scholarly journals A general framework of cysteine-proteinase mechanism deduced from studies on enzymes with structurally different analogous catalytic-site residues Asp-158 and −161 (papain and actinidin), Gly-196 (cathepsin B) and Asn-165 (cathepsin H). Kinetic studies up to pH 8 of the hydrolysis of N-α-benzyloxycarbonyl-l-arginyl-l-arginine 2-naphthylamide catalysed by cathepsin B and of l-arginine 2-naphthylamide catalysed by cathepsin H.

1985 ◽  
Vol 227 (2) ◽  
pp. 521-528 ◽  
Author(s):  
F Willenbrock ◽  
K Brocklehurst
Keyword(s):  
Catalytic Activity ◽  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Ion Pairs ◽  
Preceding Paper ◽  
Structural Features ◽  
Catalytic Site ◽  
Cysteine Proteinases ◽  
Cathepsin H ◽  
Ph Range

The pH-dependences of kcat, Km and kcat./Km for the hydrolysis at 25 degrees C at I 0.1 of L-arginine 2-naphthylamide catalysed by cathepsin H from bovine spleen were determined in the pH range approx. 4-8. The pH-dependences of these kinetic parameters were determined also for the hydrolysis at 25 degrees C at I 0.1 of N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide catalysed by cathepsin B (EC 3.4.22.1) from bovine spleen in the pH range 7-8, which extends the studies in acidic media reported by Willenbrock & Brocklehurst [(1984) Biochem. J. 222, 805-814]. These results are discussed and related to those from the reactivity-probe kinetics reported in the preceding paper [Willenbrock & Brocklehurst (1985) Biochem. J. 227, 511-519] and to known structural features present in rat liver cathepsins B and H and in papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14). Consideration of the kinetic data leads to the suggestion that in the cysteine proteinases rearrangement of intimate S-/ImH+ ion-pairs in catalytic sites is brought about by a combination of field effects in the immediate vicinity of the ion-pair and consequences of protonic dissociation of a group with pKa 5-6 remote from the catalytic site. The contributions of the two types of effect seem to differ from enzyme to enzyme. Of the four cysteine proteinases considered, only cathepsin B exerts an absolute requirement for the proton-deficient form of a group with pKa 5-6 for catalytic activity. Protonic dissociation with pKa 5-6 enhances catalytic activity in cathepsin H and in actinidin and appears to have little or no effect in papain. Only cathepsin B lacks a polar or negatively charged side chain in the residue analogous to Asp-158 in papain, and this is suggested to account for its total dependence on a protonic dissociation remote from the catalytic site.

scholarly journals Preparation of cathepsins B and H by covalent chromatography and characterization of their catalytic sites by reaction with a thiol-specific two-protonic-state reactivity probe. Kinetic study of cathepsins B and H extending into alkaline media and a rapid spectroscopic titration of cathepsin H at pH 3-4

1985 ◽  
Vol 227 (2) ◽  
pp. 511-519 ◽  
Author(s):  
F Willenbrock ◽  
K Brocklehurst
Keyword(s):  
Cathepsin B ◽  
Thiol Group ◽  
Catalytic Site ◽  
Alkaline Media ◽  
Cysteine Proteinases ◽  
Acidic Media ◽  
Active Centre ◽  
Ph Values ◽  
Cathepsin H ◽  
Covalent Chromatography

