of reducing agents. A second enzyme with pI 6.5 and subunit Mr 36 000, was also purified. It is very similar in substrate specificity to the major enzyme but has lower specific activity, and may be an autoproteolysis product. The major proteinase has pH optimum 8, a Ca2+-dependence maximum of 1 mM, and was inhibited by serine-proteinase inhibitors, especially tetrapeptidyl chloromethane derivatives with hydrophobic residues at the P-1 site. Kinetic studies also showed that tetrapeptides containing aromatic or hydrophobic residues at P-1 were the best substrates. A kcat./Km of 27 000 M-1 X S-1 was calculated for the peptide 3-carboxypropionyl-Ala-Ala-Pro-Phe-p-nitroanilide. The enzyme has significant activity against keratin, elastin and denatured type I collagen (Azocoll).     

Biochemical characterization and ultrastructural localization of two extracellular trypsins produced by Metarhizium anisopliae in infected insect cuticles.

Proteinase 2 (Pr2) is a fungal (Metarhizium anisopliae) serine proteinase which has a tryptic specificity for basic residues and which may be involved in entomopathogenicity. Analytical and preparative isoelectric focusing methods were used to separate two trypsin components, produced during growth on cockroach cuticle, with isoelectric points of 4.4 (molecular mass, 30 kDa) and 4.9 (27 kDa). The catalytic properties of the proteases were analyzed by their kinetic constants and by a combination of two-dimensional gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme overlay membranes. Both Pr2 isoforms preferentially cleave at the carboxyl sides of positively charged amino acids, preferring arginine; the pI 4.4 Pr2 isoform also possessed significant activity against lysine. Compared with the pathogen's subtilisin-like enzyme (Pr1), the pI 4.4 Pr2 isoform shows low activity against insoluble proteins in a host (Manduca sexta) cuticle. However, it degrades most cuticle proteins when they are solubilized, with high-molecular-weight basic proteins being preferentially hydrolyzed. Polyclonal antibodies raised against each Pr2 isoform were isotype specific. This allowed us to use ultrastructural immunocytochemistry to independently visualize each isoform during penetration of the host (M. sexta) cuticle. Both isoforms were secreted by infection structures (appressoria) on the cuticle surface and by the penetrant hyphae within the cuticle. The extracellular sheath, which is commonly observed around fungal cells, often contained Pr2 molecules. Intracellular labelling was sparse.     

Purification and characterization of three beta-glycosidases from midgut of the sugar cane borer,Diatraea saccharalis

Three beta-glycosidases, named betaGly1, betaGly2 and betaGly3, were isolated from midgut tissues of the sugar cane borer, Diatraea saccharalis Fabricius (Lepidoptera: Pyralidae). The three enzymes have similar Mr (58,000; 61,000; 61,000), pI (7.5, 7.4, and 7.4) and optimum pH (6.7, 6.3, and 7.2) and were resolved by hydrophobic chromatography. The beta-glycosidases prefer beta-glucosides to beta-galactosides, have four subsites for glucose binding and hydrolyse glucose-glucose beta-1,3 linkages better than beta-1, 4- or beta-1,6 linkages. betaGly1 and 2 were completely purified, whereas betaGly3 was isolated with a contaminant peptide that has no activity upon beta-glycosides.By using competing substrates, it was shown that betaGly 1 and 3 have one active site, whereas betaGly2 has two, one hydrolyzing natural and the other synthetic substrates. betaGly2 is the only D. saccharalis beta-glycosidase that can efficiently hydrolyse prunasin, the glycoside remaining after glucose removal from the plant glycoside amygdalin and that liberates the cyanogenic mandelonitrile. As shown elsewhere, betaGly2 activity is reduced when D. saccharalis is reared in amygdalin containing diets. From the results, we propose that the physiological role of betaGly 1 and 3 is the digestion of oligo- and disaccharides derived from hemicelluloses and of betaGly2 is glycolipid hydrolysis.Free energy relationships showed that D. saccharalis betaGly3 and Tenebrio molitor (Coleoptera) betaGly1 have active sites that bind similarly the transition states formed with different substrates. The same is also true for the active sites of D. saccharalis betaGly1 and T. molitor betaGly2. This suggests that active sites of similar enzymes are probably homologous, displaying nearly identical bonds between active site amino acids and substrate moieties.     

