The Function of Propeptide Domains of Cysteine Proteinases

The interactions between egg-white cystatin and the cysteine proteinases papain, human cathepsin B and bovine dipeptidyl peptidase I were studied. Cystatin was shown to be a competitive reversible inhibitor of cathepsin B (Ki 1.7 nM, k-1 about 2.3×10(-3) s-1). The inhibition of dipeptidyl peptidase I was shown to be reversible (Ki(app.) 0.22 nM, k-1 about 2.2×10(-3) s-1). Cystatin bound papain too tightly for Ki to be determined, but an upper limit of 5 pM was estimated. The association was a second-order process, with k+1 1.0×10(7) M-1×s-1. Papain was shown to form equimolar complexes with cystatin. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of complexes formed between papain or cathepsin B and an excess of cystatin showed no peptide bond cleavage after incubation for 72 h. The reaction of the active-site thiol group of papain with 5,5′-dithiobis-(2-nitrobenzoic acid) at pH 8 and 2,2′-dithiobispyridine at pH 4 was blocked by complex-formation. Dipeptidyl peptidase I and papain were found to compete for binding to cystatin, contrary to a previous report. The two major isoelectric forms of cystatin were found to have similar specific inhibitory activities for papain, and similar affinities for papain, cathepsin B and dipeptidyl peptidase I. This, together with specific oxidation of the N-terminal serine residue with periodate, showed the N-terminal amino group of cystatin 1 to be unimportant for inhibition. General citraconylation of amino groups resulted in a large decrease in the affinity of cystatin for dipeptidyl peptidase I. It is concluded that the interaction of cystatin with cysteine proteinases has many characteristics similar to those of an inhibitor such as aprotinin with serine proteinases.

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Grafting of Features of Cystatins C or B into the N-Terminal Region or Second Binding Loop of Cystatin A (Stefin A) Substantially Enhances Inhibition of Cysteine Proteinases†

Biochemistry ◽

10.1021/bi030119v ◽

2003 ◽

Vol 42 (38) ◽

pp. 11326-11333 ◽

Cited By ~ 18

Author(s):

Alona Pavlova ◽

Ingemar Björk

Keyword(s):

Terminal Region ◽

Cysteine Proteinases ◽

Stefin A ◽

Cystatin A ◽

Binding Loop

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Quantification of the expression levels of lysosomal cysteine proteinases in purified human osteoclastic cells by competitive RT-PCR

Calcified Tissue International ◽

10.1007/bf02678149 ◽

2001 ◽

Vol 68 (2) ◽

pp. 109-116 ◽

Cited By ~ 14

Author(s):

O. Ishibashi ◽

T. Inui ◽

Y. Mori ◽

T. Kurokawa ◽

T. Kokubo ◽

...

Keyword(s):

Cysteine Proteinases ◽

Rt Pcr ◽

Expression Levels

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Use of inhibitors to identify essential cysteine proteinases of Trichomonas vaginalis

FEMS Microbiology Letters ◽

10.1111/j.1574-6968.1997.tb10306.x ◽

2006 ◽

Vol 149 (1) ◽

pp. 45-50 ◽

Cited By ~ 12

Author(s):

Joseph W Irvine ◽

Michael J North ◽

Graham H Coombs

Keyword(s):

Trichomonas Vaginalis ◽

Cysteine Proteinases

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Inhibition of bone resrption by selctive inactivators of cysteine proteinases

Journal of Cellular Biochemistry ◽

10.1002/jcb.240560116 ◽

1994 ◽

Vol 56 (1) ◽

pp. 118-130 ◽

Cited By ~ 89

Author(s):

Peter A. Hill ◽

David J. Buttle ◽

Sheila J. Jones ◽

Alan Boyde ◽

Mitsuo Murata ◽

...

Keyword(s):

Cysteine Proteinases

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The structure and mechanism of action of papain

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1970.0023 ◽

1970 ◽

Vol 257 (813) ◽

pp. 237-248 ◽

Cited By ~ 61

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.

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