A procedure for the isolation of cathepsin B (EC 3.4.22.1) and of cathepsin H from bovine spleen involving covalent chromatography by thiol-disulphide interchange and ion-exchange chromatography was devised. The stabilities of both cathepsins in alkaline media are markedly temperature-dependent, and reliable kinetic data can be obtained at pH values up to 8 by working at 25 degrees C with a continuous spectrophotometric assay. Both enzyme preparations contain only one type of thiol group as judged by reactivity characteristics towards 2,2′-dipyridyl disulphide at pH values up to 8; in each case this thiol group is essential for catalytic activity. Cathepsin H was characterized by kinetic analysis of the reactions of its thiol group with 2,2′-dipyridyl disulphide in the pH range approx. 2-8 and the analogous study on cathepsin B [Willenbrock & Brocklehurst (1984) Biochem. J. 222, 805-814] was extended to include reaction at pH values up to approx. 8. Cathepsin H, like the other cysteine proteinases, was shown to contain an interactive catalytic-site system in which the nucleophilic character of the sulphur atom is maintained in acidic media. The considerable differences in catalytic site characteristics detected by this two-protonic-state reactivity probe between cathepsin B, cathepsin H, papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) are discussed. Reaction with 2,2′-dipyridyl disulphide in acidic media, which is known to provide a rapid spectrophotometric active centre titration for many cysteine proteinases, is applicable to cathepsin H. This is useful because other active-centre titrations have proved unsuitable in view of the relatively low reactivity of the thiol group in cathepsin H.

scholarly journals A marked gradation in active-centre properties in the cysteine proteinases revealed by neutral and anionic reactivity probes. Reactivity characteristics of the thiol groups of actinidin, ficin, papain and papaya peptidase A towards 4,4′-dipyridyl disulphide and 5,5′-dithiobis-(2-nitrobenzoate) dianion

1983 ◽  
Vol 209 (3) ◽  
pp. 873-879 ◽  
Author(s):  
K Brocklehurst ◽  
S M Mushiri ◽  
G Patel ◽  
F Willenbrock
Keyword(s):  
Negative Charge ◽  
Cysteine Proteinase ◽  
Catalytic Site ◽  
Side Chain ◽  
Cysteine Proteinases ◽  
Thiol Groups ◽  
Active Centre ◽  
Ph Range ◽  
Thiolate Anions ◽  
Kinetics Of

1. The kinetics of the reactions of the catalytic-site thiol groups of actinidin (the cysteine proteinase from Actinidia chinensis), ficin (EC 3.4.22.3), papain (EC 3.4.22.2) and papaya peptidase A (the other monothiol cysteine proteinase component of Carica papaya) with 4,4′-dipyridyl disulphide (4-Py-S-S-4-Py) and with 5,5′-dithiobis-(2-nitrobenzoate) dianion (Nbs22-) were studied in the pH range approx. 6-10. These studies provided the pH-independent second-order rate constants (k) for the reactions of the two probe reagents with the catalytic-site thiolate anions each in the environment of a neutral histidine side chain where an active-centre carboxy group would be ionized. 2. The ratio R equal to kNbs22-/k4-Py-S-S-4-Py provides an index of the catalytic-site solvation properties of the four cysteine proteinases and varies markedly from one enzyme to another, being 0.80 for papaya peptidase A (0.86 for the model thiol, 2-mercaptoethanol), 29 for actinidin, 0.18 for ficin and 0.015 for papain. These differences appear to derive mainly from the response of the enzyme to the negative charge on Nbs22-. 3. Possible implications of these results for (a) mechanisms of cysteine proteinase catalysis and (b) the possibility of using series of functionally related enzymes in the study of mechanism are discussed.

scholarly journals Chemical evidence for the pH-dependent control of ion-pair geometry in cathepsin B. Benzofuroxan as a reactivity probe sensitive to differences in the mutual disposition of the thiolate and imidazolium components of cysteine proteinase catalytic sites

1986 ◽  
Vol 238 (1) ◽  
pp. 103-107 ◽  
Author(s):  
F Willenbrock ◽  
K Brocklehurst
Keyword(s):  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Ph Dependence ◽  
Thiol Group ◽  
Ion Pair ◽  
Catalytic Site ◽  
Alkaline Media ◽  
Weakly Alkaline ◽  
Stem Bromelain ◽  
Ph Range