Molecular characterization of BjussuSP-I, a new thrombin-like enzyme with procoagulant and kallikrein-like activity isolated from Bothrops jararacussu snake venom

A thrombin-like enzyme, named BjussuSP-I, isolated from Bothrops jararacussu snake venom, is an acidic single-chain glycoprotein with M(r)=61,000, pI approximately 3.8 and 6% sugar. BjussuSP-I shows high proteolytic activity upon synthetic substrates, such as S-2238 and S-2288. It also shows procoagulant and kallikrein-like activity, but is unable to act on platelets and plasmin. These activities are inhibited by specific inhibitors of this class of enzymes. The complete cDNA sequence of BjussuSP-I with 696bp encodes open reading frames of 232 amino acid residues, which conserve the common domains of thrombin-like serine proteases. BjussuSP-I shows a high structural homology with other thrombin-like enzymes from snake venoms where common amino acid residues are identified as those corresponding to the catalytic site and subsites S1, S2 and S3 already reported. In this study, we also demonstrated the importance of N-linked glycans to improve thrombin-like activity of BjussuSP-I toxin.     

Influence of charge distribution at the active site surface on the substrate specificity of human neutrophil protease 3 and elastase. A kinetic and molecular modeling analysis

The biological functions of human neutrophil protease 3 (Pr3) differ from those of neutrophil elastase despite their close structural and functional resemblance. Although both proteases are strongly cationic, their sequences differ mainly in the distribution of charged residues. We have used these differences in electrostatic surface potential in the vicinity of their active site to produce fluorescence resonance energy transfer (FRET) peptide substrates for investigating individual Pr3 subsites. The specificities of subsites S5 to S3' were investigated both kinetically and by molecular dynamic simulations. Subsites S2, S1', and S2' were the main definers of Pr3 specificity. Combinations of results for each subsite were used to deduce a consensus sequence that was complementary to the extended Pr3 active site and was not recognized by elastase. Similar sequences were identified in natural protein substrates such as NFkappaB and p21 that are specifically cleaved by Pr3. FRET peptides derived from these natural sequences were specifically hydrolyzed by Pr3 with specificity constants k(cat)/K(m) in the 10(6) m(-1) s(-1) range. The consensus Pr3 sequence may also be used to predict cleavage sites within putative protein targets like the proform of interleukin-18, or to develop specific Pr3 peptide-derived inhibitors, because none is available for further studies on the physiopathological function of this protease.     

Complementary substrate specificities of class I and class II collagenases from Clostridium histolyticum

The substrate specificities of three class I (beta, gamma, and eta) and three class II (sigma, epsilon, and zeta) collagenases from Clostridium histolyticum have been investigated by quantitating the kcat/KM values for the hydrolysis of 53 synthetic peptides with collagen-like sequences covering the P3 through P3 subsites of the substrate. For both classes of collagenases, there is a strong preference for Gly in subsites P1' and P3. All six enzymes also prefer substrates that contain Pro and Ala in subsites P2 and P2' and Hyp, Ala, or Arg in subsite P3'. This agrees well with the occupancies of these sites by these residues in type I collagen. However, peptides with Glu in subsites P2 or P2' are not good substrates, even though Glu occurs frequently in these positions in collagen. Conversely, all six enzymes prefer aromatic amino acids in subsite P1, even though such residues do not occur in this position in type I collagen. In general, the class II enzymes have a broader specificity than the class I enzymes. However, they are much less active toward sequences containing Hyp in subsites P1 and P3'. Thus, the two classes of collagenases have similar but complementary sequence specificities. This accounts for the ability of the two classes of enzymes to synergistically digest collagen.     

绿僵菌分解昆虫外壳蛋白酶MAP-21的纯化与特性

以蝉蜕为底物诱导绿僵菌产生分解昆虫外壳蛋白酶 。发酵液经超滤、Ultrogel AcA 54凝胶层析、制备IEF电泳,纯化了一种蛋白酶MAP-21,SDS-PAGE电泳后经银染色呈单带。该酶的Mr为27kD左右,pI为76。它的特异识别氨基酸为Arg,其活性可被PMSF和TLCK抑制,表明其活性中心有Ser和His残基。它还可被胰蛋白酶的典型抑制剂Leupeptin、Antipain及STI等所抑制,而胰凝乳蛋白酶抑制剂TPCK和胰凝乳弹性蛋白酶抑制剂TEI对其活性无影响。专一底物和抑制剂特性试验结果表明MAP-21是类胰蛋白酶。此外,该酶还可被EDTA所抑制,表明金属离子为其活性所必需。另外还研究了MAP-21的最适作用温度和pH,以及温度耐受性等特性。     

Structural basis for inhibition promiscuity of dual specific thrombin and factor Xa blood coagulation inhibitors

