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The Effect of C-terminal Domain Deletion on the Catalytic Activity of Leishmania Donovani Surface Proteinase GP63: Role of Ser446 in Proteolysis

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Title The Effect of C-terminal Domain Deletion on the Catalytic Activity of Leishmania Donovani Surface Proteinase GP63: Role of Ser446 in Proteolysis
 
Creator Mazumder, Saumyabrata
Ganguly, Agneyo
Ali, Nahid
 
Subject Infectious Diseases and Immunology
 
Description The kinetoplastid protozoan Leishmania encodes major surface glycoprotein GP63, a zinc metallopeptidase
(EC.3.4.24.36) expressed both in promastigote and amastigote life stages. In the present study,
we explored for the first time the role of C-terminal domain (CTD) in proteinase activity by
serial truncation of Leishmania donovani GP63 (LdGP63) from carboxyl terminal end (CTend). Deletion of
180e211 amino acids from CTend (D420 and D389) resulted in almost 50% loss of catalytic activity
against azocasein, casein and gelatin. Moreover, all the truncated constructs showed reduced activity
towards immunoglobulin (IgG). Upon homology modeling, we identified two residues, S446, and F448 in
CTD, conserved in different Leishmania species, which were positioned 6.8e11 Å apart from the active
site. To ascertain the role of S446 and F448 in catalysis, we replaced S446 with Ala and Thr, and F448
with Val and Tyr by site-directed mutagenesis. The variant enzymes (S446T, F448V, and F448Y) maintained
near wild-type activity, whereas S446A demonstrated 50% loss of catalytic activity towards the
cleavage of various biological substrates. Kinetic analysis of S446A resulted in a 2.6-fold decrease in
the affinity, 10-fold decrease in turn-over rates, and large increase in transition-state binding energy
(1.4 kcal/mol) for the quenched peptide substrates. These results emphasize the relevance of CTD in the
proteolytic activity of LdGP63. Fluorescence spectroscopy, and CD analysis however, indicated that the
reduced activities showed by D389 and S446A were not due to global changes in the enzyme structures.
Indeed, identification of S446 and its possible role in the stabilization of transition-state binding between
enzyme and substrate can be exploited towards understanding of structureefunction relationship of
GP63.
 
Date 2010
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/960/1/4_bio_2010.pdf
Mazumder, Saumyabrata and Ganguly, Agneyo and Ali, Nahid (2010) The Effect of C-terminal Domain Deletion on the Catalytic Activity of Leishmania Donovani Surface Proteinase GP63: Role of Ser446 in Proteolysis. Biochimie, 92 (12). pp. 1876-1885.
 
Relation http://dx.doi.org/10.1016/j.biochi.2010.07.014
http://www.eprints.iicb.res.in/960/