Benzofuroxan reacts with the catalytic-site thiol group of cathepsin B (EC 3.4.22.1) to produce stoichiometric amount of the chromophoric reduction product, o-benzoquinone dioxime. In a study of the pH-dependence of the kinetics of this reaction, most data were collected for the bovine spleen enzyme, but the more limited data collected for the rat liver enzyme were closely similar both in the magnitude of the values of the second-order rate constants (k) and in the shape of the pH-k profile. In acidic and weakly alkaline media, the reaction is faster than the reactions of benzofuroxan with some other cysteine proteinases. For example, in the pH region around 5-6, the reaction of cathepsin B is about 10 times faster than that of papain, 15 times faster than that of stem bromelain and 6 times faster than that of ficin. The pH-dependence of k for the reaction of cathepsin B with benzofuroxan was determined in the pH range 2.7-8.3. In marked contrast with the analogous reactions of papain, ficin and stem bromelain [reported by Shipton & Brocklehurst (1977) Biochem. J. 167, 799-810], the pH-k profile for the cathepsin B reaction contains a sigmoidal component with pKa 5.2 in which k increases with decrease in pH. This modulation of the reactivity of the catalytic-site -S-/-ImH+ ion-pair state of cathepsin B (produced by protonic dissociation from -SH/-ImH+ with pKa approx. 3) towards a small, rigid, electrophilic reagent, in a reaction that appears to involve both components of the ion-pair for efficient reaction, suggests that the state of ionization of a group associated with a molecular pKa of approx. 5 may control ion-pair geometry. This might account for the remarkable finding [reported by Willenbrock & Brocklehurst (1984) Biochem. J. 222, 805-814] that, although the ion-pair appears to be generated in cathepsin B as the pH is increased across pKa 3.4, catalytic competence is not generated until the pH is increased across pKa 5-6.

The structure and mechanism of action of papain

1970 ◽  
Vol 257 (813) ◽  
pp. 237-248 ◽  
Keyword(s):  
Mechanism Of Action ◽  
Catalytic Mechanism ◽  
Proteolytic Enzymes ◽  
Cysteine Proteinase ◽  
Preceding Paper ◽  
Cysteine Residue ◽  
Enzymic Activity ◽  
Critical Survey ◽  
Cysteine Proteinases ◽  
Stem Bromelain

The cysteine proteinases form a group of enzymes which depend for their enzymic activity on the thiol group of a cysteine residue. Several which occur in plants have been investigated extensively and include papain, ficin and stem bromelain (Smith & Kimmel i960). Although the term papain, introduced last century to describe the proteolytic principle in papaya latex (Wurtz & Bouchut 1879) is still used to describe crude dried latex, the crystalline enzyme is readily obtained (Kimmel & Smith 1954). Ficin is known to consist of several closely related enzymes which have been resolved (Sgarbieri, Gupte, Kramer & Whitaker 1964), but for most structural and mechanistic studies the unresolved mixture of enzymes has been used. Stem bromelain also appears to be a mixture of at least two proteolytic enzymes which have not yet been resolved (Ota, Moore & Stein 1962; Murachi 1964). In spite of the recognized heterogeneity of ficin and stem bromelain, it does seem that both structurally and mechanistically they are similar to papain. Only one bacterial cysteine proteinase has received a detailed study, namely, streptococcal proteinase, and it appears to have little or no relation in its amino acid sequence with the plant enzymes (Liu, Stein, Moore & Elliott 1965). The functional groups involved in the catalytic mechanism are apparently the same as in the plant proteinases (Gerwin, Stein & Moore 1966; Liu 1967; Husain & Lowe 1968 a , c ), but the mechanism of action has not been extensively studied. It may well be however that the plant and bacterial cysteine proteinases have converged onto a similar mechanism of action by two independent evolutionary pathways, as now seems apparent for the animal and bacterial serine proteinases (Alden, Wright & Kraut, this volume, p. 119). Because the tertiary crystal structure of papain (Drenth, Jansonius, Koekoek, Swen & Wolthers 1968; see also the preceding paper, p. 231) is now known, a critical survey of this enzyme is apposite.

scholarly journals Demonstration of cathepsins B, H and L in xenografts of normal and Duchenne-muscular-dystrophy muscles transplanted into nude mice