BACKGROUND: A major current focus of pharmaceutical research is the development of selective inhibitors of the blood coagulation enzymes thrombin or factor Xa to be used as orally bioavailable anticoagulant drugs in thromboembolic disorders and in the prevention of venous and arterial thrombosis. Simultaneous direct inhibition of thrombin and factor Xa by synthetic proteinase inhibitors as a novel approach to antithrombotic therapy could result in potent anticoagulants with improved pharmacological properties. RESULTS: The binding mode of such dual specific inhibitors of thrombin and factor Xa was determined for the first time by comparative crystallography using human alpha-thrombin, human des-Gla (1--44) factor Xa and bovine trypsin as the ligand receptors. The benzamidine-based inhibitors utilize two different conformations for the interaction with thrombin and factor Xa/trypsin, which are evoked by the steric requirements of the topologically different S2 subsites of the enzymes. Compared to the unliganded forms of the proteinases, ligand binding induces conformational adjustments of thrombin and factor Xa active site residues indicative of a pronounced induced fit mechanism. CONCLUSION: The structural data reveal the molecular basis for a desired unselective inhibition of the two key components of the blood coagulation cascade. The 4-(1-methyl-benzimidazole-2-yl)-methylamino-benzamidine moieties of the inhibitors are able to fill both the small solvent accessible as well as the larger hydrophobic S2 pockets of factor Xa and thrombin, respectively. Distal fragments of the inhibitors are identified which fit into both the cation hole/aromatic box of factor Xa and the hydrophobic aryl binding site of thrombin. Thus, binding constants in the medium-to-low nanomolar range are obtained against both enzymes.     

Investigation of the active site of human salivary alpha-amylase from the modes of action on modified maltooligosaccharides

The modes of action of two isozymes of human salivary alpha-amylase on phenyl alpha-maltopentaoside, phenyl alpha-maltotetraoside, and their derivatives which have an iodo or an amino or a carboxyl group at their first or penultimate glucopyranosyl residues from the non-reducing-end were examined. It is conceivable that the active site of this enzyme is composed of tandem subsites (S4,S3,S2,S1,S1',S2', and S3') geometrically complementary to several glucose residues, and that the glucosidic bonds of the substrates are split between S1 and S1'. Product analysis of each digest strongly suggested the presence of a hydrophobic amino acid residue at subsite S3 in the active site of the enzyme. No difference in the modes of action on the substrates was found between the two isozymes, indicating that the three-dimensional structures of their active site areas are, at the least, similar.     

Characterization of a beta-glycosidase highly active on disaccharides and of a beta-galactosidase from Tenebrio molitor midgut lumen

The midgut of the yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae) larvae has four beta-glycosidases. The properties of two of these enzymes (betaGly1 and betaGly2) have been described elsewhere. In this paper, the characterization of the other two glycosidases (betaGly3 and betaGly4) is described. BetaGly3 has one active site, hydrolyzes disaccharides, cellodextrins, synthetic substrates and beta-glucosides produced by plants. The enzyme is inhibited by amygdalin, cellotriose, cellotetraose and cellopentaose in high concentrations, probably due to transglycosylation. betaGly3 hydrolyzes beta 1,4-glycosidic linkages with a catalytic rate independent of the substrate polymerization degree (k(int)) of 11.9 s(-1). Its active site is formed by four subsites, where subsites +1 and -1 bind glucose residues with higher affinity than subsite +2. The main role of betaGly3 seems to be disaccharide hydrolysis. BetaGly4 is a beta-galactosidase, since it has highest activity against beta-galactosides. It can also hydrolyze fucosides, but not glucosides, and has Triton X-100 as a non-essential activator (K(a)=15 microM, pH 4.5). betaGly4 has two active sites that can hydrolyze p-nitrophenyl beta-galactoside (NPbetaGal). The one hydrolyzing NPbetaGal with more efficiency is also active against methylumbellipheryl beta-D-galactoside and lactose. The other active site hydrolyzes NPbetaFucoside and binds NPbetaGal weakly. BetaGly4 hydrolyzes hydrophobic substrates with high catalytical efficiency and is able to bind octyl-beta-thiogalactoside in its active site with high affinity. The betaGly4 physiological role is supposed to be the hydrolysis of galactolipids that are found in membranes from vegetal tissues. As the enzyme has a hydrophobic site where Triton X-100 can bind, it might be activated by membrane lipids, thus becoming fully active only at the surface of cell membranes.     

Protein products of the rat kallikrein gene family. Substrate specificities of kallikrein rK2 (tonin) and kallikrein rK9.

