PepV from Lactobacillus delbrueckii, a dinuclear zinc peptidase, has been characterized as an unspecific amino dipeptidase. The crystal structure of PepV in complex with the phosphinic inhibitor AspPsi[PO(2)CH(2)]AlaOH, a dipeptide substrate mimetic, reveals a "catalytic domain" and a "lid domain," which together form an internal active site cavity that traps the inhibitor. The catalytic domain is topologically similar to catalytic domains from amino- and carboxypeptidases. However, the lid domain is unique among the related enzymes. In contrast to the other related exopeptidases, PepV recognizes and fixes the dipeptide backbone, while the side chains are not specifically probed and can vary, rendering it a nonspecific dipeptidase. The cocrystallized inhibitor illustrates the two roles of the two catalytic zinc ions, namely stabilization of the tetrahedral intermediate and activation of the catalytic water molecule.
Human anaphylatoxin C3a, derived from complement component C3 has been crystallized and its crystal structure determined at 3.2 A resolution based on multiple isomorphous replacement. The electron density map was interpretable in terms of the known chemical sequence and a molecular model constructed. The molecule has dimensions of 42 22 16 A. It resembles a drumstick. It is constructed from two helical segments Tyr 15 to Met 27 and Gly 46 to Ser 71 connected by a loop. Residues 1 to 14 are flexible. The C-terminal residues are in irregular conformation. The crystal structure analysis establishes the disulphide linkages as Cys 22 --Cys 49 , Cys 23 -Cys 56 , Cys 36 -Cys 57 . Kristallstrukturanalyse und Molekülmodell des menschlichen C3a-Anaphylatoxins Zusammenfassung: Menschliches Anaphylatoxin C3a, gewonnen von der Komplement-Komponente C3, wurde kristallisiert und die Kristallstruktur bei 3.2 Ä Auflösung durch multiplen isomorphen Ersatz bestimmt. Die Elektronendichtekarte konnte nach der bekannten chemischen Aminosäuresequenz interpretiert werden. Ein Molekularmodell wurde aufgebaut. Das Molekül hat Dimensionen von 42 22 16 Ä. Es ähnelt einem Trommelstock. Es besteht aus zwei helikalen Segmenten Tyr 15 bis Met 27 und Gly 46 bis Ser 71 , die durch eine Peptidschleife verbunden sind. Die Reste l bis 14 sind flexibel. Die C-terminalen Reste sind unregelmäßig gefaltet. Die Kristallstrukturanalyse erlaubt die Festlegung der Disulfidbrücken: Cys 22 -Cys 49 , Cys 23 -Cys 56 , Cys 36 --Cys 57 .
Based on the Entamoeba histolytica genome project (www.sanger.ac.uk/Projects/E_histolytica/) we have identified a cysteine protease inhibitor, EhICP1 (amoebiasin 1), with significant homology to chagasin. Recombinant EhICP1 inhibited the protease activity of papain and that of a trophozoite lysate with K i Õs in the picomolar range. By immunocytology, we localized the endogenous $13 kDa EhICP1 in a finely dotted subcellular distribution discrete from the vesicles containing the amoebic cysteine protease, EhCP1 (amoebapain). In an overlay assay, we observed binding of recombinant EhICP1 to EhCP1. As a heptapeptide (GNPTTGF) corresponding to the second conserved chagasin motif inhibited the protease activity of both papain (K i 1.5 lM) and trophozoite extract (K i in sub-mM range), it may be a candidate for the rational development of anti-amoebiasis drugs.
The action of the major protease from the parasitic protozoon Entamoeba histolytica, a cysteine protease of Mr 27,000-29,000, on some important proteins of the extracellular matrix has been studied. The isolated protease degraded the extracellular matrix proteins from human tissue collagen type IV and V as well as laminin and fibronectin with different velocities and specificities under native conditions. Whereas the degradation of fibronectin and laminin proceeded rapidly, yielding distinct fragment patterns, the breakdown of the collagen types happened more slowly and incompletely. The digestion of the denatured isolated alpha 2-chain of bovine collagen type I was very fast and unspecific requiring only 1/10 of the enzyme activities as compared with the other substrates mentioned above. Nearly 85% of the overall proteolytic activity of a soluble fraction of E. histolytica was strongly inhibited by antibodies against the purified histolytic protease as well as by cystatin from chicken egg white, a specific protein inhibitor of cysteine proteases. We conclude that the histolytic protease represents by far the highest portion of soluble proteolytic activity in E. histolytica which is sufficient to destroy the extracellular matrix of the host.
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