1992 ◽  
Vol 288 (2) ◽  
pp. 643-648 ◽  
Author(s):  
A Takeda ◽  
T Jimi ◽  
Y Wakayama ◽  
N Misugi ◽  
S Miyake ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Nude Mice ◽  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Normal Subjects ◽  
Muscle Fibres ◽  
Cysteine Proteinase Inhibitor ◽  
Normal Muscle ◽  
Cathepsin H

The activities and contents of the lysosomal cysteine proteinases cathepsins B, H and L were examined in xenografts of biopsied muscles transplanted from age-matched normal subjects and Duchenne-muscular-dystrophy (DMD) patients into nude mice. The activity of cathepsin B increased 9-fold and that of B-plus-L increased 24-fold in the first week after transplantation in normal muscle xenografts. By the third week, the activity of cathepsin B increased a total of 20-fold and B-plus-L increased to 36-fold the original level. The activity levels of cathepsin B, B-plus-L, H and D, and acid phosphatase in normal and DMD xenografts were not significantly different when compared 2 weeks after transplantation. However, the protein content of cathepsin B in DMD muscle xenografts was more than 3-fold that of normal xenografts at 2 weeks. The profile of cathepsin H activity in normal muscle xenografts was different than those of cathepsins B and B-plus-L. In the first week, the cathepsin H diminished sharply to about one-third of the biopsied muscle level and then, by 3 weeks after transplantation, it had increased slightly to about half the original level. The amount of endogenous cysteine-proteinase inhibitor changed in parallel with the activity of cathepsins B and B-plus-L. Cathepsins B and H, but not cathepsin L, were found immunohistochemically in regenerating muscle fibres of normal and DMD xenografts 2 weeks after transplantation. Staining of cathepsin B in DMD xenografts was slightly stronger than that in normal subjects. There was no immunostaining in degenerating or necrotic muscle fibres 2 weeks after transplantation. Western-blot analysis revealed that the cathepsin B band at 29 kDa was increased in normal xenografts 2 and 3 weeks after transplantation. Also, 2 weeks after transplantation the staining intensity of this band was slightly stronger in DMD xenografts than in normal xenografts. These results suggest that cathepsin B participates in the regeneration of transplanted muscle, both normal and DMD, and in the DMD muscle fibre-wasting processes, during regeneration.

scholarly journals Natural structural variation in enzymes as a tool in the study of mechanism exemplified by a comparison of the catalytic-site structure and characteristics of cathepsin B and papain. pH-dependent kinetics of the reactions of cathepsin B from bovine spleen and from rat liver with a thiol-specific two-protonic-state probe (2,2′-dipyridyl disulphide) and with a specific synthetic substrate (N-α-benzyloxycarbonyl-l-arginyl-l-arginine 2-naphthylamide)

1984 ◽  
Vol 222 (3) ◽  
pp. 805-814 ◽  
Author(s):  
F Willenbrock ◽  
K Brocklehurst
Keyword(s):  
Liver Enzyme ◽  
Rat Liver ◽  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Ion Pair ◽  
Catalytic Site ◽  
Site Structure ◽  
Catalytic Sites ◽  
Kinetics Of ◽  
Hydrolysis Of

Cathepsin B (EC 3.4.22.1) from bovine spleen and the analogous enzyme from rat liver were investigated at 25 degrees C at I0.1 in acidic media by kinetic study of (a) the reactions of their catalytic-site thiol groups towards the two-protonic-state reactivity probe 2,2′-dipyridyl disulphide and (b) their catalysis of the hydrolysis of N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide. Reactivity-probe kinetics showed that nucleophilic character is generated in the sulphur atom of cathepsin B by protonic dissociation with pKa 3.4, presumably to form an S-/ImH+ ion-pair. Substrate-catalysis kinetics showed that ion-pair formation is not sufficient to generate catalytic competence in cathepsin B, because catalytic activity is not generated as the pH is raised across pKa 3.4 but rather as it is raised across pKa 5-6 (5.1 for kcat; 5.6 for kcat./Km for the bovine spleen enzyme and 5.8 for kcat./Km for the rat liver enzyme). The implications of these results and of known structural differences between the catalytic sites of the rat liver enzyme and papain (EC 3.4.22.2) for the mechanism of cysteine-proteinase-catalysed hydrolysis are discussed.