Two closely related kallikrein-like proteinases having little activity toward the standard synthetic amide substrates of tissue kallikreins were isolated from the rat submandibular gland. They were found to be the protein products of the rKlk2 (tonin) and the rKlk9 genes by amino acid sequence analysis (nomenclature of the genes and proteins of the kallikrein family is according to the proposal of the discussion panel from the participants of the KININ '91 meeting held Sept. 8-14, 1991, in Munich, Germany). These two proteinases of similar structure also had very similar physicochemical properties. They differed from other kallikrein-related proteinases in having high pHi values of 6.20 (rK2) and 6.85 (rK9). Kallikrein rK2 was purified as a single peptide chain, whereas rK9 appeared as a two-chain protein after reduction. Their enzymatic properties were also very similar and differed significantly from those of other rat kallikrein-related proteinases. Unlike the five other kallikrein-related proteinases we have purified so far, kallikrein rK9 was not inhibited by aprotinin. rK9 also differed from rK2 by its tissue localization. The prostate gland contained only rK9 where it was the major kallikrein-like component. The amino acids preferentially accommodated by the proteinase S3 to S2' subsites were identified using synthetic amide and protein substrates. Unlike other kallikrein-related proteinases, rK2 had a prevalent chymotrypsin-like specificity, whereas rK9 had both chymotrypsin-like and trypsin-like properties. Both rK2 and rK9 preferred a prolyl residue in position P2 of the substrate and did not accommodate bulky and hydrophobic residues at that position, as did most of the other kallikrein-related proteinases. This P2-proline-directed specificity is necessary for processing the precursors of several biologically active peptides. Subsites accommodating residues COOH-terminal to the scissile bond were also important in determining the overall substrate specificity of these proteinases. rK2 and rK9 both showed a preference for hydrophobic residues in P2'. Other subsites upstream of the S3 subsite were found to intervene in substrate binding and hydrolysis. The restricted specificity of rK2 and rK9 is consistent with the presence of an extended substrate binding site, and hence with a processing enzyme function. Their P1 specificities enabled both proteinases to release angiotensin II from angiotensinogen and from angiotensinogen I, but rK9 was at least 100 times less active than rK2 on both substrates. The substrate specificities of rK2 and rK9 were correlated with key amino acids defining their substrate binding site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and characterization of an elastinolytic proteinase secreted by cercariae of Schistosoma mansoni

An elastinolytic proteinase secreted by tissue-invasive larvae of Schistosoma mansoni has been purified to homogeneity. Size-exclusion chromatography and chromatofocusing were used to purify the enzyme 18-fold from crude larval secretions. The native enzyme has a molecular weight of 30,000, a pI of 8, a pH optimum of 9, and a calcium dependence of 2 mM. A second Mr 17,000 form of the enzyme was present in crude secretions and appears to be an autoproteolysis product. The enzyme is a serine proteinase that preferentially binds tetrapeptide inhibitors or substrates with an aromatic or hydrophobic residue at the P-1 site. In addition to being active against elastin, the enzyme degrades Azocoll, gelatin, laminin, fibronectin, keratin, and type IV collagen.     

Digestive peptidase evolution in holometabolous insects led to a divergent group of enzymes in Lepidoptera

Trypsins and chymotrypsins are well-studied serine peptidases that cleave peptide bonds at the carboxyl side of basic and hydrophobic l-amino acids, respectively. These enzymes are largely responsible for the digestion of proteins. Three primary processes regulate the activity of these peptidases: secretion, precursor (zymogen) activation and substrate-binding site recognition. Here, we present a detailed phylogenetic analysis of trypsins and chymotrypsins in three orders of holometabolous insects and reveal divergent characteristics of Lepidoptera enzymes in comparison with those of Coleoptera and Diptera. In particular, trypsin subsite S1 was more hydrophilic in Lepidoptera than in Coleoptera and Diptera, whereas subsites S2–S4 were more hydrophobic, suggesting different substrate preferences. Furthermore, Lepidoptera displayed a lineage-specific trypsin group belonging only to the Noctuidae family. Evidence for facilitated trypsin auto-activation events were also observed in all the insect orders studied, with the characteristic zymogen activation motif complementary to the trypsin active site. In contrast, insect chymotrypsins did not seem to have a peculiar evolutionary history with respect to their mammal counterparts. Overall, our findings suggest that the need for fast digestion allowed holometabolous insects to evolve divergent groups of peptidases with high auto-activation rates, and highlight that the evolution of trypsins led to a most diverse group of enzymes in Lepidoptera.     