scholarly journals Activity of lysosomal cysteine proteinase during differentiation of rat skeletal muscle

1983 ◽  
Vol 214 (3) ◽  
pp. 871-877 ◽  
Author(s):  
H Kirschke ◽  
L Wood ◽  
F J Roisen ◽  
J W C Bird
Keyword(s):  
Cysteine Proteinase ◽  
Petri Dish ◽  
Cysteine Proteinases ◽  
Endogenous Inhibitor ◽  
Rat Skeletal Muscle ◽  
L6 Cells ◽  
Tissue Extracts ◽  
Cell Extracts ◽  
Cathepsin H ◽  
Diameter Petri Dish

Cysteine-proteinase activities were measured in extracts of pre- and post-fusion populations of rat myogenic line L6 cells and in extracts of whole rat muscle. Activities of cathepsins B, L and H were compared. The substrates used included Z-Phe-Arg-NMec (cathepsins B and L), Z-Arg-Arg-NMec (cathepsin B), and Arg-NMec (cathepsin H) (where Z = benzyloxycarbonyl, and NMec = 4-methyl-7-coumarylamide); the enzyme activities were more specifically differentiated by appropriate concentrations of the inhibitors Z-Phe-Phe-CHN2 (CHN2 = diazomethane), bestatin and E-64 [L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane]. These experiments have demonstrated the feasibility of determining the cysteine-proteinase activities of myoblasts from a single (60 mm-diameter) Petri dish, with enzyme concentrations in the range of 5-20 ng/ml. Specific activities of the enzymes in L6 cells increased 2-20-fold after fusion. Concentrations of cysteine proteinases in extracts from cultured myoblasts were two orders of magnitude greater than those in muscle-tissue extracts. Cultured-cell extracts contained endogenous inhibitor(s) to purified rat cathepsins B, L and H.

scholarly journals Biotin-labelled peptidyl diazomethane inhibitors derived from the substrate-like sequence of cystatin: targeting of the active site of cruzipain, the major cysteine proteinase of Trypanosoma cruzi

1996 ◽  
Vol 318 (2) ◽  
pp. 395-399 ◽  
Author(s):  
Gilles LALMANACH ◽  
Roger MAYER ◽  
Carole SERVEAU ◽  
Julio SCHARFSTEIN ◽  
Francis GAUTHIER
Keyword(s):  
Trypanosoma Cruzi ◽  
Cathepsin B ◽  
Active Sites ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Cysteine Proteinases ◽  
Irreversible Inhibitors ◽  
Human Cystatin C ◽  
Spacer Arm ◽  
Biotin Labelling

Biotin-labelled peptidyl diazomethane inhibitors of cysteine proteinases, based on the N-terminal substrate-like segment of human cystatin C, a natural inhibitor of cysteine proteinases, were synthesized. These synthetic derivatives were tested as irreversible inhibitors of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, to compare the kinetics of the inhibition of the parasite proteinase with that of the mammalian cathepsins B and L. The accessibility of the active sites of these proteinases to these probes was also investigated. The inhibition of cruzipain by Biot-LVG-CHN2 (where Biot represents biotinyl and L,V and G are single-letter amino acid residue abbreviations) and Biot-Ahx-LVG-CHN2 (where Ahx represents 6-aminohexanoic acid) was similar to that of unlabelled inhibitor. Biotin labelling of the inhibitor slowed the inhibition of both cathepsin B and cathepsin L. Adding a spacer arm (Ahx) between the biotin and the peptide moiety of the derivative increased the inhibition of cathepsin B but not that of cathepsin L. The discrimination provided by this spacer is probably due to differences in the topologies of the binding sites of proteinases, a feature that can be exploited to improve targeting of individual cysteine proteinases. Analysis of the blotted proteinases revealed marked differences in the accessibility of extravidin–peroxidase conjugate to the proteinase-bound biotinylated inhibitor. Cruzipain molecules exposed to Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 were readily identified, but the reaction was much stronger when the enzyme was treated with the spacer-containing inhibitor. In contrast with the parasite enzyme, rat cathepsin B and cathepsin L treated with either Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 produced no detectable bands. Papain, the archetype of this family of proteinases, was poorly labelled with Biot-LVG-CHN2, but strong staining was obtained with Biot-Ahx-LVG-CHN2. These findings suggest that optimized biotinylated diazomethanes might considerably improve their selectivity for the T. cruzi target enzyme.

scholarly journals Peptide glyoxals: a novel class of inhibitor for serine and cysteine proteinases

1993 ◽  
Vol 293 (2) ◽  
pp. 321-323 ◽  
Author(s):  
B Walker ◽  
N McCarthy ◽  
A Healy ◽  
T Ye ◽  
M A McKervey
Keyword(s):  
Inhibitory Activity ◽  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Cysteine Proteinases ◽  
Reversible Inhibitors ◽  
Pancreatic Elastase ◽  
Porcine Pancreatic Elastase

A series of novel synthetic dipeptides, containing a C-terminal glyoxal grouping (-COCHO), have been tested as inhibitors against typical members of the serine- and cysteine-proteinase families. For example, the sequences benzyloxycarbonyl (Cbz)-Pro-Phe-CHO (I) and Cbz-Phe-Ala-CHO (II), which fulfil the known primary and secondary specificity requirements of chymotrypsin and cathepsin B respectively, have been found to be potent reversible inhibitors of their respective target proteinase. Thus I was found to inhibit chymotrypsin with a Ki of approximately 0.8 microM, whereas II exhibits a Ki of approximately 80 nm against cathepsin B. These Ki values are some 10-fold and 3-fold lower than those reported for the corresponding peptide-aldehyde inhibitors of chymotrypsin and cathepsin B upon which the peptidyl-glyoxals were fashioned. Unexpectedly, the sequence Cbz-Pro-Ala-CHO, which was designed to inhibit elastase-like proteinases, exhibited no inhibitory activity towards porcine pancreatic elastase, even when used at concentrations as high as 200 microM.

scholarly journals Coexistence of renin and cathepsin B in secretory granules of granular duct cells in male mouse submandibular gland.

1993 ◽  
Vol 41 (3) ◽  
pp. 433-438 ◽  
Author(s):  
K Sano ◽  
S Waguri ◽  
N Sato ◽  
E Kominami ◽  
Y Uchiyama
Keyword(s):  
Submandibular Gland ◽  
Golgi Complex ◽  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Secretory Granules ◽  
Interstitial Cells ◽  
Double Immunostaining ◽  
Duct Cells ◽  
Cathepsin H ◽  
Rat Pituitary

Cathepsin B, a representative lysosomal cysteine proteinase, has been demonstrated to coexist with renin in secretary granules of rat pituitary LH/FSH cells and renal juxtaglomerular cells. We investigated immunocytochemically the localization of cathepsins B, H, and L in the submandibular gland of male mice, in which active renin is also produced. By light microscopy, granular immunodeposits for cathepsin B were detected in epithelial cells of the gland, particularly in granular duct cells and interstitial cells. Immunoreactivity for cathepsins H and L was mainly found in interstitial cells, although that for cathepsin H was weakly seen in acinar cells. By electron microscopy, immunogold particles indicating cathepsin B intensely labeled small granules near the Golgi complex of granular duct cells and weakly labeled large secretory granules, whereas those showing renin labeled both granules. Double immunostaining co-localized immunogold particles showing renin and cathepsin B in small perinuclear granules near the Golgi complex. Some immunopositive granules seemed to be closely associated with the Golgi elements. These results indicate that the co-localization of renin and cathepsin B is also seen in secretory granules of granular duct cells in the mouse submandibular gland, as seen in rat juxtaglomerular and LH/FSH cells. This suggests that cathepsin B is one of the possible candidates for the renin-processing enzyme.

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