Human leukocyte cathepsin G. Subsite mapping with 4-nitroanilides, chemical modification, and effect of possible cofactors

The extended substrate binding site of cathepsin G from human leukocytes has been mapped by using a series of peptide 4-nitroanilide substrates. The enzyme has a significant preference for substrates with a P1 Phe over those with the other aromatic amino acids Tyr and Trp. The S2 subsite was mapped with the substrates Suc-Phe-AA-Phe-NA where AA was 13 of the 20 amino acid residues commonly found in proteins. The best residues were Pro and Met. The S3 subsite was mapped with the sequence Suc-AA-Pro-Phe-NA by using 14 different amino acid residues for AA. The two best residues were the isosteric Val and Thr. No significant improvement in reactivity was obtained by extending the substrate to include seven different P4 residues. The kinetic parameters for cathepsin G are significantly slower than those for many other serine proteases. Changes in the reaction conditions and addition of possible cofactors or ligands were in general found to have little effect on the enzymatic activity, while chemical modifications and proteolysis destroyed the activity of cathepsin G. Cathepsin G hydrolyzed peptides containing model desmosine residues and prefers the hydrophobic picolinoyllysine derivative over lysine by substantial margins at both the S4 and S2 subsites but will not tolerate it at S3. Substrates with sequences related to the cathepsin G cleavage site in angiotensin I and angiotensinogen, and the reactive site of alpha 1-antichymotrypsin, were hydrolyzed effectively by enzyme, but with unexceptional rates. Our results indicate that the natural substrate(s) and function(s) of cathepsin G still remain to be discovered.     

Defining the substrate specificity of mouse cathepsin P

Cathepsin P is a recently discovered placental cysteine protease that is structurally related to the more ubiquitously expressed, broad-specificity enzyme, cathepsin L. We studied the substrate specificity requirements of recombinant mouse cathepsin P using fluorescence resonance energy transfer (FRET) peptides derived from the lead sequence Abz-KLRSSKQ-EDDnp (Abz, ortho-aminobenzoic acid and EDDnp, N-[2,4-dinitrophenyl]ethylenediamine). Systematic modifications were introduced resulting in five series of peptides to map the S(3) to S(2)(') subsites of the enzyme. The results indicate that the subsites S(1), S(2), S(1)('), and S(2)('), present a clear preference for hydrophobic residues. The specificity requirements of the S(2) subsite were found to be more restricted, preferring hydrophobic aliphatic amino acids. The S(3) subsite of the enzyme presents a broad specificity, accepting negatively charged (Glu), positively charged (Lys, Arg), and hydrophobic aliphatic or aromatic residues (Val, Phe). For several substrates, the activity of cathepsin P was markedly regulated by kosmotropic salts, particularly Na(2)SO(4). No significant effect on secondary or tertiary structure could be detected by either circular dichroism or size exclusion chromatography, indicating that the salts most probably disrupt unfavorable ionic interactions between the substrate and enzyme active site. A substrate based upon the preferred P(3) to P(2)(') defined by the screening study, ortho-aminobenzoic-Glu-Ile-Phe-Val-Phe-Lys-Gln-N-(2,4-dinitrophenyl)ethylenediamine (cleaved at the Phe-Val bond) was efficiently hydrolyzed in the absence of high salt. The k(cat)/K(m) for this substrate was almost two orders of magnitude higher than that of the original parent compound. These results show that cathepsin P, in contrast to other mammalian cathepsins, has a restricted catalytic specificity.     

Compared action of neutrophil proteinase 3 and elastase on model substrates. Favorable effect of S'-P' interactions on proteinase 3 catalysts

Neutrophil proteinase 3 (Pr3) and elastase (NE) may cause lung tissue destruction in emphysema and cystic fibrosis. These serine proteinases have similar P(1) specificities. We have compared their catalytic activity using acyl-tetrapeptide-p-nitroanilides, which occupy the S(5)-S'(1) subsites of their substrate binding site, and intramolecularly quenched fluorogenic heptapeptides, which bind at S(5)-S'(4). Most p-nitroanilide substrates are turned over slowly by Pr3 as compared with NE. These differences disappear with the fluorogenic heptapeptides, some of which are hydrolyzed even faster by Pr3 than by NE. Elongation of substrates strongly increases the catalytic efficiency of Pr3, whereas it has little effect on NE catalysis. These different sensitivities to S'-P' interactions show that Pr3 and NE are not interchangeable enzymes despite their similar P(1) specificity.